EP3749328A1 - Préparation pour nourrissons fermentée comportant des oligosaccharides non digestibles - Google Patents

Préparation pour nourrissons fermentée comportant des oligosaccharides non digestibles

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Publication number
EP3749328A1
EP3749328A1 EP19703731.0A EP19703731A EP3749328A1 EP 3749328 A1 EP3749328 A1 EP 3749328A1 EP 19703731 A EP19703731 A EP 19703731A EP 3749328 A1 EP3749328 A1 EP 3749328A1
Authority
EP
European Patent Office
Prior art keywords
nutritional composition
oligosaccharide
intestinal
human subjects
metabolome
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP19703731.0A
Other languages
German (de)
English (en)
Inventor
Sebastian TIMS
Akhtar Raish OOZEER
Jan Knol
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nutricia NV
Original Assignee
Nutricia NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nutricia NV filed Critical Nutricia NV
Publication of EP3749328A1 publication Critical patent/EP3749328A1/fr
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7016—Disaccharides, e.g. lactose, lactulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs

Definitions

  • the present invention relates to the field of nutrition for infants and young children for improving intestinal microbiota.
  • the intestinal microbiota In human adults the intestinal microbiota is considered to be a stable ecosystem, hence the microbial colonization process in early-life, which is heavily intertwined with the maturation of the gastrointestinal tract itself, can be considered as fundamental step in healthy development.
  • Several environmental factors that can occur in early life have been shown to have long-lasting impact on the intestinal microbiota and its activity, thereby increasing the risk of diseases in later life.
  • Early-life nutrition is a major factor that impacts the developing intestinal microbiota community. Bifidobacterium species typically dominate the intestinal microbiota of breastfed infants, while an intestinal microbiota that is richer in Firmicutes members is typically observed in infants that are fed conventional formulae.
  • the composition of the intestinal microbiota that is relevant. Also to a large extent it is the function of the microbiota and/or the presence of metabolites that will impact the intestinal physiology and being and have an effect on health now and later in life. Differences are observed between breast fed infants and conventional formula fed infants in these respects, which may explain the why breast fed infants have an improved health outcome on many aspects relating to gut, immune system, brain and metabolic health.
  • Formulae supplemented with a prebiotic scGOS/lcFOS mixture have been shown to modulate the gut microbiota composition and function, i.e. the metabolic activity and fermentation profile was promoted towards what is found in human milk fed infants (Knol et al, 2005, JPGN 40:36- 42).
  • the effects of this scGOS/lcFOS mixture on the metabolic activity and fermentation profile was previously shown to be maintained to a large extent when added to a partly fermented, using Bifidobacterium breve and Streptococcus thermophilus, infant milk formulae (Huet, F. et al, 2016, JPGN 63:e43-53).
  • WO 2017/021476 relates to a nutritional composition comprising fucosylated and N-acetylated oligosaccharides for promoting or inducing a global gut microbiota that is closer in function to the one of infants fed exclusively with human breast milk, in comparison to infants fed with a conventional nutritional composition.
  • Metabolites portray a more final phenotype that are the results of all the genetically encoded functions in an ecosystem, as they represent the final sum of all activated genes, epigenetic expression modifications and other transcriptional regulations, posttranslational protein modifications, and environmental factors (both biotic and abiotic).
  • This untargeted data set showed that the infant gut ecosystem functioning was highly dependent on and reactive to the diet.
  • One example was the different profile in secondary bile salts, where the pattern in the experimental groups remained more similar to, or deviated less from, the pattern of the breastfed reference group.
  • the invention concerns a method for promoting the development in human subjects with an age of 36 months or below of an intestinal microbiota that is in function closer to the intestinal microbiota- function of human subjects at the same age fed with human milk, comprising administering a nutritional composition that comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, when compared to the intestinal microbiota-function of human subjects with the same age fed a nutritional composition not comprising non- digestible oligosaccharides.
  • the method according to the invention can be seen as a non-medical method for promoting the development of an intestinal microbiota-function.
  • the invention can also be worded as the use of non-digestible oligosaccharide for the manufacture of a nutritional composition that comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, for use in promoting the development in human subjects with an age of 36 months or below of an intestinal microbiota that is in function closer to the intestinal microbiota-function of human subjects at the same age fed with human milk when compared to the intestinal microbiota-function of human subjects with the same age fed a nutritional composition not comprising non-digestible oligosaccharides.
  • the invention can also be worded as a nutritional composition that comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, for use in promoting the development in human subjects with an age of 36 months or below of an intestinal microbiota that is in function closer to the intestinal microbiota-function of human subjects at the same age fed with human milk when compared to the intestinal microbiota- function of human subjects with the same age fed a nutritional composition not comprising non-digestible oligosaccharides.
  • the nutritional composition is at least partly fermented by lactic acid producing bacteria comprises 0.02 to 1.5 wt% based on dry weight of the sum of lactic acid and lactate.
  • the promoting the development of an intestinal microbiota-function is when compared to the intestinal microbiota-function of human subjects with the same age fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight.
  • the invention also concerns a method for promoting the development in human subjects with an age of 36 months or below of an intestinal microbiota that is in function closer to the intestinal microbiota-function of human subjects at the same age fed with human milk, comprising administering a nutritional composition that is at least partly fermented by lactic acid producing bacteria wherein the nutritional composition comprises 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and wherein the nutritional composition comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, when compared to the intestinal microbiota-function of human subjects with the same age fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides.
  • the invention can also be worded as the use of a fermented composition and non-digestible oligosaccharide for the manufacture of a nutritional composition that is at least partly fermented by lactic acid producing bacteria wherein the nutritional composition comprises 0.02 to 1.5 wt% based on dry weight of the sum of lactic acid and lactate and wherein the nutritional composition comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, for use in promoting the development in human subjects with an age of 36 months or below of an intestinal microbiota that is in function closer to the intestinal microbiota-function of human subjects at the same age fed with human milk when compared to the intestinal microbiota- function of human subjects with the same age fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides.
  • the invention can also be worded as a nutritional composition that is at least partly fermented by lactic acid producing bacteria wherein the nutritional composition comprises 0.02 to 1.5 wt% based on dry weight of the sum of lactic acid and lactate and wherein the nutritional composition comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, for use in promoting the development in human subjects with an age of 36 months or below of an intestinal microbiota that is in function closer to the intestinal microbiota-function of human subjects at the same age fed with human milk when compared to the intestinal microbiota- function of human subjects with the same age fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides.
  • the nutritional composition in the methods or uses according to the present invention is preferably at least party fermented.
  • a partly fermented nutritional composition comprises at least for a part a composition that was fermented by lactic acid producing bacteria. It was shown that the presence of fermented composition in the final nutritional composition results, upon administration, in an intestinal microbiota-function more similar to the intestinal microbiota-function of breastfed infants.
  • the fermentation preferably takes place during the production process of the nutritional composition.
  • the nutritional composition does not contain significant amounts of viable bacteria in the final product due to heat inactivation after fermentation or inactivation by other means.
  • the fermented composition is a milk-derived product, which is a milk substrate that is fermented by lactic acid producing bacteria, wherein the milk substrate comprises at least one selected from the group consisting of milk, whey, whey protein, whey protein hydrolysate, casein, casein hydrolysate or mixtures thereof.
  • nutritional compositions comprising fermented compositions and non-digestible oligosaccharide and their way of producing them are described in WO 2009/151330, WO 2009/151331 and
  • the fermented composition preferably comprises bacterial cell fragments like glycoproteins, glyco lipids, peptidoglycan, lipoteichoic acid (LTA), lipoproteins, nucleotides, and/or capsular polysaccharides.
  • LTA lipoteichoic acid
  • bio-active compounds can be formed, such as short chain fatty acids, bioactive peptides and/or oligosaccharides, and other metabolites, which may also result in an intestinal microbiota- function more similar to the intestinal microbiota-function of breastfed infants.
  • bioactive compounds that that are produced during fermentation by lactic acid producing bacteria or other food grade bacteria may also be referred to as post-biotics.
  • a composition comprising such post-biotics is thought to be advantageously closer to breast milk, as breast milk is not a clean synthetic formula, but contains metabolites, bacterial cells, cell fragments and the like. Therefore the fermented composition, in particular fermented milk-derived product, is believed to have an improved effect compared to non-fermented milk-derived product without or with merely lactic acid producing bacteria on the intestinal microbiota-function.
  • the final nutritional composition comprises 5 to 97.5 wt% of the fermented composition based on dry weight, more preferably 10 to 90 wt% , more preferably 20 to 80 wt%, even more preferably 25 to 60 wt%.
  • the level of the sum of lactic acid and lactate in the final nutritional composition can be taken, as this is the metabolic end product produced by the lactic acid producing bacteria upon fermentation.
  • the present final nutritional composition comprises 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight of the composition, more preferably 0.05 to 1.0 wt%, even more preferably 0.1 to 0.5 wt%.
  • At least 50 wt%, even more preferably at least 90 wt%, of the sum of lactic acid and lactate is in the form of the L(+)-isomer.
  • the sum of L(+)-lactic acid and L(+)-lactate is more than 50 wt%, more preferably more than 90 wt%, based on the sum of total lactic acid and lactate.
  • L(+)- lactate and L(+)-lactic acid is also referred to as L-lactate and L-lactic acid.
  • Lactic acid producing bacteria used for producing the fermented ingredient
  • Lactic acid producing bacteria used for preparing the fermented ingredient, in particular for fermentation of the milk substrate are preferably provided as a mono- or mixed culture.
  • Lactic acid producing bacteria consists of the genera Bifidobacterium, Lactobacillus, Carnobacterium, Enterococcus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella.
  • the lactic acid producing bacteria used for fermentation comprises bacteria of the genus Bifidobacterium and/or Streptococcus.
  • the Streptococcus is a strain of S. thermophilus .
  • Selection of a suitable strain of S. thermophilus is described in example 2 of EP 778885 and in example 1 of FR 2723960.
  • the nutritional composition comprises 10 2 - 10 5 cfu living bacteria of S thermophilus, per g dry weight of the final nutritional composition, preferably the final nutritional composition comprises 10 3 - 10 4 living bacteria of S. thermophilus per g dry weight.
  • S. thermophilus to prepare the fermented ingredient for the purpose of the present invention have been deposited by Compagnie Gervais Danone at the Collection Nationale de Cultures de Microorganismes (CNCM) run by the Institut Pasteur, 25 rue du Dondel Roux, Paris, France on 23 August 1995 under the accession number 1-1620 and on 25 August 1994 under the accession number 1-1470.
  • Other S. thermophilus strains are commercially available.
  • thermophilus does not survive the stomach it is not considered a probiotic bacterium.
  • Bifidobacteria are Gram-positive, anaerobic, rod-shaped bacteria.
  • Preferred Bifidobacterium species to prepare the fermented ingredient for the purpose of the present invention preferably have at least 95 % identity of the 16 S rRNA sequence when compared to the type strain of the respective Bifidobacterium species, more preferably at least 97% identity as defined in handbooks on this subject for instance Sambrook, L, Fritsch, E.F., and Maniatis, T. (1989), Molecular Cloning, A Faboratory Manual, 2nd ed., Cold Spring Harbor (N.Y.) Faboratory Press.
  • the Bifodobacteria preferably used are also described by Scardovi, V.
  • the lactic acid producing bacteria used for fermentation comprises or is at least one Bifidobacterium selected from the group consisting of B. breve, B. infantis, B. bfidum, B. catenulatum, B. adolescentis, B. thermophilum, B. gallicum, B. animalis or lactis, B. angulatum, B.
  • the B. breve is B. breve M-16V (Morinaga) or B. breve 1-2219, even more preferably B. breve 1-2219.
  • the nutritional composition of the invention comprises fermented composition that is fermented by lactic acid producing bacteria comprising both B. breve and S. thermophilus.
  • the fermentation by lactic acid producing bacteria is fermentation by Streptococcus thermophilus and Bifidobacterium breve.
  • the final nutritional composition comprises fermented composition wherein the lactic acid producing bacteria are inactivated after fermentation.
  • the fermented composition is not fermented by Lactobacillus bulgaricus.
  • L. bulgaricus fermented products are considered not suitable for infants, since in young infants the specific dehydrogenase that converts D-lactate to pyruvate is far less active than the dehydrogenase which converts L-lactate.
  • the nutritional composition of the invention comprises inactivated lactic acid producing bacteria and/or bacterial fragments derived from lactic acid producing bacteria obtained from more than 1x10 4 cfu lactic acid producing bacteria per g based on dry weight of the final composition, more preferably 1x10 5 cfu, even more preferably 1x10 6 cfu.
  • the inactivated bacteria or bacterial fragments are obtained from less than 1x10 12 cfu lactic acid producing bacteria per g based on dry weight of the final composition, more preferably 1x10 10 cfu, even more preferably 1x10 9 cfu.
  • the correlation of inactivated lactic acid bacteria and cfu can be determined by molecular techniques, known in the art, or by checking the production process.
  • the fermented composition is a milk-derived product, which is a milk substrate that is fermented by lactic acid producing bacteria, and said milk substrate comprising at least one selected from the group consisting of milk, whey, whey protein, whey protein hydrolysate, casein, casein hydrolysate or mixtures 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 selected from the group consisting of milk, whey, whey protein, whey protein hydrolysate, casein, casein hydrolysate or mixtures thereof.
  • Milk can be whole milk, semi-skimmed milk and/or skimmed milk.
  • the milk substrate to be fermented comprises skimmed milk.
  • Whey can be sweet whey, and/or acid whey.
  • the whey is present in a concentration of 3 to 80 g dry weight per 1 aqueous medium containing milk substrate, more preferably 40 to 60 g per 1.
  • whey protein hydrolysate is present in 2 to 80 g dry weight per 1 aqueous medium containing milk substrate, more preferably 5 to 15 g/l.
  • lactose is present in 5 to 50 g dry weight per 1 aqueous substrate, more preferably 1 to 30 g/l.
  • the aqueous medium containing milk substrate comprises buffer salts in order to keep the pH within a desired range.
  • sodium or potassium dihydrogen phosphate is used as buffer salt, preferably in 0.5 to 5 g/l, more preferably 1.5 to 3 g per 1.
  • the aqueous medium containing milk substrate comprises cysteine in amount of 0.1 to 0.5 g per 1 aqueous substrate, more preferably 0.2 to 0.4 g/l. The presence of cysteine results in low redox potential of the substrate which is advantageous for activity of lactic acid producing bacteria, particularly bifidobacteria.
  • the aqueous medium containing milk substrate comprises yeast extract in an amount of 0.5 to 5 g/l aqueous medium containing milk substrate, more preferably 1.5 to 3 g/l.
  • Yeast extract is a rich source of enzyme co-factors and growth factors for lactic acid producing bacteria.
  • the presence of yeast extract will enhance the fermentation by lactic acid producing bacteria.
  • the milk substrate in particular the aqueous medium containing milk substrate, is pasteurised before the fermentation step, in order to eliminate the presence of unwanted living bacteria.
  • the product is pasteurised after fermentation, in order to inactivate enzymes.
  • the enzyme inactivation takes place at 75 °C for 3 min.
  • the aqueous medium containing milk substrate is homogenised before and/or the milk-derived product is homogenised after the fermentation. Homogenisation results in a more stable substrate and/or fermented product, especially in the presence of fat.
  • the inoculation density is preferably between 1x10 2 to 5x10 10 , preferably between 1x10 4 to 5x10 9 cfu lactic acid producing bacteria/ml aqueous medium containing milk substrate, more preferably between 1x10 7 to 1x10 9 cfu lactic acid producing bacteria/ml aqueous medium containing milk substrate.
  • the final bacteria density after fermentation is preferably between 1x10 3 to 1x10 10 , more preferably between 1x10 4 to 1x10 9 cfu/ml aqueous medium containing milk substrate.
  • the fermentation is preferably performed at a temperature of approximately 20 °C to 50 °C, more preferably 30 °C to 45 °C, even more preferably approximately 37 °C to 42 °C.
  • the optimum temperature for growth and/or activity for lactic acid producing bacteria, more particularly lactobacilli and/or bifidobacteria is between 37 °C and 42 °C.
  • the incubation is preferably performed at a pH of 4 to 8, more preferably 6 to 7.5. This pH does not induce protein precipitation and/or an adverse taste, while at the same time lactic acid producing bacteria such as lactobacilli and/or bifidobacteria are able to ferment the milk substrate.
  • the incubation time preferably ranges from 10 minutes to 48 h, preferably from 2 h to 24 h, more preferably from 4 h to 12 h.
  • a sufficient long time enables fermentation and the concomitant production of immunogenic cell fragments such as glycoproteins, glycolipids, peptidoglycan, lipoteichoic acid (LTA), flagellae, lipoproteins, DNA and/or capsular polysaccharides and metabolites (postbio tics) to take place at a sufficient or higher extent, whereas the incubation time needs not be unnecessarily long for economical reasons.
  • a milk derived product or milk substrate preferably skimmed milk
  • one or more lactic acid producing strains preferably a strain of S. thermophilus
  • a second milk-derived product is prepared in a similar way using one or more Bifidobacterium species for fermentation.
  • the two fermented products are preferably mixed together and mixed with other components making up an infant formula, except the fat component.
  • the mixture is preheated, and subsequently fat is added in-line, homogenized, pasteurized and dried.
  • the fermentation takes place having both Bifidobacterium, preferably B. breve, and S. thermophilus in the fermentation tank.
  • thermophilus for example with 5% of a culture containing 10 6 to 10 10 bacteria per ml.
  • this milk substrate comprises milk protein peptides.
  • Temperature and duration of fermentation are as mentioned above.
  • the fermented ingredient may be pasteurised or sterilized and for example spray dried or lyophilised to provide a form suitable to be formulated in the end product.
  • a preferred method for preparing the fermented composition to be used in the nutritional composition of invention is disclosed in WO 01/01785, more particular in examples 1 and 2.
  • a preferred method for preparing the fermented composition to be used in the nutritional composition of invention is described in WO 2004/093899, more particularly in example 1.
  • Living cells of lactic acid producing bacteria in the fermented composition are after fermentation preferably eliminated, for example by inactivation and/or physical removal.
  • the cells are preferably inactivated.
  • the lactic acid producing bacteria are heat killed after fermentation of the milk substrate.
  • Preferable ways of heat killing are (flash) pasteurization, sterilization, ultra-high temperature treatment, high temperature/short time heat treatment, and/or spray drying at temperatures bacteria do not survive.
  • Cell fragments are preferably obtained by heat treatment. With this heat treatment preferably at least 90 % of living microorganisms are inactivated, more preferably at least 95 %, even more preferably at least 99 %.
  • the fermented nutritional composition comprises less than 1x10 5 colony forming units (cfu) living lactic acid bacteria per g dry weight.
  • the heat treatment preferably is performed at a temperature ranging from 70 to 180 °C, preferably from 80 to 150 °C, preferably for about 3 minutes to 2 hours, preferably in the range of 80 to 140 °C for 5 minutes to 40 minutes.
  • Inactivation of the lactic acid bacteria advantageously results in less post acidification and a safer product. This is especially advantageous when the nutritional composition is to be administered to infants or toddlers.
  • the fermented ingredient may be pasteurised or sterilized and for example spray dried or lyophilised to provide a form suitable to be formulated in the end product.
  • the present nutritional composition comprises non-digestible oligosaccharide and preferably comprises at least two different non-digestible oligosaccharides, in particular two different sources of non-digestible oligosaccharide. It was shown that the presence of non-digestible oligosaccharides improve the functioning of intestinal microbiota in making it more similar to the functioning of intestinal microbiota of breastfed infants. Hence, the presence of both the non-digestible oligosaccharide and the fermented composition, in particular the milk-derived product obtained by fermentation with lactic acid producing bacteria, synergistically and advantageously results in intestinal microbiota that is in function more similar to the intestinal microbiota-function of infants that are predominantly or exclusively breastfed.
  • oligosaccharide refers to saccharides with a degree of polymerization (DP) of 2 to 250, preferably a DP 2 to 100, more preferably 2 to 60, even more preferably 2 to 10. If oligosaccharide with a DP of 2 to 100 is included in the present nutritional composition, this results in compositions that may contain oligosaccharides with a DP of 2 to 5, a DP of 50 to 70 and a DP of 7 to 60.
  • DP degree of polymerization
  • non-digestible oligosaccharide refers to oligosaccharides which are not digested in the intestine by the action of acids or digestive enzymes present in the human upper digestive tract, e.g. small intestine and stomach, but which are preferably fermented by the human intestinal microbiota.
  • sucrose, lactose, maltose and maltodextrins are considered digestible.
  • the present non-digestible oligosaccharide is soluble.
  • soluble when having reference to a polysaccharide, fibre or oligosaccharide, means that the substance is at least soluble according to the method described by L. Prosky et al, J. Assoc. Off. Anal. Chem. 71, 1017-1023 (1988).
  • the non-digestible oligosaccharide included in the present nutritional compositions in the methods or uses according to the present invention preferably include a mixture of non- digestible oligosaccharides.
  • the non-digestible oligosaccharide is preferably selected from the group consisting of fructo-oligosaccharide, such as inulin, non-digestible dextrins, galactooligosaccharide, such as transgalacto-oligosaccharide, xylo-oligosaccharide, arabino- oligosaccharide, arabinogalacto-oligosaccharide, gluco-oligosaccharide, gentio- oligosaccharide, glucomanno-oligosaccharide, galactomannooligosaccharide, mannan- oligosaccharide, isomalto-oligosaccharide, nigero-oligosaccharide, glucomanno- oli
  • non-digestible oligosaccharides share many biochemical properties and have similar functional benefits including improving the intestinal microbiota- function. Yet is understood that some non-digestible oligosaccharides and preferably some mixtures have an even further improved effect. Therefore more preferably the non-digestible oligosaccharides are selected from the group consisting of fructo-oligosaccharide, such as inulin, galacto-oligosaccharide, such as betagalacto-oligosaccharide, and mixtures thereof, even more preferably betagalacto- oligosaccharide and/or inulin, most preferably betagalacto-oligosaccharide.
  • fructo-oligosaccharide such as inulin
  • galacto-oligosaccharide such as betagalacto-oligosaccharide
  • mixtures thereof even more preferably betagalacto- oligosaccharide and/or inulin, most preferably betagalacto-oligosacchari
  • the non-digestible oligosaccharide is selected from the group consisting of galacto-oligosaccharide, fructo- oligosaccharide and mixtures of thereof, more preferably betagalacto-oligosaccharides, fructo- oligosaccharides and mixtures thereof.
  • the non-digestible oligosaccharide is preferably selected from the group consisting of b- galacto-oligosaccharide, ⁇ -galacto-oligosaccharide, and galactan.
  • non-digestible oligosaccharide is b-galactooligosaccharide.
  • the non-digestible oligosaccharide comprises galactooligosaccharide with ⁇ (1 ,4), ⁇ (1 ,3) and/or ⁇ (1 ,6) glycosidic bonds and a terminal glucose.
  • Transgalacto-oligosaccharide is for example available under the trade name Vivinal®GOS (Domo FrieslandCampina Ingredients), Bi2muno (Clasado), Cup-oligo (Nissin Sugar) and Oligomate55 (Yakult). These oligosaccharides improve the intestinal microbiota- function to a larger extent.
  • the non-digestible oligosaccharide preferably comprises fructo-oligosaccharide.
  • a fructo- oligosaccharide may in other context have names like fructopolysaccharide, oligofructose, polyfructose, polyffuctan, inulin, levan and fructan and may refer to oligosaccharides comprising b-linked fructose units, which are preferably linked by ⁇ (2,1) and/or ⁇ (2,6) glycosidic linkages, and a preferable DP between 2 and 200.
  • the fructo- oligosaccharide contains a terminal ⁇ (2,1) glycosidic linked glucose.
  • the fructo- oligosaccharide contains at least 7 b-linked fructose units.
  • inulin is used.
  • Inulin is a type of fructo-oligosaccharide wherein at least 75% of the glycosidic linkages are ⁇ (2,1) linkages.
  • inulin has an average chain length between 8 and 60 monosaccharide units.
  • a suitable fructo-oligosaccharide for use in the compositions of the present invention is commercially available under the trade name Raftiline®HP (Orafti).
  • Other suitable sources are Raftilose (Orafti), Fibrulose and Fibruline (Cosucra) and Frutafit and Frutalose (Sensus).
  • the present nutritional composition comprises a mixture of galacto-oligosaccharide and fructo-oligosaccharide.
  • the mixture of galacto-oligosaccharide and fructo- oligosaccharide is present in a weight ratio of from 1/99 to 99/1, more preferably from 1/19 to 19/1, more preferably from 1/1 to 19/1, more preferably from 2/1 to 15/1, more preferably from 5/1 to 12/1, even more preferably from 8/1 to 10/1, even more preferably in a ratio of about 9/1.
  • This weight ratio is particularly advantageous when galacto-oligosaccharide has a low average DP and fructo-oligosaccharide has a relatively high DP.
  • the present nutritional composition comprises a mixture of short chain fructo- oligosaccharide and long chain fructo-oligosaccharide.
  • the mixture of short chain fructo-oligosaccharide and long chain fructo-oligosaccharide 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 is a mixture of short chain fructo-oligosaccharide with an average DP below 10, preferably below 6 and a fructo- oligosaccharide with an average DP above 7, preferably above 11, even more preferably above 20.
  • the present nutritional composition comprises a mixture of short chain fructo- oligosaccharide and short chain galacto-oligosaccharides.
  • the mixture of short chain fructo-oligosaccharide and short chain galacto-oligosaccharides 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 is a mixture of short chain fructo-oligosaccharide and galacto-oligosaccharides with an average DP below 10, preferably below 6.
  • the present nutritional composition comprises 2.5 to 20 wt% total non-digestible oligosaccharide, more preferably 2.5 to 15 wt%, even more preferably 3.0 to 10 wt%, most preferably 5.0 to 7.5 wt%, based on dry weight of the nutritional composition. Based on 100 ml the present nutritional composition preferably comprises 0.35 to 2.5 wt% total non-digestible oligosaccharide, more preferably 0.35 to 2.0 wt%, even more preferably 0.4 to 1.5 wt%, based on 100 ml of the nutritional composition. A lower amount of non-digestible oligosaccharide will be less effective in improving the intestinal microbiota- function, whereas a too high amount will result in side-effects of bloating and abdominal discomfort.
  • the nutritional composition used according to the present invention may also be considered as being a pharmaceutical composition and is preferably suitable for administration to infants.
  • the present nutritional composition is preferably for enteral administration, more preferably for oral administration.
  • the nutritional composition used according to the present invention is not a probiotic composition or a composition comprising probiotics.
  • the lactic acid producing bacteria are preferably either rendered non-replicating or inactivated during the production and/or do not survive under conditions present in the human upper gastro-intestinal tract.
  • the present nutritional composition is preferably an infant formula, follow on formula, toddler milk or toddler formula, or growing up milk intended for young children.
  • the present nutritional composition can be advantageously applied as a complete nutrition for infants.
  • the present nutritional composition is an infant formula.
  • An infant formula is defined as a formula for use in infants and can for example be a starter formula, intended for infants of 0 to 6 or 0 to 4 months of age.
  • a follow on formula is intended for infants of 4 or 6 months to 12 months of age. At this age infants start weaning on other food.
  • a toddler or growing up milk or formula is intended for children of 12 to 36 months of age.
  • the present composition preferably comprises a lipid component, protein component and carbohydrate component and is preferably administered in liquid form.
  • the present nutritional composition may also be in the form of a dry food, preferably in the form of a powder which is accompanied with instructions as to mix said dry food, preferably powder, with a suitable liquid, preferably water.
  • the nutritional composition used according to the invention preferably comprises other fractions, such as vitamins, minerals, trace elements and other micronutrients in order to make it a complete nutritional composition.
  • infant formulas comprise vitamins, minerals, trace elements and other micronutrients according to international directives.
  • the present nutritional composition preferably comprises lipid, protein and digestible carbohydrate wherein the lipid provides 5 to 50% of the total calories, the protein provides 5 to 50% of the total calories, and the digestible carbohydrate provides 15 to 90% of the total calories.
  • the lipid provides 35 to 50% of the total calories
  • the protein provides 7.0 to 12.5% of the total calories
  • the digestible carbohydrate provides 40 to 55% of the total calories.
  • the lipid provides 3 to 7 g lipid per 100 kcal, preferably 4 to 6 g per 100 kcal, the protein provides 1.6 to 4 g per 100 kcal, preferably 1.7 to 2.5 g per 100 kcal and the digestible carbohydrate provides 5 to 20 g per 100 kcal, preferably 8 to 15 g per 100 kcal of the nutritional composition.
  • the present nutritional composition comprises lipid providing 4 to 6 g per 100 kcal, protein providing 1.6 to 2.0 g per 100 kcal, more preferably 1.7 to 1.9 g per 100 kcal and digestible carbohydrate providing 8 to 15 g per 100 kcal of the nutritional composition.
  • the lipid provides 3 to 7 g lipid per 100 kcal, preferably 4 to 6 g per 100 kcal, the protein provides 1.6 to 2.1 g per 100 kcal, preferably 1.6 to 2.0 g per 100 kcal and the digestible carbohydrate provides 5 to 20 g per 100 kcal, preferably 8 to 15 g per 100 kcal of the nutritional composition and wherein preferably the digestible carbohydrate component comprises at least 60 wt% lactose based on total digestible carbohydrate, more preferably at least 75 wt%, even more preferably at least 90 wt% lactose based on total digestible carbohydrate.
  • the amount of total calories is determined by the sum of calories derived from protein, lipids, digestible carbohydrates and non-digestible oligosaccharide.
  • the present nutritional composition 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 present nutritional composition preferably comprises lactose. As the present nutritional composition comprises a fermented composition that is obtained by fermentation by lactic acid producing bacteria, the amount of lactose is reduced compared to its source due to the fermentation whereby lactose is converted into lactate and/or lactic acid.
  • lactose is preferably added.
  • the present nutritional composition does not comprise high amounts of carbohydrates other than lactose.
  • sugar other than lactose Compared to digestible carbohydrates such as maltodextrin, sucrose, glucose, maltose and other digestible carbohydrates with a high glycemic index, lactose has a lower glycemic index and is therefore preferred.
  • the present nutritional composition 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. Based on dry weight the present nutritional composition preferably comprises at least 25 wt% lactose, preferably at least 40 wt%, more preferably at least 50 wt% lactose.
  • the present nutritional composition preferably comprises at least one lipid selected from the group consisting of animal lipid (excluding human lipids) and vegetable lipids.
  • the present composition comprises a combination of vegetable lipids and at least one oil selected from the group consisting of fish oil, animal oil, algae oil, fungal oil, and bacterial oil.
  • the lipid of the present nutritional composition preferably provides 3 to 7 g per 100 kcal of the nutritional composition, preferably the lipid provides 4 to 6 g per 100 kcal.
  • the nutritional composition When in liquid form, e.g. as a ready-to-feed liquid, the nutritional composition preferably comprises 2.1 to 6.5 g lipid per 100 ml, more preferably 3.0 to 4.0 g per 100 ml.
  • the present nutritional composition preferably comprises 12.5 to 40 wt% lipid, more preferably 19 to 30 wt%.
  • the lipid comprises the essential fatty acids alpha- lino lenic acid (ALA), linoleic acid (LA) and/or long chain polyunsaturated fatty acids (LC-PUFA).
  • ALA alpha- lino lenic acid
  • LA linoleic acid
  • LC-PUFA long chain polyunsaturated fatty acids
  • the LC-PUFA, LA and/or ALA may be provided as free fatty acids, in triglyceride form, in diglyceride form, in monoglyceride form, in phospholipid form, or as a mixture of one of more of the above.
  • the present nutritional composition comprises at least one, preferably at least two lipid sources selected from the group consisting of rape seed oil (such as colza oil, low erucic acid rape seed oil and canola oil), high oleic sunflower oil, high oleic safflower oil, olive oil, marine oils, microbial oils, coconut oil, palm kernel oil.
  • rape seed oil such as colza oil, low erucic acid rape seed oil and canola oil
  • high oleic sunflower oil high oleic safflower oil
  • olive oil marine oils
  • microbial oils coconut oil
  • palm kernel oil lipid sources
  • the present nutritional composition preferably comprises protein.
  • the protein used in the nutritional composition is preferably selected from the group consisting of non-human animal proteins, preferably milk proteins, vegetable proteins, such as preferably soy protein and/or rice protein, and mixtures thereof.
  • the present nutritional composition preferably contains casein, and/or whey protein, more preferably bovine whey proteins and/or bovine casein.
  • the protein in the present nutritional composition comprises protein selected from the group consisting of whey protein and casein, preferably whey protein and casein, preferably the whey protein and/or casein is from cow’s milk.
  • the protein comprises less than 5 wt% based on total protein of free amino acids, dipeptides, tripeptides or hydrolyzed protein.
  • the present nutritional composition preferably comprises casein and whey proteins in a weight ratio casein : whey protein of 10 : 90 to 90 : 10, more preferably 20 : 80 to 80 : 20, even more preferably 35 : 65 to 55 : 45
  • the wt% protein based on dry weight of the present nutritional composition is calculated according to the Kjeldahl-method by measuring total nitrogen and using a conversion factor of 6.38 in case of casein, or a conversion factor of 6.25 for other proteins than casein.
  • protein or‘protein component’ as used in the present invention refers to the sum of proteins, peptides and free amino acids.
  • the present nutritional composition preferably comprises protein providing 1.6 to 4.0 g protein per 100 kcal of the nutritional composition, preferably providing 1.6 to 3.5 g, even more preferably 1.75 to 2.5 g per 100 kcal of the nutritional composition.
  • the present nutritional composition comprises protein providing 1.6 to 2.1 g protein per 100 kcal of the nutritional composition, preferably providing 1.6 to 2.0 g, more preferably 1.75 to 2.1 g, even more preferably 1.75 to 2.0 g per 100 kcal of the nutritional composition.
  • the present nutritional composition comprises protein in an amount of less than 2.0 g per 100 kcal, preferably providing 1.6 to 1.9 g, even more preferably 1.75 to 1.85 g per
  • the nutritional composition When in liquid form, e.g. as a ready-to-feed liquid, the nutritional composition preferably comprises 0.5 to 6.0 g, more preferably 1.0 to 3.0 g, even more preferably 1.0 to 1.5 g protein per 100 ml, most preferably 1.0 to 1.3 g protein per 100 ml.
  • the present nutritional composition 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%, even more preferably 8 to 9.5 wt% protein based on dry weight of the total nutritional composition.
  • the nutritional composition preferably comprises 45 to 200 kcal/100 ml liquid.
  • the nutritional composition has more preferably 60 to 90 kcal/100 ml liquid, even more preferably 65 to 75 kcal/100 ml liquid. This caloric density ensures an optimal ratio between water and calorie consumption.
  • the nutritional composition more preferably has a caloric density between 45 and 65, even more preferably between 50 and 60 kcal/100 ml.
  • the osmolarity of the present composition is preferably between 150 and 420 mOsmol/l, more preferably 260 to 320 mOsmol/l.
  • the low osmolarity aims to further reduce the gastrointestinal stress.
  • the preferred volume administered on a daily basis is in the range of about 80 to 2500 ml, more preferably about 200 to 1200 ml per day.
  • the number of feedings per day is between 1 and 10, preferably between 3 and 8.
  • the nutritional composition is administered daily for a period of at least 2 days, preferably for a period of at least 4 weeks, preferably for a period of at least 8 weeks, more preferably for a period of at 25 least 12 weeks, in a liquid form wherein the total volume administered daily is between 200 ml and 1200 ml and wherein the number of feedings per day is between 1 and 10.
  • the present nutritional composition when in liquid form, preferably has a viscosity between 1 and 60 mPa.s, preferably between 1 and 20 mPa.s, more preferably between 1 and 10 mPa.s, most preferably between 1 and 6 mPa.s.
  • the low viscosity ensures a proper administration of the liquid, e.g. a proper passage through the whole of a nipple. Also this viscosity closely resembles the viscosity of human milk. Furthermore, a low viscosity results in a normal gastric emptying and a better energy intake, which is essential for infants which need the energy for optimal growth and development.
  • the present nutritional composition alternatively is in powder form, suitable for reconstitution with water to a ready to drink liquid.
  • the present nutritional composition is preferably prepared by admixing a powdered composition with water. Normally infant formula is prepared in such a way.
  • the present invention thus also relates to a packaged power composition wherein said package is provided with instructions to admix the powder with a suitable amount of liquid, thereby resulting in a liquid composition with a viscosity between 1 and 60 mPa.s.
  • the viscosity of the liquid is determined using a Physica Rheometer MCR 300 (Physica Messtechnik GmbH, Ostfilden, Germany) at a shear rate of 95 s -1 at 20 °C.
  • prevention of a disease or certain disorder also means ‘reduction of the risk’ of a disease or certain disorder and also means‘treatment of a person at risk’ of said disease or said certain disorder.
  • the methods according to the present invention comprising administering the present nutritional composition also refer to administering an effective amount of the nutritional composition to an individual in need of such treatment.
  • the metabolome refers to the complete set of small- molecule chemicals found within a biological sample.
  • the small molecule chemicals found in a given metabolome may include both endogenous metabolites that are naturally produced by an organism (such as amino acids, organic acids, nucleic acids, fatty acids, amines, sugars, vitamins, co-factors, pigments, antibiotics, etc.) as well as exogenous chemicals.
  • endogenous metabolites that are naturally produced by an organism
  • a small molecule must typically have a molecular weight ⁇ 1500 Da. Metabolomics could contribute considerably to the functional picture of the gut microbiota.
  • the characteristics of the faecal samples in this study reflected that the infants consuming the formula with non-digestible oligosaccharides, preferably consuming partly fermented formula with non-digestible oligosaccharides, have a more breastfed like microbiota composition and more breastfed like physiological conditions in the gut as compared to the control group.
  • the more in-depth untargeted analyses showed that the gut microbiota functioning in infants fed the inventive, preferably partly fermented, formula deviated less from the breastfed reference group than in infants fed control formula, suggesting that the formula with non-digestible oligosaccharides, preferably the partly fermented formula with non-digestible oligosaccharides, drives the gut ecosystem towards a more breastfed-like situation.
  • Superpathways relating to amino acid, lipid, xenobiotics, carbohydrates, nucleotides, cofactors and vitamins, energy and peptide were found to be affected.
  • the inventors have found that that upon consumption of the nutritional composition of the present invention the intestinal metabolome of infants is more similar to the intestinal metabolome of breastfed infants.
  • the intestinal metabolome was more similar to breastfed infants’ intestinal metabolome when compared to the intestinal metabolome of infants fed formula without non-digestible oligosaccharides and when compared to the intestinal metabolome of infants fed non-fermented formula without non-digestible oligosaccharides.
  • promoting the development of an intestinal microbiota-function refers an intestinal metabolome that is more similar to the intestinal metabolome of human subjects at the same age fed with human milk when compared to the intestinal metabolome of human subjects with the same age fed a nutritional composition not comprising non-digestible oligosaccharides.
  • promoting the development of an intestinal microbiota-function refers an intestinal metabolome that is more similar to the intestinal metabolome of human subjects at the same age fed with human milk when compared to the intestinal metabolome of human subjects with the same age fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides.
  • the present invention concerns a method for establishing a metabolome in human subjects with an age of 36 months or below that is more similar to the intestinal metabolome of human subjects at the same age fed with human milk, comprising administering a nutritional composition that comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, when compared to the intestinal metabolome of human subjects with the same age fed a nutritional composition not comprising non-digestible oligosaccharides.
  • the method according to the above aspect invention can be seen as a non medical method for establishing a metabolome.
  • the invention can also be worded as the use of non-digestible oligosaccharide for the manufacture of a nutritional composition that comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, for use in establishing a metabolome in human subjects with an age of 36 months or below that is more similar to the intestinal metabolome of human subjects at the same age fed with human milk when compared to the intestinal metabolome of human subjects with the same age fed a nutritional composition not comprising non-digestible oligosaccharides.
  • the invention can also be worded as a nutritional composition that comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, for use in establishing a metabolome in human subjects with an age of 36 months or below that is more similar to the intestinal metabolome of human subjects at the same age fed with human milk when compared to the intestinal metabolome of human subjects with the same age fed a nutritional composition not comprising non-digestible oligosaccharides.
  • the present invention concerns a method for establishing a metabolome in human subjects with an age of 36 months or below that is more similar to the intestinal metabolome of human subjects at the same age fed with human milk, comprising administering a nutritional composition that is at least partly fermented by lactic acid producing bacteria wherein the nutritional composition comprises 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and wherein the nutritional composition comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, when compared to the intestinal metabolome of human subjects with the same age fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides.
  • the invention can also be worded as the use of a fermented composition and non-digestible oligosaccharide for the manufacture of a nutritional composition that is at least partly fermented by lactic acid producing bacteria wherein the nutritional composition comprises 0.02 to 1.5 wt% based on dry weight of the sum of lactic acid and lactate and wherein the nutritional composition comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, for use in establishing a metabolome in human subjects with an age of 36 months or below that is more similar to the intestinal metabolome of human subjects at the same age fed with human milk when compared to the intestinal metabolome of human subjects with the same age fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides.
  • the invention can also be worded as a nutritional composition that is at least partly fermented by lactic acid producing bacteria wherein the nutritional composition comprises 0.02 to 1.5 wt% based on dry weight of the sum of lactic acid and lactate and wherein the nutritional composition comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, for use in establishing a metabolome in human subjects with an age of 36 months or below that is more similar to the intestinal metabolome of human subjects at the same age fed with human milk when compared to the intestinal metabolome of human subjects with the same age fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides.
  • the intestinal metabolic profile or metabolome observed in infants consuming the nutritional composition according to the present invention has less than 45 % of metabolites that are significantly different in level, when compared to that observed for breastfed infants, more preferably less than 40 %, even more preferably less than 35 % of the metabolites are at a significantly different level.
  • the intestinal metabolic profile or metabolome observed in infants consuming the nutritional composition according to the present invention has more than 10 % of metabolites that are significantly different in level, when compared to control formula fed infants, more preferably more than 15%, even more preferably more than 20% of the metabolites are at a significantly different level.
  • the intestinal metabolome has less than 45 % of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects at the same age fed with human milk, more preferably less than 40 %, even more preferably less than 35 % of the metabolites are at a significantly different level, and has more than 10 % of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects with the same age fed a nutritional composition not comprising non-digestible oligosaccharides, more preferably more than 15%, even more preferably more than 20% of the metabolites are at a significantly different level.
  • the intestinal metabolic profile or metabolome observed in infants consuming the nutritional composition according to the present invention has at least 5 % less metabolites that are significantly different in level related to the intestinal metabolome of human subjects at the same age fed with human milk, when compared to the metabolites that are significantly different in level in human subjects with the same age fed a nutritional composition not comprising non-digestible oligosaccharides related to the intestinal metabolome of human subjects at the same age fed with human milk, more preferably the intestinal metabolome has at least 10 % less metabolites that are significantly different in level.
  • the present invention concerns a method for establishing a metabolome in human subjects with an age of 36 months or below that has at least 5 % less metabolites that are significantly different in level related to the intestinal metabolome of human subjects at the same age fed with human milk, comprising administering a nutritional composition that comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, when compared to the metabolites that are significantly different in level in human subjects with the same age fed a nutritional composition not comprising non-digestible oligosaccharides related to the intestinal metabolome of human subjects at the same age fed with human milk, more preferably the intestinal metabolome has at least 10 % less metabolites that are significantly different in level.
  • the invention can also be worded as the use of and non-digestible oligosaccharide for the manufacture of a nutritional composition that comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, for use in establishing a metabolome in human subjects with an age of 36 months or below that has at least 5 % less metabolites that are significantly different in level related to the intestinal metabolome of human subjects at the same age fed with human milk, when compared to the metabolites that are significantly different in level in human subjects with the same age fed a nutritional composition not comprising non-digestible oligosaccharides related to the intestinal metabolome of human subjects at the same age fed with human milk, more preferably the intestinal metabolome has at least 10 % less metabolites that are significantly different in level.
  • the present invention can also be worded as a nutritional composition that comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, for use in establishing a metabolome in human subjects with an age of 36 months or below that has at least 5 % less metabolites that are significantly different in level related to the intestinal metabolome of human subjects at the same age fed with human milk, when compared to the metabolites that are significantly different in level in human subjects with the same age fed a nutritional composition not comprising non-digestible oligosaccharides related to the intestinal metabolome of human subjects at the same age fed with human milk, more preferably the intestinal metabolome has at least 10 % less metabolites that are significantly different in level.
  • the present invention concerns a method for establishing a metabolome in human subjects with an age of 36 months or below that has less than 45 % of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects at the same age fed with human milk, more preferably less than 40 %, even more preferably less than 35 % of the metabolites are at a significantly different level, comprising administering a nutritional composition that comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide.
  • the invention can also be worded as the use of non-digestible oligosaccharide for the manufacture of a nutritional composition that comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, for use in establishing a metabolome in human subjects with an age of 36 months or below that has less than 45 % of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects at the same age fed with human milk, more preferably less than 40 %, even more preferably less than 35 % of the metabolites are at a significantly different level.
  • the invention can also be worded as a nutritional composition that comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, for use in establishing a metabolome in human subjects with an age of 36 months or below that has less than 45 % of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects at the same age fed with human milk, more preferably less than 40 %, even more preferably less than 35 % of the metabolites are at a significantly different level.
  • the present invention concerns a method for establishing a metabolome in human subjects with an age of 36 months or below that has more than 10% of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects with the same age fed a nutritional composition not comprising non-digestible oligosaccharides, more preferably more than 15%, even more preferably more than 20% of the metabolites are at a significantly different level.
  • the invention can also be worded as the use of non-digestible oligosaccharide for the manufacture of a nutritional composition that comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, for use in establishing a metabolome in human subjects with an age of 36 months or below that has more than 10 % of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects with the same age fed a nutritional composition not comprising non-digestible oligosaccharides, more preferably more than 15%, even more preferably more than 20% of the metabolites are at a significantly different level.
  • the invention can also be worded as a nutritional composition that comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide for use in establishing a metabolome in human subjects with an age of 36 months or below that has more than 10 % of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects with the same age fed a nutritional composition not comprising non-digestible oligosaccharides, more preferably more than 15%, even more preferably more than 20% of the metabolites are at a significantly different level.
  • a metabolome is established that has less than 45 % of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects at the same age fed with human milk, more preferably less than 40 %, even more preferably less than 35 % of the metabolites are at a significantly different level, and has more than 10 % of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects with the same age fed a nutritional composition not comprising non-digestible oligosaccharides, more preferably more than 15%, even more preferably more than 20% of the metabolites are at a significantly different level.
  • the intestinal metabolome has less than 45 % of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects at the same age fed with human milk, more preferably less than 40 %, even more preferably less than 35 % of the metabolites are at a significantly different level, and has more than 10 % of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects with the same age fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides, more preferably more than 15%, even more preferably more than 20% of the metabolites are at a significantly different level.
  • the intestinal metabolic profile or metabolome observed in infants consuming the nutritional composition according to the present invention has at least 5 % less metabolites that are significantly different in level related to the intestinal metabolome of human subjects at the same age fed with human milk, when compared to the metabolites that are significantly different in level in human subjects with the same age fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides related to the intestinal metabolome of human subjects at the same age fed with human milk, more preferably the intestinal metabolome has at least 10 % less metabolites that are significantly different in level.
  • the present invention concerns a method for establishing a metabolome in human subjects with an age of 36 months or below that has at least 5 % less metabolites that are significantly different in level related to the intestinal metabolome of human subjects at the same age fed with human milk, comprising administering a nutritional composition that is at least partly fermented by lactic acid producing bacteria wherein the nutritional composition comprises 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and wherein the nutritional composition comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, when compared to the metabolites that are significantly different in level in human subjects with the same age fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides related to the intestinal metabolome of human subjects at the same age fed with human milk, more preferably the
  • the invention can also be worded as the use of a fermented composition and non-digestible oligosaccharide for the manufacture of a nutritional composition that is at least partly fermented by lactic acid producing bacteria wherein the nutritional composition comprises 0.02 to 1.5 wt% based on dry weight of the sum of lactic acid and lactate and wherein the nutritional composition comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, for use in establishing a metabolome in human subjects with an age of 36 months or below that has at least 5 % less metabolites that are significantly different in level related to the intestinal metabolome of human subjects at the same age fed with human milk, when compared to the metabolites that are significantly different in level in human subjects with the same age fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides
  • the present invention can also be worded as a nutritional composition that is at least partly fermented by lactic acid producing bacteria wherein the nutritional composition comprises 0.02 to 1.5 wt% based on dry weight of the sum of lactic acid and lactate and wherein the nutritional composition comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, for use in establishing a metabolome in human subjects with an age of 36 months or below that has at least 5 % less metabolites that are significantly different in level related to the intestinal metabolome of human subjects at the same age fed with human milk, when compared to the metabolites that are significantly different in level in human subjects with the same age fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides related to the intestinal metabolome of human subjects at the same age fed with human milk, more preferably
  • the present invention concerns a method for establishing a metabolome in human subjects with an age of 36 months or below that has less than 45 % of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects at the same age fed with human milk, more preferably less than 40 %, even more preferably less than 35 % of the metabolites are at a significantly different level, comprising administering a nutritional composition that is at least partly fermented by lactic acid producing bacteria wherein the nutritional composition comprises 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and wherein the nutritional composition comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide.
  • the invention can also be worded as the use of a fermented composition and non-digestible oligosaccharide for the manufacture of a nutritional composition that is at least partly fermented by lactic acid producing bacteria wherein the nutritional composition comprises 0.02 to 1.5 wt% based on dry weight of the sum of lactic acid and lactate and wherein the nutritional composition comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, for use in establishing a metabolome in human subjects with an age of 36 months or below that has less than 45 % of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects at the same age fed with human milk, more preferably less than 40 %, even more preferably less than 35 % of the metabolites are at a significantly different level.
  • the invention can also be worded as a nutritional composition that is at least partly fermented by lactic acid producing bacteria wherein the nutritional composition comprises 0.02 to 1.5 wt% based on dry weight of the sum of lactic acid and lactate and wherein the nutritional composition comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, for use in establishing a metabolome in human subjects with an age of 36 months or below that has less than 45 % of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects at the same age fed with human milk, more preferably less than 40 %, even more preferably less than 35 % of the metabolites are at a significantly different level.
  • the present invention concerns a method for establishing a metabolome in human subjects with an age of 36 months or below that has more than 10% of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects with the same age fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides, more preferably more than 15%, even more preferably more than 20% of the metabolites are at a significantly different level.
  • the invention can also be worded as the use of a fermented composition and non-digestible oligosaccharide for the manufacture of a nutritional composition that is at least partly fermented by lactic acid producing bacteria wherein the nutritional composition comprises 0.02 to 1.5 wt% based on dry weight of the sum of lactic acid and lactate and wherein the nutritional composition comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, for use in establishing a metabolome in human subjects with an age of 36 months or below that has more than 10 % of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects with the same age fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides, more preferably more than 15%, even more preferably more than 20% of the metabolites are at
  • the invention can also be worded as a nutritional composition that is at least partly fermented by lactic acid producing bacteria wherein the nutritional composition comprises 0.02 to 1.5 wt% based on dry weight of the sum of lactic acid and lactate and wherein the nutritional composition comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide for use in establishing a metabolome in human subjects with an age of 36 months or below that has more than 10 % of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects with the same age fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides, more preferably more than 15%, even more preferably more than 20% of the metabolites are at a significantly different level.
  • a metabolome is established that has less than 45 % of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects at the same age fed with human milk, more preferably less than 40 %, even more preferably less than 35 % of the metabolites are at a significantly different level, and has more than 10 % of metabolites that are significantly different in level, when compared to the intestinal metabolome of human subjects with the same age fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides, more preferably more than 15%, even more preferably more than 20% of the metabolites are at a significantly different level.
  • the % of metabolites that differ in level is expressed based on total metabolites tested.
  • the nutritional composition according to the invention is a nutritional composition that comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide, preferably the nutritional composition according to the invention is a nutritional composition that is at least partly fermented by lactic acid producing bacteria wherein the nutritional composition comprises 0.02 to 1.5 wt% based on dry weight of the sum of lactic acid and lactate and wherein the nutritional composition comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide.
  • Control formula are formula that do not comprise non-digestible oligosaccharides.
  • control formula are formula that do not comprise fermented composition and do not comprise non-digestible oligosaccharides.
  • the intestinal metabolic profile or metabolome observed in infants consuming the nutritional composition according to the present invention has less than 350 metabolites that are significantly different in level, when compared to that observed for infants fed with human milk, more preferably less than 300 metabolites are at a significantly different level.
  • the intestinal metabolic profile or metabolome observed in infants consuming the nutritional composition according to the present invention has more than 150 metabolites that are significantly different in level, when compared to control formula fed infants, more preferably more than 200 metabolites are at a significantly different level.
  • the intestinal metabolic profile or metabolome observed in infants consuming the nutritional composition according to the present invention has at least 75 more metabolites, preferably at least 100, more preferably at least 125, that are not statistically different related to infants fed with human milk, compared to the number of metabolites that is not statistically different in control formula fed infants related to infants fed with human milk.
  • the invention further is for use in promoting the development in human subjects with an age of 36 months or below of an intestinal microbiota that is in composition closer to the intestinal microbiota of human subjects at the same age fed with human milk when compared to the intestinal microbiota of human subjects with the same age fed a nutritional composition not comprising non-digestible oligosaccharides.
  • the promoting the development of an intestinal microbiota refers to an intestinal microbiota that has a lower alpha-diversity, preferably as determined by Chao-l index, compared to the intestinal microbiota of the human subject fed a nutritional composition not comprising non-digestible oligosaccharides.
  • an intestinal microbiota refers to an intestinal microbiota that has a lower abundance of Blautia and/or Erysipelotrichales and/or an increased abundance of Lactobacillus, preferably refers to an intestinal microbiota that has a lower abundance of Blautia and/or Erysipelotrichales, more preferably refers to an intestinal microbiota that has a lower abundance of Blautia, compared to the intestinal microbiota of the human subject fed a nutritional composition not comprising non-digestible oligosaccharides.
  • the invention further is for use in promoting the development in human subjects with an age of 36 months or below of an intestinal microbiota that is in composition closer to the intestinal microbiota of human subjects at the same age fed with human milk when compared to the intestinal microbiota of human subjects with the same age fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides.
  • an intestinal microbiota refers to an intestinal microbiota that has a lower alpha-diversity, preferably as determined by Chao- 1 index, compared to the intestinal microbiota of the human subject fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides.
  • an intestinal microbiota refers to an intestinal microbiota that has a lower abundance of Blautia and/or Erysipelotrichales and/or an increased abundance of Lactobacillus, preferably refers to an intestinal microbiota that has a lower abundance of Blautia and/or Erysipelotrichales, more preferably refers to an intestinal microbiota that has a lower abundance of Blautia, compared to the intestinal microbiota of the human subject fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides.
  • intestinal metabolome or‘intestinal microbiota- function’ equally refers to 'large intestinal metabolome’ or 'large intestinal microbiota-function’ or ‘colonic metabolome’ or ‘colonic microbiota-function’ or ‘faecal metabolome’ or‘faecal microbiota function’.
  • Secondary bile acids are generated from primary bile acids via enzymes of the intestinal bacteria and therefore reflect the functioning of the intestinal microbiota. It was found after the intervention periods that the pattern of secondary bile acids in the experimental group was more similar to the pattern in the breastfed reference group than the control group. In general, the secondary bile acids were lower in the breastfed group and in the experimental group when compared to the control group.
  • the promoting the development of an intestinal microbiota-function refers to the profile of bile acids, preferably of secondary bile acids.
  • the promoting the development of an intestinal microbiota- function refers to the level of bile acids, preferably the level of secondary bile acids, that is reduced compared to the level of bile acids of the human subject fed a nutritional composition not comprising non-digestible oligosaccharides.
  • the promoting the development of an intestinal microbiota- function refers to the level of bile acids, preferably the level of secondary bile acids, that is reduced compared to the level of bile acids of the human subject fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides.
  • GABA intestinal gamma-aminobutyrate
  • Intestinal GABA is considered beneficial and to have a beneficial effect on visceral sensitivity and pain perception. GABA is thought to beneficially effect the enteral and central nervous system.
  • the promoting the development of an intestinal microbiota-function refers to the level of gamma- aminobutyrate (GABA).
  • the promoting the development of an intestinal microbiota-function refers to the level of gamma- amino butyrate (GABA) that is increased compared to the level of GABA of the human subject fed a nutritional composition not comprising non-digestible oligosaccharides.
  • GABA gamma- amino butyrate
  • the promoting the development of an intestinal microbiota-function refers to the level of gamma- amino butyrate (GABA) that is increased compared to the level of GABA of the human subject fed a nutritional composition not being at least partly fermented by lactic acid producing bacteria and comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight and not comprising non-digestible oligosaccharides.
  • GABA gamma- amino butyrate
  • the effects described herein i.e. the promoting, developing, improving, inducing, maintaining or driving the intestinal microbiota-function towards an intestinal microbiota-function more similar to the intestinal microbiota-function found in breastfed infants, are observed when compared to the intestinal microbiota-function of infants having been administered a nutritional composition not comprising a fermented composition and the non-digestible oligosaccharide, preferably not comprising the combination of fermented composition and the non-digestible oligosaccharide.
  • the present nutritional composition is used for improving the intestinal microbiota- function in a human subject with an age of 0 to 36 months. In one embodiment the present nutritional composition is used for improving the intestinal microbiota-function in a human subject of 0 to 18 months, even more preferably an infant with an age of 12 months of age or below, even more preferably an infant with an age of 0 to 6 months, most preferably an infant of 0 to 4 months. In one embodiment the present nutritional composition is used for improving the intestinal microbiota-function in a toddler of 12 to 36 months, most preferably a toddler with an age of 18 to 30, or 24 months. Preferably the present nutritional composition is further used for providing 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.
  • the methods or uses according to the present invention are for use in vaginally delivered infants. In a preferred embodiment, the methods or uses according to the present invention are for use in term infants, preferably for healthy term infants. In a preferred embodiment, the methods or uses according to the present invention are for use in healthy vaginally delivered infants. In a preferred embodiment, the methods or uses according to the present invention are for use in healthy infants bom by Caesarean section.
  • the methods or uses according to the present invention are for use in human subjects with an age of 36 months or below that have a fragile or unbalanced intestinal microbiota or have intestinal microbial dysbiosis or human subjects with an age of 36 months or below that are at risk of having a fragile or unbalanced intestinal microbiota or intestinal microbial dysbiosis, preferably human subjects with an age of 36 months or below selected from the group consisting of preterm infants, infants bom small for gestational age, infants with low birth weight, infants or toddlers treated or having been treated by antibiotics, infants bom by Caesarean section, or infants or toddlers suffering or having suffered from an intestinal inflammation or intestinal infection or infants form mothers having been treated with antibiotics peri-natally.
  • Microbial dysbiosis includes and preferably is dysbacteriosis.
  • the microbial dysbiosis or dysbacteriosis is the dysbiosis in the colon.
  • the nutritional composition of the present invention is for use in providing a healthy intestinal function and/or for use in preventing and/or treating intestinal microbiota dysbiosis in human subjects with an age of 36 months or below.
  • the final nutritional composition comprises 2.5 to 15 wt% based on dry weight of non-digestible oligosaccharide an optionally comprises a fermented composition as defined herein.
  • Figure 1 shows numeric information on metabolites that are significantly different in level per visit and when comparing different diets groups.
  • Figure 2 show the distribution of significantly changing metabolites per arm per visit, per metabolic superpathway; A: number of metabolites; B: percentage of metabolites based on total number of metabolites analysed; upper panels: breastfed versus control group; middle panels: breastfed versus experimental group; lower panels: experimental versus control group.
  • Example 1 Effect of partly fermented infant formula with non-digestible oligosaccharides or control formula on intestinal microbiota function compared with a breastfed reference group Growth and safety of an experimental formula (formula 1) versus control formula (formula 2) was investigated in an explorative clinical study, using a 3-4 months intervention in healthy, term infants. In a randomized, controlled, multi-centre, double-blinded, prospective clinical trial, infants were enrolled before 28 days of age and assigned to receive one of two formulae until 17 weeks of age.
  • Experimental infant formula 1 is an infant formula containing 0.8g/l00ml non-digestible oligosaccharides of scGOS (source Vivinal® GOS) and lcFOS (source RaftilinHP®) in a 9: 1 wt ratio.
  • scGOS source Vivinal® GOS
  • lcFOS source RaftilinHP®
  • 30% based on dry weight was derived from LactofidusTM, a commercially available infant formula marketed under brand name Gallia.
  • LactofidusTM is a fermented milk derived composition and is produced by fermenting with S. thermophilus and comprises B. breve. A mild heat treatment is employed.
  • the infant formula 1 comprised about 0.33 wt% (lactic acid + lactate) based on dry weight, of which at least 95% is L-lactic acid + L- lactate.
  • the level of colony forming units of lactic acid producing bacteria, S. thermophilus, in infant formula 1 was about 2x10 4 cfu/g dry weight and was derived from the fermented composition LactofidusTM.
  • Control infant formula 2 is a commercially available non-fermented infant formula without scGOS/lcFOS.
  • the composition of the two formulae was similar in energy and macronutrient composition (per 100 ml: 66 kcal, 1.2 g protein (bovine whey protein/casein in 1/1 weight ratio), 7.7 g digestible carbohydrate (of which 7.6 g lactose), 3.4 g fat (mainly vegetable fat).
  • the two infant formula further comprised vitamins, minerals, trace elements and other micronutrients according to international directive 2006/14 l/EC for infant formula.
  • the ITT population consisted of 94 subjects in the experimental group, 105 subjects in the control group.
  • Faecal samples were collected at of randomization or the day thereafter (baseline), at 8 weeks of age and at 16-17 weeks of age no later than one day after the last intake of study product.
  • the faecal parameters were analysed in a subgroup of infants, which were selected on: natural birth (vaginal delivery), no use of probiotics, thickeners, antibiotics or other medication that could influence the microbiota from birth until the end of study participation, no laxatives three days or less prior to faecal sampling.
  • This subgroup consisting of 30 subjects from each of the three study arms - a total of 90 subjects, resulting in a total of 270 stool samples.
  • DNA extraction from stools samples was performed with QIAmp DNA Stool Mini Kit (Qiagen) according to the manufacturer’s protocol except for the addition of two bead-beating steps.
  • Qiagen QIAmp DNA Stool Mini Kit
  • To 0.2 - 0.3 g of faecal sample 300 mg of 0.1 mm glass beads together with 1.4 mL of ASL (lysis) buffer and on this suspension the first bead-beating step was applied for 3x 30 sec (FastPrep- 24 instrument program 5.5).
  • the second bead-beating step was applied for 3x 30 sec (FastPrep-24 instrument program 5.5) to homogenize the sample. Following each bead-beating step samples were cooled for 5 min on ice. Extracted DNA purity was checked using the NanoDropTM spectrophotometer (Thermo Fisher Scientific Inc.), whereas DNA quality and concentration was measured using the Quant-iTTM 193 dsDNA BR Assay kit (InvitrogenTM). DNA aliquots were stored at -80°C until use.
  • Quantitative Insights Into Microbial Ecology (QIIME) pipeline version 1.8.0 was used to analyse the sequence data.
  • Quality control filters were set to discard sequences with a length below 200 bases, with a length above 1000 bases, with a mean sequence quality score of less than 25, with any ambiguous bases, or contained homopolymer stretches of more than 6 bases.
  • Chimeric sequences were filtered with QIIME’s own ChimeraS layer.
  • OTU Operational Taxonomic Unit
  • the relative abundances of each taxon is primarily subjected to the Two-part statistics test (Wagner et al., 2011). In this test first the proportions of zeros in the two groups are compared, and then the medians of the non-zero data in the two groups are compared. The two parts are combined and one p-value is obtained for each taxon at each visit.
  • pFDR positive false discovery rate
  • Targeted microbiota quantification by qPCR showed that in comparison to the control the samples from the experimental group at 8 and 17 weeks of age showed statistically significant increased amounts of Bifidobacterium and a lower amount of the Clostridium difficile and Clostridium perfringens group, while at baseline these measurements showed no significant differences between the treatment groups. No significant effects on the total amount of bacteria was observed.
  • the overall microbiota profile diversity can be summarized into one diversity index per sample.
  • a diversity index better known as alpha-diversity
  • the Chao-l index an estimate on species richness based on abundance data
  • was and remained low for the breastfed reference group at 4 months median Chao 1 index was 91.37, Q1-Q3 66.71- 119.2; mean 90.17, 95% Confidence Interval (Cl) range 79.48-100.9
  • Cl Confidence Interval
  • Table 1 Genera with p ⁇ 0.05 and q ⁇ 0.05 (Two-part statistics) for the difference between Experimental and Control.
  • the number of metabolites that were significantly different between the between breastfed group vs control group and experimental group and control group, but at the same time not signifivcantly different diff between breastfed group vs experimental group was 58 at 8 weeks and 91 at 17 weeks.
  • the detected metabolites showed that there are significant differences at baseline between the breastfed infants and either the control or experimental group, 268 (34.7%) or 250 (32.6%) respectively, whereas between the two treatment arms no major difference was observed (i.e. only 16 (2.1%) metabolites were significantly different; Figure 1).
  • This can be explained by the fact that the breastfed reference group upon inclusion was already completely breastfed, whereas the infants that were enrolled into the groups fed on the two formula were already (in part) formula fed.
  • the number of significantly different metabolites between the breastfed group and control group increased in time in the control arm up to 404 (51.9%) metabolites, while the number of differential metabolites between the experimental and breastfed reference arm remain more or less constant, i.e.
  • the characteristics of the faecal samples in this study reflected that the infants consuming the partly fermented formula have a more breastfed like microbiota composition and more breastfed like physiological conditions in the gut as compared to the control group. Moreover, the more in depth untargeted analyses showed that the gut microbiota functioning in infants on partly fermented formula deviates less from the breastfed reference group than in infants on control formula, suggesting that the partly fermented formula drives the gut ecosystem towards a more breastfed-like situation. Superpathways relating to amino acid, lipid, xenobiotics, carbohydrates, nucleotides, cofactors and vitamins, energy and peptide were involved (see figure 2).
  • GABA intestinal gamma-aminobutyrate
  • Example 2 Consumption of formula with non-digestible oligosaccharides improves the intestinal microbiota
  • Test group 1 Infant formula 1 comprising per 100 ml: 66 kcal, 1.35 g protein (bovine whey protein/casein in 1/1 weight ratio), 8.2 g digestible carbohydrate (of which 5.6 g lactose, and 2.1 g maltodextrin), 3.0 g fat (mainly vegetable fat), 0.8 g non-digestible oligosaccharides comprising scGOS (source Vivinal® GOS) and lcFOS (source RaftilinHP®) in a 9:1 wt ratio.
  • scGOS source Vivinal® GOS
  • lcFOS source RaftilinHP®
  • infant formula about 50 % based on dry weight is derived from LactofidusTM.
  • the infant formula comprised about 0.55 wt% lactic acid + lactate based on dry weight, of which at least 95% is L(+)- lactic acid/lactate.
  • the composition further comprised vitamins, minerals, trace elements and other micronutrients according to international directive 2006/14 l/EC for infant formula.
  • Test group 2 Infant formula 2, similar as formula 1 of which about 15% based on dry weight was derived from LactofidusTM.
  • the infant formula comprised about 0.17 wt% lactic acid + lactate based on dry weight, of which at least 95% is L(+)- lactic acid/lactate.
  • Test group 3 Infant formula 3, similar to infant formula 1, but without the non-digestible oligosaccharides scGOS and lcFOS.
  • Test group 4 Infant formula 4, a non-fermented infant formula with 0.8 g non-digestible oligosaccharides comprising scGOS (source Vivinal® GOS) and lcFOS (source RaftilinHP®) in a 9: 1 wt ratio, but not comprising LactofidusTM and for the remainder similar in composition as infant formula 1.
  • scGOS source Vivinal® GOS
  • lcFOS source RaftilinHP®
  • the overall microbiota profile diversity can be summarized into one diversity index per sample.
  • the Chao-l Index was calculated at a depth of 1496 sequences per sample (deepest measurement possible with no missing values).
  • the Chao-l estimate for group 1 had a lower value, so a indicative of a lower diversity (median 70.68, Q1-Q3 52.5- 145.9; mean 73.11, 95 % Cl interval 60.34 - 85.89) than group 3 without non-digestible oligosaccharides (median 104.2, Q1-Q3 77.69-139.8; mean 104.8, 95 % Cl interval 88.97- 120.7) and also lower than group 4 without fermented composition (median 97.32, Q1-Q3 68.99-120.5; mean 96.04, 95 % Cl 82.71-109.4).
  • the control group and the breastfed reference group are present. It can therefore be deduced that an improved effect on the intestinal microbiota function is observed in infants fed a formula with non-digestible oligosaccharides which is more similar to the breastfed reference group, when compared to formula without non-digestible oligosaccharides. Further improved effects are observed when the nutritional composition is partly fermented. The improved effects are observed when compared to formula without fermented composition and without non-digestible oligosaccharides.

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Abstract

L'administration aux nourrissons d'une préparation pour nourrissons comprenant des oligosaccharides non digestibles qui est de préférence également partiellement fermentée, permet d'obtenir un profil métabolomique intestinal et une fonction de microbiote qui est plus similaire à celui des nourrissons nourris au sein par rapport aux nourrissons nourris avec une préparation classique pour nourrissons.
EP19703731.0A 2018-02-09 2019-02-11 Préparation pour nourrissons fermentée comportant des oligosaccharides non digestibles Pending EP3749328A1 (fr)

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FR2723960B1 (fr) 1994-08-31 1996-10-11 Gervais Danone Co Cultures de streptococcus thermophilus a activite beta-galactosidase elevee, leur procede d'obtention, et leurs utilisations
FR2723963B1 (fr) 1994-08-31 1997-01-17 Gervais Danone Co Preparation de produits fermentes par streptococcus thermophilus, enrichis en galacto-oligosaccharides et en beta-galactosidase
FR2795917B1 (fr) 1999-07-06 2001-08-31 Gervais Danone Sa Procede de preparation d'un produit lacte immunostimulant et ses applications
FR2853908B1 (fr) 2003-04-16 2005-06-03 Gervais Danone Sa Produit immunomodulateur obtenu a partir d'une culture de bifidobacterium et compositions le contenant
PL2351492T3 (pl) * 2005-02-28 2015-02-27 Nutricia Nv Kompozycja odżywcza z probiotykami
US20110034407A1 (en) * 2007-12-21 2011-02-10 N. V. Nutricia Use of sphingomyelin and non-digestible carbohydrates for improving intestinal microbiota
RU2543634C2 (ru) 2008-06-13 2015-03-10 Н.В. Нютрисиа Питание, стимулирующее иммунную систему
WO2010002241A1 (fr) * 2008-06-30 2010-01-07 N.V. Nutricia Composition nutritive pour bébés nés par césarienne
WO2013187755A1 (fr) * 2012-06-14 2013-12-19 N.V. Nutricia Formule fermentée avec oligosaccharides non digestibles pour nourrissons
BR112015014420A2 (pt) * 2012-12-18 2017-07-11 Abbott Lab oligossacarídeos do leite humano para melhorar sintomas de estresse
EP3079502A1 (fr) * 2013-11-04 2016-10-19 N.V. Nutricia Préparation fermentée avec des oligosaccharides non digestibles
ES2772685T3 (es) 2015-08-04 2020-07-08 Nestle Sa Composiciones nutricionales con 2FL y LNNT para inducir una microbiota intestinal similar a la de los bebés lactantes
EP3493817A1 (fr) * 2016-08-04 2019-06-12 Nestec S.A. Compositions nutritionnelles contenant du 2fl et du lnnt destinées à être utilisées dans la prévention et/ou le traitement de la diarrhée non-rotavirus agissant sur la dysbiose du microbiote intestinal

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