WO2025229071A1

WO2025229071A1 - Synbiotic combination

Info

Publication number: WO2025229071A1
Application number: PCT/EP2025/061855
Authority: WO
Inventors: Justyna Urszula POLKA; Ana María GIL RODRÍGUEZ; Harm Johannes WOPEREIS
Original assignee: Nutricia NV
Current assignee: Nutricia NV
Priority date: 2024-04-30
Filing date: 2025-04-30
Publication date: 2025-11-06
Anticipated expiration: 2026-10-30
Also published as: WO2025229072A1

Abstract

The invention concerns the specific combination of non-digestible saccharides and lactic acid producing bacteria that was found to be especially suitable for young children, facilitating the most favorable development of the microbiota from an solely breast fed infant-like to an adult-like type taking into account the composition as well as the metabolic activity, on top of that, enabling a high level of butyrate formation, resulting in anti-inflammatory effects and improved gut barrier function.

Description

SYMBIOTIC COMBINATION

FIELD OF THE INVENTION

The present invention is in the field of nutritional compositions for young children comprising mixtures of dietary fibers and probiotics that have a beneficial effect on the development of the microbiota.

BACKGROUND OF THE INVENTION

Fibers are an important part of the diet for young and old. They have many beneficial effects, in particular on gut health. For infants a source of dietary fibers are the human milk oligosaccharides (HMOS) as found in human milk. Infant formulas have been designed to mimic functionally and/or structurally the human milk oligosaccharides. For example, mixtures of galactooligosaccharides (GOS) and polyfructose or long-chain fructooligosaccharides (IcFOS) were found to reduce the number of hard stools in infants (Moro et al., J Pediatr Gastroenterol Nutr. 2002;34(3):291-295). Infant formulas with molecules structurally identical to human milk oligosaccharides are also known in the art. In adult nutrition the dietary fibers are mainly derived from plant sources and consist of a mixture of soluble and insoluble fiber, a mixture of indigestible poly- and oligosaccharides, and fermentable and non- fermentable fibers. EP0756828 describes a fiber mix with a composition representing the dietary fiber in a typical adult Western diet.

However, research on the effect of fibres and probiotics in young children is scarce. Young children have specific nutritional requirements. Their immune system and intestinal microbiota are still in a transitional phase from an infant gut microbiota predominant in Bifidobacterium, a genus of the phylum Actinomycetota (formerly Actinobacteria) and an intestinal environment high in lactic acid, acetic acid and an acidic pH, towards a more complex and diversified microbiota, with increased levels of the phyla Bacteroidota (formerly Bacteroidetes) and Bacillota (formerly Firmicutes), towards adult-level abundances and an intestinal environment with a mildly acidic pH and with increased levels of propionic acid and butyric acid and no detectable or reduced levels of lactic acid. The microbiota of young children still differs from that of adults, which is mainly marked by more abundant levels of Bifidobacterium indicating that this genus has an important role in the gradual maturation of gut microbiota into adulthood.

The gut immune system transitions from an inflammatory-prone state in infancy to a balanced and regulated state in adulthood. The interplay between gut microbiota, diet, and immune cells contributes to this maturation process. While in infants the interaction between the gut microbiome and the immune system is essential for tolerance induction to food antigens while also preventing pathogens from invading the gut mucosa, the adult gut microbiome maintains a balance between tolerance and defense and is generally less pro-inflammatory. During childhood and adolescence, the cytokine balance generally shifts towards a profile wherein anti-inflammatory cytokines become more prominent.

Ensuring in an infant aged 4 to 6 months and older and in a young child that the transition from infant to adult microbiota follows the right progression, can provide long lasting health benefits. Infants aged 4 to 6 months and older are transitioning from exclusive breastfeeding or formula feeding to solid foods, which is a critical period to ensure age-appropriate gut maturation and immune system development. Furthermore, unhealthy eating habits when infants start weaning from 4 or 6 months of age or are toddler-age are quite prevalent in this age category leading to several health and nutritional challenges. Many toddlers do not consume a sufficient amount of fibers. This may result in an increased risk for functional gastrointestinal disorders. Constipation for example is one of the most prevalent functional gastrointestinal disorders in young children. The occurrence of constipation in toddlers and children can be high and was reported to be 10% in the second year of life (Loehning-Baucke, J Pediatr 2005; 146:359-363). Another report mentions prevalence up to 27%. Functional constipation, also known as chronic idiopathic constipation, has a large impact on the quality of life and healthcare costs. Lactulose or poly-ethylene glycol are commonly prescribed laxatives in case of chronic childhood constipation. However, these are fibers not naturally occurring in food, having their effect mainly in the proximal colon and having no additional benefits for the child. In addition, in infants and young children the immune system is still developing. Building tolerance to harmless substances, fighting infections, and building immune memory to fight infections is extremely important.

WO 2022/122958 discloses a mixture of beta-galactooligosaccharides, inulin, soluble soy fiber and resistant starch suitable for young children.

WO 2022/103321 and WO 2022/103320 disclose a composition for children with B. breve, oat betaglucan, inulin, and resistant starch.

CN109349618 discloses a medicinal composition with oat beta-glucan, inulin, resistant dextrin and L. acidophilus for use in constipation.

WO2021/149663 discloses nutritional compositions comprising LNnT, LNT, 2’-FL, 3’-SL and 6’-SL together with Bifidobacterium longum ssp infantis.

WO2021/116236 discloses nutritional compositions comprising HMOs and optionally the probiotic comprises Bifidobacterium longum subsp. infantis, Lactobacillus rhamnosus GG (ATCC number 53103), or a combination thereof, for providing nutrition to infants at different age stages ranging from 0 to 12 months of age.

Wopereis et al, 2018, J. Allergy Clin. Immunol. 141 , p 1334-1342. e5 showed that a reduced level of intestinal butyrate and butyrate producing bacteria in the microbiota of infants of 26-week-old was associated with a higher incidence of atopic dermatis when assessed at 18 months of age.

However, further improvements can still be made in the design of optimal mixes of fibers and probiotics for young children to enable a smooth transition from solely breast- or formula fed infant-type to adulttype gut microbiome and to improve gut health. Therefore, there is a need for an improved tailored and age-adapted mixture of non-digestible saccharides and lactic acid producing bacteria, to be applied in nutritional composition for young children.

SUMMARY OF THE INVENTION

Based on preclinical data obtained with a faecal slurry fermentation model using faecal material from young children the inventors found after extensive testing of numerous combinations of non-digestible saccharides (NDS) and lactic acid producing bacteria (LAPB) that a specific NDS mixture comprising galactooligosaccharides, inulin, and 5 human milk oligosaccharides (HMOs) 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3’-sialyllactose (3’-SL) and 6’-sialyllactose (6’-SL) in combination with a mixture of Lactobacillus acidophilus and at least one Bifidobacterium allowed for beneficial intestinal metabolite production profiles and microbiota formed, wherein the combinations results in a profile that lies between a NDS mixture developed for starter formulas, comprising GOS and IcFOS with or without HMOS, and a an adult intestinal metabolite and microbiota profile. When looking at specific metabolites, surprisingly a significantly higher butyrate production was found with the specific synbiotic combination of the NDS mixture with a mixture of Lactobacillus acidophilus and at least one Bifidobacterium selected from the group consisting of B. breve, B. bifidum, and B. longum subsp. longum, when compared to the mixture of NDS without LAPB or combination with other LAPB. This butyrate production was best increased when L. acidophilus was combined with Bifidobacterium longum subsp. longum. The increased butyrate production was associated with a specific increase of lactate- utilizing and butyrate producing bacterial species. In addition, it was found that the NDS mixture comprising beta-galactooligosaccharides, inulin, and 5 HMOs in combination with a mixture of Lactobacillus acidophilus and at least one Bifidobacterium provided beneficial effects on inflammatory parameters. It was found that the metabolites formed upon fermentation by the combination of the NDS mixture and LAPB mixture specifically beneficially improved the intestinal barrier function.

Therefore, the specific combination of the NDS mixture of beta-galactooligosaccharides, inulin, a mixture of 5 human milk oligosaccharides consisting of 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL and the LAPB mixture of Lactobacillus acidophilus and at least one Bifidobacterium selected from the group consisting of B. breve, B. bifidum, and B. longum subsp. longum is especially suitable for infants of 6 months and older and young children, preferably young children, facilitating the most favorable development of the microbiota from an infant-like to an adult-like type taking into account the composition as well as the metabolic activity, on top of that, enabling a high level of butyrate formation, resulting in antiinflammatory effects and improved gut barrier function.

DETAILED DESCRIPTION OF THE INVENTION

The invention thus concerns a combination of a mixture of non-digestible saccharides and a mixture of lactic acid producing bacteria comprising

- as non-digestible saccharides a mixture of beta-galactooligosaccharides, inulin, a mixture of 5 HMOs consisting of 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL, and

- as lactic acid producing bacteria a mixture of Lactobacillus acidophilus and at least one Bifidobacterium selected from the group consisting of B. breve, B. bifidum, and B. longum subsp. longum.

Definitions

The term HMO or HMOs refer to human milk oligosaccharide(s). HMOs are complex carbohydrates found in human breast milk ((Urashima et al.: Milk Oligosaccharides. Nova Science Publisher (2011); Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)). These carbohydrates are resistant to enzymatic hydrolysis by digestive enzymes. Each oligosaccharide is based on a combination of lactose and one or more of four monosaccharides (N-acetyl-D-glucosamine, D-galactose, sialic acid and/or L- fucose) to for an oligosaccharide. HMOs can be divided in neutral or non-acidic HMOs which can either be fucosylated or non-fucosylated, and acidic HMOs that have at least one sialyl residue in their structure. In the context of the present invention lactose is not regarded as an HMO species. HMOs can be manufactured by means known in the art.

As used herein, the term "degree of polymerization" (DP) means the number of monomer units joined together in a poly- or oligomer.

The term "probiotic" refers to microorganisms, such as bacteria or yeast, which have been shown to exert a beneficial effect on the health of a host subject. Probiotics can usually be classified as ‘viable’ or ‘non-viable’. The term ‘viable probiotics’ refers to living microorganisms, with the amount of a viable probiotic being detailed in colony-forming units (CFU). Probiotics that have been heat-killed, or otherwise inactivated, are termed ‘non-viable probiotics’ i.e. non-living microorganisms. Non-viable probiotics may still retain the ability to favourably influence the health of the host even though they may have been heat-killed or otherwise inactivated.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

Mixtures of non-digestible saccharides

The present invention concerns a combination of a mixture of specific non-digestible saccharides (NDS) and a mixture of lactic acid producing bacteria (LAPB) and supplements and compositions comprising such a combination. Non-digestible saccharides for the purpose of the present invention are synonym with non-digestible carbohydrates. Non-digestible saccharides are saccharides that are resistant to digestion and absorption in the human stomach and small intestine and enter the colon intact. So, compounds like lactose, maltose, glucose, standard maltodextrin, and standard starch are regarded as digestible. NDS can be soluble or insoluble in water. The term "soluble" in the present context, when having reference to the present NDS, means that the substance is water soluble according to the method described by L. Prosky et al., J. Assoc. Off. Anal. Chem. 71 , 1017-1023 (1988). If a NDS is not water soluble according to the method described by Prosky, the NDS is considered insoluble. NDS can be fermentable in the colon, or non-fermentable. The term “fermentable” refers to the capability to undergo (anaerobic) breakdown by micro-organisms in the lower part of the gastro-intestinal tract, e.g. colon, to smaller molecules, in particular short chain fatty acids and lactate. The fermentability may be determined by the method described in Titgemeyer et al. Am. J. Clin. Nutr. 53, 1418-1424 (1991). NDS can be as short as a dimer of 2 monomeric carbohydrate moieties but can also have an average degree of polymerization well above 1500. NDS with a degree of polymerization 2 - 9 are considered oligosaccharides, whereas NDS with a degree of polymerization of 10 or above are considered polysaccharides. The present NDS mixture comprises beta-galactooligosaccharides, inulin and a mixture of 5HMO s 2’- fucosyllactose (2-’FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3’-sialyllactose (3’-SL) and 6'- sialyllactose (6’-SL).

Beta-qalactooliciosaccharides

Beta-galactooligosaccharides (bGOS) as used in the present invention refers to oligosaccharides composed of more than 50%, preferably more than 65% galactose units based on total monomeric units of the beta-galactooligosaccharides, with an average degree of polymerization (DP) of 2 - 9, in which at least 50%, more preferably at least 75%, even more preferably at least 90%, of the galactose units are linked together via a beta-glycosidic linkage, preferably a beta-1 ,4-glycosidic linkage, a beta-1 ,6- glycosidic linkage and/or a beta-1 ,3-glycosidic linkage. The average DP is preferably in the range of 3 - 6. Beta-galactooligosaccharides are non-digestible, water soluble and fermentable. A glucose unit may be present at the reducing end of the chain of galactose units. Beta-galactooligosaccharides are sometimes also referred to as trans-galactooligosacchariodes (TOS). Beta-galactooligosaccharides can be analyzed according to AOAC method 2001 .02. A suitable source of beta-galactoologosaccharides is VivinalOGOS (commercially available from Borculo Domo Ingredients, Zwolle, Netherlands). Other suitable sources are Oligomate® (Yakult), Cupoligo® (Nissin) and Bi2muno® (Classado).

Beta-galactooligosaccharides are reminiscent to human milk oligosaccharides in that human milk oligosaccharides also comprise beta-glycosidic linkages and comprise galactose as a monomeric unit. For a NDS mixture adapted for young children it is beneficial to have as part of the NDS mixture NDS that will promote the composition and activity of bacteria that are typically found in human milk fed infant microbiota. So, a high amount of beta-galactooligosaccharides is advantageous for children, in particular young children, in particular paediatric patients and/or constipated children, since it favourably stimulates the intestinal bifidobacteria, the intestinal production of the organic acids and stimulates the immune system. The use of bGOS together with the other NDS and in combination with the mixture of LAPB according to the invention, results in an intestinal microbiota rich in bifidobacteria, which is beneficial for young children. The use of bGOS together with the other NDS and in combination with the mixture of LAPB according to the invention, results in an intestinal microbiota intermediate between infant-type and adult-type.

Inulin

Inulin as used in the present invention refers to carbohydrates composed of more than 50%, preferably more than over 65% fructose units based on total monomeric units of the inulin, in which at least 50%, more preferably at least 75%, even more preferably at least 90%, of the fructose units are linked together via a beta-glycosidic linkage, preferably a beta-2, 1-glycosidic linkage. A glucose unit may be present at the reducing end of the chain of fructose units. Inulin can be analyzed according to AOAC method 997.08. Inulin is a water soluble and fermentable non-digestible polysaccharide. Preferably an inulin is used that has an average DP of at least 10. Preferably an inulin is used that has an average DP of 10 - 60. More preferably the inulin has an average DP of between 20 and 40. Suitable sources of inulin are Raftiline GR (Beneo, Orafti), Raftiline HP (Beneo, Orafti) and Fibruline (Cosucra). A sufficient amount of inulin is advantageous for children, in particular young children, pediatric patients and/or constipated children since it favorably stimulates the intestinal bifidobacteria and/or the intestinal production of the organic acids. The use of inulin, such as inulin with an average DP above 20, together with bGOS has a synergistic effect in respect of stimulation of bifidobacteria, lactobacilli and production of organic acids. The use of inulin, together with the other NDS and in combination with the mixture of LAPB according to the invention, results in an intestinal microbiota intermediate between infant-type and adult-type, which is beneficial for young children.

Mixture of 5 human milk oligosaccharides

The mixture of 5 HMOs as used in the present invention refers to a specific combination of HMOs consisting of 2’-fucosyllactose (2-’FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3’-sialyllactose (3’-SL) and 6'-sialyllactose (6’-SL).

The above HMOs may be isolated by chromatography or filtration technology from a natural source such as animal milks. Alternatively, they may be produced by biotechnological means using specific enzymes such as fucosyltransferases and/or fucosidases to produce 2’-FL and 3-FL, sialidases and glycosyltransferases for SLs and LNT, either using enzyme-based fermentation technology (recombinant or natural enzymes) or microbial fermentation technology known in the art. In the latter case, microbes may either express their natural enzymes and substrates or may be engineered to produce respective substrates and enzymes. Single microbial cultures and/or mixed cultures may be used. Alternatively, these HMOs may be produced by chemical synthesis, for example from lactose and free monomers such as fucose, sialic acid, N acetyl glucosamine, galactose. These HMOs are commercially available, for example from Kyowa Hakko, Japan, FrieslandCampina, The Netherlands, Glycom/DSM, Denmark, Chr. Hansen, Denmark, and Sigma- Aldrich.

The amount of each specific HMO is preferably, based on total weight of the 5 HMOs, 42 to 62 wt% 2’- FL, 10 to 16 wt% 3-FL, 21 to 31 wt% LNT, 3 to 5 wt% 3’-SL, 4 to 6 wt% 6’-SL, the sum of 2’-FL, 3-FL, LNT, 3’ SL and 6’-SL being 100 %, more preferably 47 to 57 wt% 2’-FL, 11 to 15 wt% 3-FL, 23 to 28 wt% LNT, 3.5 to 4.5 wt% 3’-SL and 4.5 to 5.5 wt% 6’-SL. Such ratios of a combination of 5 HMOs were found to provide beneficial effects on fermentation and gut microbiota.

IMPS mixture: mixture of bGOS, inulin and 5 HMOs

The NDS of the present invention comprises a mixture of non-digestible oligosaccharides consisting of bGOS, inulin, 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL.

Preferably the combination of the mixture of NDS and mixture of LAPB or the nutritional composition comprising combination of the mixture of NDS and mixture of LAPB does not comprise other NDS than the mixture of bGOS-inulin and 5 HMOs according to the invention. Preferably the NDS in the combination of NDS and LAPB according to the invention consists of at least 90 wt%, more preferably 95 wt% even more preferably at least 98 wt% of the NDS consisting of a mixture of bGOS-inulin and 5 HMOs according to the invention. Preferably the NDS in the nutritional composition comprising the combination of NDS and LAPB according to the invention consist of at least 90 wt%, more preferably 95 wt% even more preferably at least 98 wt% of the NDS according to the invention. Preferably the NDS in the combination of NDS and LAPB or in the nutritional composition comprising the combination of NDS and LAPB consists of the mixture of NDS consisting of a mixture of GOS-FOS and 5 HMOs according to the invention. Having a substantial amount of other NDS present besides the NDS consisting of a mixture of bGOS-inulin and 5 HMOs of the present invention may not result in the same effects on the microbiota composition, immune effects, activity, and butyrate formation in young children. Preferably the combination of the mixture of NDS and mixture of LAPB or the nutritional composition comprising combination of the mixture of NDS and mixture of LAPB according to the present invention comprises 50 to 97.5 wt% of bGOS and inulin based on total NDS and 2.5 to 50 wt% of 5HMOs based on total NDS, the sum of bGOS, inulin and 5 HMOs being 100 % of the total NDS. More preferably the present nutritional composition comprises 60 to 95 wt% of bGOS and inulin based on total NDS and 5 to 40 wt% of 5 HMOs based on total non-digestible oligosaccharides, the sum of bGOS, inulin and 5 HMOs being 100 %.

Preferably the weight ratio of bGOS and inulin to 5HMOs ranges from 20 to 1 , more preferably ranges from 10 to 1 , more preferably ranges from 5 to 1 , even more preferably ranges from 2 to 1 . Such ratios will result in further improved effects on the gut microbiota, gut immunity, and fermentation profiles. Preferably the NDS mixture comprises a mixture of bGOS, inulin and 5 HMOs consisting of 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL in a weight ratio of bGOS : inulin : 2’-FL : 3-FL : LNT : 3’-SL : 6’-SL of 1 : 0.2 - 0.26 : 0.3 - 0.4 : 0.07 - 0.12 : 0.16 - 0.2 : 0.01 - 0.05 : 0.02 - 0.06. More preferably the mixture of NDS consists of bGOS, inulin, 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL in a weight ratio of bGOS : inulin : 2’-FL : 3-FL : LNT : 3’-SL : 6’-SL of 1 : 0.22 - 0.24 : 0.32 - 0.38 : 0.08 - 0.1 : 0.17 - 0.19 : 0.02 - 0.04 : 0.03 - 0.05.

Worded alternatively, preferably, based on total weight, the mixture of NDS consists of 40 to 65 wt% bGOS, 10 to 15 wt% inulin, 15 to 22 wt% 2’-FL, 3 to 7 wt% 3-FL, 7.5 to 12.5 wt% LNT, 1 .0 to 1 .8 wt% 3’-SL, and 1 .4 to 2.2 wt% 6’-SL, the sum being 100 %. More preferably, based on total weight, the mixture of NDS consists of 45 to 60 wt% bGOS, 11 to 14 wt% inulin, 17.5 to 20 wt% 2’-FL, 4 to 6 wt% 3-FL, 8 to 11 wt% LNT, 1 .2 to 1 .6 wt% 3’-SL, and 1 .6 to 2.0 wt% 6’-SL.

These ratios of bGOS, inulin and 5 HMOs ensures their specific beneficial effect, such as intestinal butyrate formation, and development of the microbiota composition and metabolic activity from infanttype to adult-type.

Lactic acid producing bacteria.

The combination of NDS and LAPB of the present invention contains at least two types of lactic acid producing bacteria (LAPB). The combination of at least two types of lactic acid producing bacteria according to the invention consists of 2 types of bacteria wherein at least one is a Lactobacillus acidophilus, and one is a Bifidobacterium selected from the group consisting of B. longum subsp. longum, B. breve and B. bifidum. Preferably the Bifidobacterium in combination with the L. acidophilus is B. longum subsp. longum.

The present combination of NDS and LAPB preferably contains 2 x 10⁴ to 3 x 10¹³ colony forming units (cfu) LAPB per gram dry weight of the combination, preferably 2 x 10⁵ to 3 x 10¹² cfu, more preferably 2 x 10⁶ to 3 x 10¹° cfu most preferably from 3 x 10⁶ to 3 x 10⁹ cfu LAPB per gram dry weight. The present nutritional composition comprising the combination of NDS and LAPB preferably contains 2 x 10³ to 3 x 10¹² colony forming units (cfu) LAPB per gram dry weight, preferably 2 x 10⁴ to 3 x 10¹¹ cfu, more preferably 2 x 10⁵ to 3 x 10⁹ cfu most preferably from 3 x 10⁵ to 3 x 10⁸ cfu LAPB per gram dry weight of nutritional composition.

Lactobacillus acidophilus

The LAPB according to the present invention comprises a strain of Lactobacilus acidophilus. A syntrophic effect was observed in combinations comprising the mixtures of NDS of the present invention and mixtures of LAPB comprising L. acidophilus. The formation of butyrate was stimulated in LAPB mixtures where a Bifidobacterium and L. acidophilus was present. This result was not, or to a lesser extent, observed when Lactobacilli belonging to L. helveticus, L. plantarum or L. paracasei were used. The effect was observed with several different strains of L. acidophilus.

L. acidophilus is a Gram-positive, anaerobic, branched rod-shaped bacterium. It is a homofermentative microorganism, producing lactate via the EMP pathway. The L. acidophilus according to the present invention preferably has at least 95 % identity with the 16S rRNA sequence when compared to the type of strain of L. acidophilus ATCC 4326, more preferably at least 97% identity (Stackebrandt & Goebel, 1994, Int. J. Syst. Bacteriol. 44:846-849). Preferred L. acidophilus strains are those isolated from the human gastro-intestinal tract. Typically, these are commercially available from producers of lactic acid bacteria, but they can also be directly isolated from faeces, identified, characterised and produced.

Suitable L. acidophilus strains are available. Examples of suitable L. acidophilus strains are L. acidophilus LA-5, DD1 (Chr Hansen). NCFM (ATCC 700396) Rhodia Inc. Especially preferred is to use L. acidophilus NCFM (ATCC strain designation SD5221). This strain is known to have health effects in children.

The present combination of a mixture of NDS and a mixture of LAPB preferably contains at least 10⁴ cfu L. acidophilus per gram dry weight, more preferably at least 10⁵ cfu, even more preferably at least 10⁶ cfu L. acidophilus per gram dry weight. The present combination preferably contains not more than 10¹³ cfu L. acidophilus per gram dry weight of the combination of a mixture of NDS and mixture of LAPB, more preferably not more than 10¹² cfu, even more preferably not more than 10¹° cfu L. acidophilus per gram dry weight. The present combination of NDS and LAPB preferably contains 10⁴ to 10¹³ colony forming units (cfu) L. acidophilus per gram dry weight, preferably 10⁵ to 10¹² cfu, more preferably 10⁶ to 1 O¹⁰ cfu most preferably from 10⁶ to 10⁹ cfu L. acidophilus per gram dry weight.

Bifidobacteria

The LAPB according to the present invention comprises a strain of Bifidobacterium. It was found that the combination of L. acidophilus and a Bifidobacterium selected from B. longum subsp. longum, B. bifidum or B. breve together with the NDS mixture of the present invention resulted in higher butyrate production. From additional experiments (combining the LAPB with NDS) it was found that it is this combination of the two LAPB that results in higher butyrate. The presence of a Bifidobacterium strongly promoted the growth of the L. acidophilus. B. longum subsp. infantis is not preferred, since this is a species that is found particularly in human breastfed infants, but to a lesser extent in healthy toddlers and adults. Preferably the mixture of LAPB does not comprise B. longum subsp. infantis. Preferably B. longum subsp. longum is present together with L. acidophilus. B. longum subsp. longum together with L. acidophilus in combination with the NDS of the present invention resulted in an even higher SCFA and especially butyrate production than in combination with one of the other bifidobacteria. Additional experiments testing the combination of 8. longum subsp. longum and 8. breve were indicative of an even further improvement on metabolite formation. The effects were not strain specific.

The present combination of NDS and LAPB preferably contains at least 10⁴ cfu of a Bifidobacterium selected from 8. longum subsp. longum, B. breve or B. bifidum per gram dry weight of the combination of NDS and LAPB, more preferably at least 10⁵ cfu, even more preferably at least 10⁶ cfu per gram dry weight. The present combination of NDS and LAPB preferably contains 10⁴ to 2.10¹³ colony forming units (cfu) Bifidobacterium selected from B. longum subsp. longum, B. breve or 8. bifidum per gram dry weight of the combination of NDS and LAPB, preferably 10⁵ to 2.10¹² cfu, more preferably 10⁶ to 2.10¹⁰ cfu most preferably from 10⁶ to 2.10⁹ cfu per gram dry weight.

Bifidobacterium longum subsp. longum

The LAPB according to the present invention preferably comprises a strain of Bifdobacterium longum subsp. longum. This subspecies was special in that it enhanced the formation of butyrate the most when the NDS mixture of the present invention and a L. acidophilus was fermented by the microbiota of young children.

Bifidobacterium longum subsp. longum (B. longum) is a Gram- positive, anaerobic, branched rod-shaped bacterium. The 8. longum subsp. longum according to the present invention preferably has at least 95 % identity of the 16S rRNA sequence when compared to the type of strain of B. longum subsp. longum ATCC 15707, more preferably at least 97% identity (Stackebrandt & Goebel, 1994, Int. J. Syst. Bacteriol. 44:846-849). Preferred B. longum subsp. longum strains are those isolated from the faeces of healthy human milk-fed infants or of toddlers. Typically, these are commercially available from producers of lactic acid bacteria, but they can also be directly isolated from faeces, identified, characterised and produced. 8. longum subsp. longum can utilize both human milk oligosaccharides and plant polysaccharides. Srutkova, et al, 2011,. Journal of Microbiological Methods. 87 (1): 10- 16. doi:10.1016/j.mimet .2011.06.014. PMID 21756944 describe a method to identify 8. longum spp longum.

Suitable B. longum subsp. longum strains are available. Examples of suitable B. longum subsp. \ongum strains are 8. longum BB356 (Mori nag a). BB536 originated from the gut of a healthy breastfed infant in 1969 and is commercially applied in many products such as probiotic supplements. BB536 is deposited at ATCC as BAA-999. Other strains are BB-46 (Chr Hansen), R0175 (Lallemand).

The present combination of NDS and LAPB preferably contains at least 10⁴ cfu 8. longum supsp. longum per gram dry weight of the combination of NDS and LAPB, more preferably at least 10⁵ cfu, even more preferably at least 10⁶ cfu per gram dry weight. The present combination of NDS and LAPB preferably contains 10⁴ to 10¹³ colony forming units (cfu) B. longum subsp. longum per gram dry weight of the combination of NDS and LAPB, preferably 10⁵ to 10¹² cfu, more preferably 10⁶ to 1 O¹⁰ cfu most preferably from 10⁶ to 10⁹ cfu per gram dry weight.

Bifidobacterium breve

The LAPB according to the present invention preferably comprises a strain of Bifidobacterium breve. A syntrophic effect was observed with L. acidophilus and the NDS mixture of the invention, especially with respect to reducing branched chain fatty acid formation related to proteolytic activity. Especially a combination of B. breve with L. acidophilus were able to form the highest amounts of butyrate in combination with the NDS mixture of the present invention.

Bifidobacterium breve is a Gram- positive, anaerobic, branched rod-shaped bacterium. The B. breve according to the present invention preferably has at least 95 % identity of the 16 S rRNA sequence when compared to the type-strain of B. breve ATCC 15700, more preferably at least 97% identity (Stackebrandt & Goebel, 1994, Int. J. Syst. Bacteriol. 44:846-849). Preferred B. breve strains are those isolated from the faeces of healthy human milk-fed infants. Typically, these are commercially available from producers of lactic acid bacteria, but they can also be directly isolated from faeces, identified, characterized and produced.

Suitable B. breve strains are available. Examples of suitable B. breve strains are B. breve UCC2003 (NCIMB 8807), C50, JCM7017, NCFB2258 and NCIMB8815, JCM7019, LMG13208, NCFB2257, NCIMB11815, ATCC 15700, M-16V (BCCM/LMG 23729, Morinaga).

Especially preferred is to use B. breve C50. B. breve C50 was deposited under deposit number CNCM 1-2219, under the Budapest Treaty at the Collection Nationale de Cultures de Microorganism, at Institut Pasteur, 25 Rue du Dr Roux, Paris, France on 31 May 1999 by Compagnie Gervais Danone. This strain was published in WO 2001/001785 and in US patent 7,410,653. Another preferred Bifidobacterium breve to use is Bifidobacterium breve CNCM 1-5177. B. breve CNCM 1-5177 was deposited under the Budapest Treaty at the Collection Nationale de Cultures de Microorganism, at Institut Pasteur, 25 Rue du Dr Roux, Paris, France on 9 March 2017 by Compagnie Gervais Danone. Especially preferred is the B. breve M-16V strain (Morinaga).

The present combination of NDS and LAPB preferably contains at least 10⁴ cfu B. breve per gram dry weight of the combination of NDS and LAPB, more preferably at least 10⁵ cfu, even more preferably at least 10⁶ cfu B. breve Per gram dry weight. The present combination preferably contains 10⁴ to 10¹³ colony forming units (cfu) B. breve per gram dry weight of the combination of NDS and LAPB, preferably 10⁵ to 10¹² cfu, more preferably 10⁶ to 10¹° cfu most preferably from 10⁶ to 10⁹ cfu B. breve per gram dry weight.

The present nutritional composition comprising the combination of NDS and LAPB preferably contains at least 10³ cfu B. breve per gram dry weight, more preferably at least 10⁴ cfu, even more preferably at least 10⁵ cfu B. breve. The present nutritional composition preferably contains 10³ to 10¹² colony forming units (cfu) B. breve per gram dry weight of the combination of NDS and LAPB, preferably 10⁴ to 10¹¹ cfu, more preferably 10⁵ to 19⁹ cfu most preferably from 10⁵ to 10⁸ cfu B. breve per gram dry weight.

The present combination of NDS and LAPB preferably contains at least 10⁴ cfu B. breve and at least 10⁴ cfu L. acidophilus per gram dry weight of the combination of NDS and LAPB more preferably at least 10⁵ cfu 8. breve and at least 10⁵ cfu L. acidophilus, even more preferably at least 10⁶ cfu 8. breve and at least 10⁶ cfu L. acidophilus. The present combination of NDS and LAPB preferably contains 10⁴ to 10¹³ cfu 8. breve and 10⁴ to 10¹³ cfu L. acidophilus per gram dry weight of the combination of NDS and LAPB, preferably 10⁵ to 10¹² cfu of each of both, more preferably 10⁶ to 1 O¹⁰ cfu of each of both most preferably from 10⁶ to 10⁹ cfu of each of both 8. breve and L. acidophilus per gram dry weight.

Bifidobacterium bifidum

The LAPB preferably comprises a strain of Bifidobacterium bifidum. This species combined with L. acidophilus and the NDS mixture of the invention showed beneficially enhanced butyrate production. A syntrophic effect was observed for Bifidobacterium bifidum with L. acidophilus and the NDS mixture of the invention, especially with respect to reducing branched chain fatty acid formation related to proteolytic activity and the boosting of operational taxonomic units (OTU) of 8. pseudocatenulatum as well as the butyrate-producing A. hallii. Additionally, a syntrophic effect was observed for Bifidobacterium bifidum with L. acidophilus and the NDS mixture of the invention on the immune system providing for a beneficial anti-inflammatory effect; it was found the combination provides for a significantly improved anti-inflammatory cytokine index.

Bifidobacterium bifidum is a Gram-positive, anaerobic, branched rod-shaped bacterium. The B. bifidum according to the present invention preferably has at least 95 % identity of the 16 S rRNA sequence when compared to the type strain of B. bifidum ATCC 29521 , more preferably at least 97% identity (Stackebrandt & Goebel, 1994, Int. J. Syst. Bacteriol. 44:846-849). Preferred B. bifidum strains are those isolated from the faeces of healthy human milk-fed infants. Typically, these are commercially available from producers of lactic acid bacteria, but they can also be directly isolated from faeces, identified, characterised and produced. Examples of suitable and available 8. bifidum strains are 8. bifidum R0071 from Lallemand or B. bifidum Bb-06 (Dupont Dansico). Most preferably, the B. bifidum is B. bifidum CNCM 1-4319. This strain was deposited under Budapest treaty at the Collection National de Cultures de Microorganisms (CNCM) at Institut Pasteur, 25 Rue de Dr Roux, 75724 Paris by Compagnie Gervais Danone on 19 May 2010. 8. bifidum CNCM 1-4319 is a strain originally isolated from the infant microbiota of a healthy baby born in the Netherlands. This strain is especially preferred because it has the ability to protect the intestinal epithelial barrier measured by transepithelial electrical resistance (TEER) in an in vitro model (WO 2011/148358) and in an animal model it was shown to restore gut integrity and functionality from stress-induced and inflammatory damage (Tondereau at al., Microorganisms, 2020, 8, 1313). This is a characteristic that is especially beneficial under conditions when the intestinal microbiota is in disbalance. 8. bifidum CNCM 1-4319 has also been disclosed in US 9,402,872.

The present combination of NDS and LAPB preferably contains at least 10⁴ cfu B. bifidum per gram dry weight, more preferably at least 10⁵ cfu, even more preferably 10⁶ cfu B. bifidum per gram dry weight. The present combination of NDS and LAPB preferably contains 10⁴ to 10¹³ colony forming units (cfu) 8. bifidum per gram dry weight, preferably 10⁵ to 10¹² cfu, more preferably 10⁶ to 10¹° cfu, most preferably 10⁶ to 10⁹ cfu 8. bifidum per gram dry weight.

Preferably the L. acidophilus and at least one Bifidobacterium selected from the group of B. longum subsp. longum, B. bifidum, and B. breve, are the sole LAPB present in the synbiotic combination with the mixture of NDS and in the nutritional composition comprising the combination of the mixture of NDS and LAPB. The presence of additional LAPB may disturb the balance and hence butyrate formation.

Nutritional composition

In one embodiment the present invention also concerns a nutritional composition comprising the specific combination of NDS and LAPB. In one embodiment the present nutritional composition is a liquid. In one embodiment, preferably the present nutritional composition is a ready-to-feed composition. Preferably the composition is administered orally. Preferably the nutritional composition of the present invention is in a powdered form, which can be reconstituted with water to form a liquid. In the context of the present invention the terms ‘powder’ and ‘dry’ are used interchangeably.

In the context of the present invention, the nutritional composition according to the invention can also be named a follow-on formula or a young child formula, preferably the nutritional composition is a young child formula. Follow-on formulas as used herein are intended for infants staring with 4 to 6 months of age to 12 months of age and are intended to be supplementary feedings for infants that start weaning on other foods. In the context of the present invention this is referred to as a nutritional composition, or follow-on formula, for the age of 6 to 12 months.

Young child formula refers to nutritional compositions, artificially made, intended for children of 12 months to 36 months, in other words for children of 1 to 3 years of age which are intended to be supplementary feedings. In the context of the present invention this is referred to as a nutritional composition, or young child formula, for the age of 12 to 36 months, for the age of 1 to 3 years.

Infant formulae and follow-on formulae are subject to strict regulations, for example for the EU regulations no. 609/2013 and no. 2016/127 and Codex Alimentarius for Infant Formula, CODEX STAN 72-1981 . Young child formulas preferably follow the directive for follow-on formula.

The nutritional composition is preferably a follow-on formula or a young child formula. In a preferred aspect the nutritional composition is a young child formula. In the context of the present invention the term ‘formula’ means that it concerns a non-natural or synthetic or artificial composition and that mammalian milk, in particular human milk, is excluded.

In order to meet the caloric requirements of a young child, the nutritional composition according to the invention preferably comprises 45 - 100 kcal per 100 ml, more preferably 50 - 75 kcal per 100 ml, even more preferably 60 -70 kcal 100 ml. This caloric density ensures an optimal ratio between water and calorie consumption and this balance is important for preventing constipation. The amount of calories is the sum of the calories provided by the protein, the lipid, and the digestible carbohydrate and NDS.

The nutritional composition according to the invention comprises lipid, protein, digestible carbohydrates, and non-digestible saccharides (NDS). The lipid preferably provides 20 - 55% of the total calories, the protein preferably provides 5 - 15% of the total calories, the digestible carbohydrate preferably provides 30 - 74% of the total calories and the NDS 1 - 15% of the total calories of the nutritional composition. Preferably nutritional composition according to the invention comprises lipid providing 25 - 50% of the total calories, protein providing 6 - 13% of the total calories, digestible carbohydrate providing 40 - 65% of the total calories and NDS providing 1.2 - 10% of the total calories of the nutritional composition. More preferably the present nutritional composition comprises lipid providing 30 - 45 % of the total calories, protein providing 7 - 10% of the total calories, digestible carbohydrate providing 45 - 55% of the total calories and NDS providing 1 .5 - 7% of the total calories of the nutritional composition.

Preferably combination of NDS and LAPB is part of a nutritional composition. When in liquid, ready to drink form, the nutritional composition according to the invention preferably comprises 0.2 - 6.0 g of the present NDS mixture per 100 ml, preferably 0.4 - 5.3 g, more preferably 0.6 - 3.3 g and even more preferably 0.8 - 2.7 g of the present NDS mixture per 100 ml nutritional composition. When in powder form, preferably the nutritional composition according to the invention comprises 1 .5 - 45 g of the present NDS mixture per 100 g dry weight, preferably 2 - 40 g, more preferably 3 - 25 g and even more preferably 3.5-20 g of the present NDS mixture per 100 g dry weight of the nutritional composition. Based on calories, preferably the nutritional composition according to the invention comprises 0.3 - 9.0 g of the present NDS mixture per 100 kcal, preferably 0.4 - 8.0 g, more preferably 0.6 - 5.0 g and even more preferably 0.7 - 4.05 g of the present NDS mixture per 100 kcal nutritional composition. Such a quantity of NDS promotes the advantageous effects of these NDS in the gastro-intestinal tract yet is suitable for young children and minimizes the risk of unwanted side effects such as bloating, abdominal pain, flatulence and/or a feeling of satiety. The amount of NDS in the nutritional composition can suitably be determined according to McCleary, Anal Bioanal Chem 2007, 389:291-308. This method suitably determines total NDS including resistant starch and non-digestible oligosaccharides. For the present invention the caloric density of the NDS is set at 2 kcal per gram.

Furthermore, the nutritional composition according to the invention preferably comprises 0.13 - 2.7 g beta-galactooligosaccharides per 100 ml, more preferably 0.20 - 2.0 g, even more preferably 0.40 - 1 .3 g beta-galactooligosaccharides per 100 ml. Based on dry weight, the nutritional composition according to the invention preferably comprises 1.0 - 20 g beta-galactooligosaccharides per 100 g, more preferably 1.5 - 15 g, even more preferably 3.0 - 10 g beta-galactooligosaccharides per 100 g. Based on calories, the nutritional composition according to the invention preferably comprises 0.2 - 4.0 g beta- galactooligosaccharides per 100 kcal, more preferably 0.3 - 3.0 g, even more preferably 0.6 - 2.0 g beta-galactooligosaccharides per 100 kcal.

Furthermore, the nutritional composition according to the invention preferably comprises 0.02 - 1.5 g inulin per 100 ml, more preferably 0.04 - 1.0 g, even more preferably 0. 08 - 0.5 g inulin per 100 ml. Based on dry weight, the nutritional composition according to the invention preferably comprises 0.1 - 2.0 g inulin per 100 g, more preferably 0.2 -1.5 g, even more preferably 0.4 - 1.0 g inulin per 100 g. Based on calories, the nutritional composition according to the invention preferably comprises 0.02 - 0.4 g inulin per 100 kcal, more preferably 0.04 - 0.6 g, even more preferably 0.06 - 0.3 g inulin per 100 kcal.

The nutritional composition according to the invention preferably comprises 20 to 400 mg of the mixture of 5 HMOs per 100 ml, more preferably 30 to 300 mg per 100 ml, even more preferably 40 to 250 mg per 100 ml. Based on dry weight of the nutritional composition the amount of human milk oligosaccharides is preferably 0.14 to 2.86 wt%, more preferably 0.21 to 2.14 wt%, even more preferably 0.28 to 1 .79 wt%. Preferably the amount of human milk oligosaccharides per 100 kcal is 30 to 600 mg, more preferably 45 to 450 mg, even more preferably 60 to 375 mg. Furthermore, the present nutritional composition comprising the combination of NDS and LAPB preferably contains 10³ to 10¹² colony forming units (cfu) L. acidophilus per gram dry weight, preferably 10⁴ to 10¹¹ cfu, more preferably 10⁵ to 10⁹ cfu most preferably from 10⁵ to 10⁸ cfu L. acidophilus per gram dry weight of the nutritional composition.

In addition to the L. acidophilus, the present nutritional composition comprising the combination of NDS and LAPB preferably contains 10³ to 2.10¹² colony forming units (cfu) of a Bifidobacterium selected from B. longum subsp longum, B. breve, and B. bifidum per gram dry weight of the nutritional composition comprising the combination of NDS and LAPB, preferably 10⁴ to 2.10¹¹ cfu, more preferably 10⁵ to 2.10⁹ cfu most preferably from 10⁵ to 2.10⁸ cfu per gram dry weight of the nutritional composition.

In addition to the L. acidophilus, the present nutritional composition comprising the combination of NDS and LAPB thus preferably contains 10³ to 10¹² colony forming units (cfu) B. longum ssp longum per gram dry weight of the nutritional composition comprising the combination of NDS and LAPB, preferably 10⁴ to 10¹¹ cfu, more preferably 10⁵ to 10⁹ cfu most preferably from 10⁵ to 10⁸ cfu per gram dry weight of the nutritional composition.

In another embodiment, in addition to the L. acidophilus, the present nutritional composition comprising the combination of NDS and LAPB preferably comprises 10³ to 10¹² colony forming units (cfu) B. breve per gram dry weight, preferably 10⁴ to 10¹¹ cfu, more preferably 10⁵ to 19⁹ cfu most preferably from 10⁵ to 10⁸ cfu B. breve per gram dry weight of the nutritional composition.

In another embodiment, in addition to the L. acidophilus, the present nutritional composition comprising the combination of NDS and LAPB preferably comprises 10³ to 10¹² colony forming units (cfu) B. bifidum per gram dry weight, preferably 10⁴ to 10¹¹ cfu, more preferably 10⁵ to 10⁹ cfu, most preferably 10⁵ to 10⁸ cfu B. bifidum per gram dry weight of the nutritional composition.

The nutritional composition preferably comprises 1 .3 - 4.7 g lipid per 100 ml, more preferably 1 .9 - 4.0 g per 100 ml, more preferably 2.1 - 3.3 g per 100 ml. Based on dry weight, the nutritional composition preferably comprises 10 - 35 g lipid per 100 g, more preferably 14 - 30 g per 100 g, more preferably 16 - 25 g lipid per 100 g dry weight of the nutritional composition. Based on calories, the nutritional composition preferably comprises 2.0 - 7.0 g lipid per 100 kcal, more preferably 2.8 - 6.0 g per 100 kcal, more preferably 3.2 - 5.0 lipid g per 100 kcal of the nutritional composition. The lipid preferably provides 20 - 55%, more preferably 25 - 50%, more preferably 30 - 45% of the total calories of the present nutritional composition.

The amount of saturated fatty acids is preferably below 45 wt.% based on total lipids more preferably below 25 wt.%. The concentration of monounsaturated fatty acids preferably ranges from 30 to 65% based on weight of total fatty acids. The concentration of polyunsaturated fatty acids preferably ranges from 15 to 60% based on weight of total fatty acids. Preferably the nutritional composition comprises the n-6 polyunsaturated fatty acid linoleic acid (LA) and the n-3 polyunsaturated fatty acid alpha-linolenic acid (ALA). LA and ALA are essential fatty acids and important for healthy growth and development of children. Preferably the nutritional composition comprises long chain poly-unsaturated fatty acids (LC- PUFA). LC-PUFA are defined in the present invention as fatty acids or acyl chains with two or more double bonds and a chain length of 20 or above. Preferably the nutritional composition comprises docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA). The nutritional composition preferably comprises 0.8 - 2.7 g protein per 100 ml, more preferably 0.9 - 2.1 g per 100 ml, more preferably 1 .1 - 1 .6 g per 100 ml nutritional composition. Based on dry weight, the nutritional composition preferably comprises 6 - 20 g protein per 100 g, more preferably 7 - 16 g per 100 g, more preferably 8 - 12 g protein per 100 g dry weight of the nutritional composition. Based on calories, the nutritional composition preferably comprises 1.2 - 4.0 g protein per 100 kcal, more preferably 1.4 - 3.2 g per 100 kcal, more preferably 1.6 - 2.4 g protein per 100 kcal of the nutritional composition. The protein preferably provides 5 - 15%, more preferably 6 - 13% even more preferably 7 - 10% based on total calories of the composition.

Protein is to be taken as the sum of proteins, peptides, and free amino acids. The amount of protein can be calculated according to the amount of nitrogen multiplied by 6.25.

The present nutritional composition preferably comprises casein and/or whey proteins. Preferably the weight ratio casein:whey protein is 0:100 to 90:10, more preferably 20:80 to 90:10, more preferably 40:60 to 80:20.

The nutritional composition preferably comprises 4.7 - 11 .2 g digestible carbohydrates per 100 ml, more preferably 6.0 - 10.7 g per 100 ml, more preferably 7.3 - 9.3 g per 100 ml nutritional composition. Based on dry weight, the nutritional composition preferably comprises 35 - 84 g digestible carbohydrates per 100 g, more preferably 45 - 80 g per 100 g, more preferably 55 - 70 g digestible carbohydrates per 100 g dry weight of the nutritional composition. Based on calories, the nutritional composition preferably comprises 7 - 17 g digestible carbohydrates per 100 kcal, more preferably 9 - 16 g per 100 kcal, more preferably 11 - 14 g digestible carbohydrates per 100 kcal of the nutritional composition. The digestible carbohydrates preferably provide 30 - 74%, more preferably 40 - 65%, more preferably 45 - 55 % of the total calories of the present nutritional composition.

Preferably the composition comprises at least one digestible carbohydrate selected from the group consisting of lactose, maltodextrin, digestible starch, saccharose, glucose, and maltose, more preferably lactose.

Preferably the nutritional composition comprises vitamins, minerals and trace elements and other micronutrients in recommended daily amounts as known in the art and according to international guidelines. The osmolarity of the present nutritional composition is preferably between 150 and 700 mOsmol/l, more preferably 200 to 400 mOsmol/l. This osmolarity advantageously reduced gastrointestinal stress, results in an optimal balance between water and nutrient uptake, which is beneficial for children suffering from or at risk of constipation.

Nutritional supplement

In one embodiment of the present invention the combination of NDS and LAPB is in the form of a supplement. Such a supplement can be packed as such in powder form, or with a suitable carrier such as maltodextrin. The powder supplement can be packed in tins or sachets or the like. Preferably the powder is protected against water and oxygen. The supplement can be added to cow’s milk, water, yoghurt and the like.

Application

The synbiotic combination of NDS and LAPB of the present invention, or supplement or nutritional composition comprising this combination, is preferably intended for administration to children from 6 months up to and including 12 years of age, more preferably young children from 1 , 2, or 3 years old, or alternatively young children from 1 to up to and including 3 years of age. Preferably the children are healthy. In particular, the combination of NDS and LAPB of the present invention, or supplement or nutritional composition comprising the combination of NDS and LAPB mixture according to the present invention is beneficial for young children from 6 months, preferably from 1 year up to and including 12 years of age, more preferably young children of 1 , 2 or 3 years old, that are at risk of or are suffering from constipation, in particular functional constipation. Such young children with or at risk of constipation may especially benefit.

In particular, the combination of NDS and LAPB of the present invention, or supplement or nutritional composition comprising the combination of NDS and LAPB of the present invention is beneficial for infants of 6 months and older, preferably young children from 1 year up to and including 12 years of age, more preferably young children of 1 , 2 or 3 years old, that are or have been treated with antibiotics. Such young children that are or have been treated with antibiotics are at risk of intestinal microbial dysbiosis and may especially benefit.

In particular, the combination of NDS and LAPB of the present invention, or supplement or nutritional composition comprising the combination of NDS and LAPB of the present invention is beneficial for infants of 6 months of age and older, preferably young children from 1 year up to and including 12 years of age, more preferably young children of 1 , 2 or 3 years old, that are at risk of or are suffering from allergy, in particular atopic dermatitis. Such young children with allergy or at risk of allergy, in particular atopic dermatitis, may especially benefit.

In particular, the combination of NDS and LAPB of the present invention, or supplement or nutritional composition comprising the NDS and LAPB mixture of the present invention is beneficial for young children from 1 year up to and including 12 years of age, more preferably young children of 1 , 2 or 3 years old, that are picky eaters. Such young children that are picky eaters, may have a too low fiber intake from the remainder of their diet and may especially benefit.

It was found that the synbiotic combination of NDS and LAPB of the present invention when fermented by microbiota of young children had an intermediate effect in relation to the profile of metabolites formed and the microbiota established. This was when compared to the metabolite product profile and microbiome of infants and that of an adult. In the present context, an infant has an age of less than 12 months, and an adult has an age of 18 years and above. Also, the present mixture of NDS was more intermediate than a prior art NDS mixture developed for toddlers. Unexpectedly the combination of the NDS mixture with a specific mixture of L. acidophilus and a Bifidobacterium selected from B. longum supsp longum, B. breve or B. bifidum, enhanced the absolute and relative amounts of butyrate formed. Of these three, combination with B. longum supsp longum resulted in the highest level of butyrate. The metabolites that were formed improved the intestinal barrier function and had an anti-inflammatory effect.

Therefore, the present synbiotic combination is beneficial for infants from 6 months of age and older, more preferably for toddlers in promoting a smooth transition from infant type microbiota to adult type microbiota. This transition applies to the composition of the intestinal microbiota as well as the activity of the intestinal microbiota.

It was found that upon fermentation of the synbiotic combination of the invention the gut barrier was improved. Therefore, the present combination can be used to improve the intestinal barrier function. An improved gut barrier function will reduce the number of infections. Therefore, the synbiotic combination of the present invention is for use in preventing infections.

Butyrate beneficially modulates visceral sensitivity and intestinal motility, which can help in conditions like constipation. Therefore, this synbiotic combination of the invention will treat or prevent constipation. It was found that that upon fermentation of the synbiotic combination of the invention the pro- inflammatory cytokines were reduced. Therefore, the synbiotic combination of the present invention is for use in treating or preventing inflammation, in particular intestinal inflammation.

An enhanced level of intestinal butyrate in young children reduces the occurrence of allergy and/or atopic dermatitis. Therefore, the synbiotic combination of the present invention is for use in treating or preventing allergy and/or atopic dermatitis.

Thus, the synbiotic combination of NDS and LAPB of the present invention, or supplement or nutritional composition comprising this combination is for use in a method of therapy.

In one embodiment, the method of therapy is preventing or treating an intestinal disorder selected from the group consisting of microbial dysbiosis, constipation, intestinal infections, intestinal inflammation, and diarrhea.

In another embodiment, the method of therapy is preventing or treating intestinal inflammation and/or increasing the intestinal barrier function in a subject.

In another embodiment, the method of therapy is improving intestinal microbiota composition and activity.

Preferably the use is in infants from 6 months and older, more preferably in a young child 1 up to and including 12 years of age, more preferably in a young child of 1 , 2, or 3 years old.

The invention can also be worded as a method for preventing or treating an intestinal disorder selected from the group consisting of microbial dysbiosis, constipation, intestinal infections, and diarrhea in a young child; intestinal inflammation and/or increasing the intestinal barrier function in a young child, allergy, preferably atopic dermatitis. comprising administering the synbiotic combination of NDS and LAPB of the present invention, or supplement or nutritional composition comprising this combination to the young child.

Alternatively, the invention can be worded as the use of non-digestible saccharides and lactic acid producing bacteria for the manufacture of a synbiotic composition for preventing or treating an intestinal disorder selected from the group consisting of microbial dysbiosis, constipation, intestinal infections, and diarrhea in a young child; intestinal inflammation and/or increasing the intestinal barrier function in a young child, allergy, preferably atopic dermatitis, wherein the non-digestible saccharides is a mixture of beta-galactooligosaccharides, inulin, 2’-FL, 3-FL, LNT, 3’-SL, 6’-SL and the lactic acid producing bacteria is a mixture of Lactobacillus acidophilus and at least one Bifidobacterium selected from the group consisting of B. breve, B. bifidum, and B. longum supsp longum.

The invention can also be worded as a method for improving intestinal microbiota composition and activity in a young child comprising administering the synbiotic combination of NDS and LAPB of the present invention, or supplement or nutritional composition comprising this combination to the young child.

The invention can be worded as the use of non-digestible saccharides and lactic acid producing bacteria for the manufacture of a synbiotic composition for improving intestinal microbiota composition and activity in a young child wherein the non-digestible saccharides is a mixture of beta- galactooligosaccharides, inulin, 2’-FL, 3-FL, LNT, 3’-SL, 6’-SL and the lactic acid producing bacteria is a mixture of Lactobacillus acidophilus and at least one Bifidobacterium selected from the group consisting of B. breve, B. bifidum, and B. longum supsp longum.

The invention also concerns a method for providing nutrition to a young child, comprising administering the synbiotic combination of NDS and LAPB of the present invention, or supplement or nutritional composition comprising this combination to the young child.

In a further embodiment, the invention concerns a method of providing nutrition to a human subject of 0.5-3 years of age comprising providing a formula comprising beta-galactooligosaccharides and inulin as defined herein above and the human milk oligosaccharides 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), 3’- sialyllactose (3’-SL), 6’-sialyllactose (6’-SL) and lacto-N-tetraose (LNT), and preferably comprising B. breve, when the subject is 0-6 months of age and providing the nutritional composition comprising the combination of a mixture of non-digestible saccharides and a mixture of lactic acid producing bacteria according to the present invention when the human subject is 6 months to 3 years of age, preferably 1-3 years of age. EXAMPLES

Material and methods

Mixtures of non-digestible saccharides consisting of beta-GOS-inulin and a mixture of 5 HMOs consisting of 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL and twelve mixtures of lactic acid producing bacteria and their combinations were tested. In addition, two reference controls were used and one blanc (NSC - No Substrate Control). This resulted in 28 different combinations that were tested based on 12 mixtures of non-digestible saccharides (NDS with lactic acid producing bacteria (LAPB) that were tested, compared to the 12 combinations of probiotics, the NDS mixture and 2 controls.

The NDS mixture FM1 of beta-galactooligosaccharides (scGOS) and long chain fructooligosaccharides (IcFOS) and five different human milk oligosaccharides without LAPD was originally developed as a mixture for infants. Benchmark 1 (BM1) containing scGOS/lcFOS in a 9:1 ratio and 2’-fucosyllactose (2’-FL), and BM2 containing scGOS/lcFOS in a 9:1 ratio, are prior art mixtures specially adapted for infants. Table 1 shows the non-digestible saccharides composition of the tested mixtures.

Table 1 : Non-digestible saccharides mixtures (weight % based on total NDS) ^a Source of scGOS is Vivinal® GOS (FrieslandCampina, Domo-Borculo NL). ^b Source of IcFOS is Raftiline HP (Beneo-Orafti).

⁰ Source of inulin is Orafti® GR (Beneo-Orafti). ^d 2’-Fucosyllactose (2’-FL) and 5 HMOS are obtained from Chr Hansen. 5 HMOS contained 2’-FL, 3-FL, 6’-SL, 3’-SL and LNT in the weight ratio: 52 : 13 : 5 : 4 : 26.

The following strains of LAPB were used:

Lactobacillus acidophilus, strain NCFM (Dupont/IFF) - also known as ATCC 700396.

Lactobacillus plantarum, 299v (Probi) DSM9843 (can also be referred to as Lactiplantibacillus plantarum 299v)

Lactobacillus paracasei Lpp1 , Danone, CNCM 1-1518

Lactobacillus helveticus R0052 (Lallemand) CNCM 1-1722

Bifidobacterium longum subspecies longum, BB536 (Morinaga) ATCC BAA-999

Bifidobacterium bifidum, (Danone) CNCM 1-4319

Bifidobacterium breve, BbC50, (Danone) CNCM 1-2219

All strains are commercially available or deposited under the Budapest treaty.

The LAPB were used in dry form and added to an end concentration of 2.5 x 10⁸ CFU/5 mL, i.e. 5 x 10⁷ CFU/mL. Table 2 shows the combinations that were tested. Table 2. List of bacterial strains, fiber mixtures and their combinations tested.

Faecal slurry fermentations were performed with samples from six healthy toddlers (1 to 3 years of age). Children were not breastfed nor antibiotics were used in the 90 days before sample collection.

Non-digestible saccharides were tested at a dose equivalent of 5 g/d and the lactic acid producing bacteria were dosed at a final concentration of 2 x 10⁷ cfu/ml. Fermentation was performed using the SIFR technology. The method is disclosed in Van den Abbeele et al. (2023) Front. Microbiol., Volume 14 | https://doi.org/10.3389/fmicb.2023.1131662, the SIFR technology has been validated by studying the impact of three structurally different carbohydrates (inulin, 2’-fucosyllactose and resistant dextrin). In short, individual bioreactors were processed in parallel in a bioreactor management device (Cryptobiotix, Ghent, Belgium). Each bioreactor contained 5 ml of nutritional medium-faecal inoculum blend supplemented with the mix of NDS and LAPB to be tested, then sealed individually, before being rendered anaerobic. After preparation, bioreactors were incubated under continuous agitation (140 rpm) at 37°C for 24 h (MaxQ 6,000, Thermo Scientific, Thermo Fisher Scientific, Merelbeke, Belgium). Samples were taken at t= 24 h and stored at -80°C.

Analysis of SCFA, BCFA, pH, lactate:

SCFA (acetate, propionate, butyrate and valerate) and branched chain fatty acids (bCFA; sum of isobutyrate, isocaproate and isovalerate) were determined via GC with flame ionization detection. (Trace 1300, Thermo Fisher Scientific, Merelbeke, Belgium), upon diethyl ether extraction as previously described (De Weirdt et al., 2010). pH was measured using an electrode (Hannah Instruments Edge HI2002, Temse, Belgium). Lactate was measured with an enzymatic method and quantified via spectrophotometry according to manufacturer’s instructions (EnzytecTM, R-Biopharm, Darmstadt, Germany).

Microbial composition 16S rRNA gene profiling:

Upon DNA extraction, library preparation and sequencing were performed on an Illumina MiSeq platform with v3 chemistry. The 16S rRNA gene V3-V4 hypervariable regions were amplified using primers 341 F (50 -CCT ACG GGN GGC WGC AG-30) and 785Rmod (50 -GAC TAC HVG GGT ATC TAA KCC-30 ). Results were analyzed at different taxonomic levels (phylum, family, and OTU level). For taxonomic analysis, the proportional data derived from sequencing (%) were corrected for the total amount of cells present in each sample (detected via flow cytometry), allowing to obtain more representative insights in the impact of interventions on the gut microbiota.

Data analysis:

For exploratory evaluation of the obtained results, a series of principal component analyses (PCA) was performed. The two principal components with the largest eigenvalues were plotted. Statistics were performed as follows:

For the statistical evaluation of the treatment effects on fundamental fermentation parameters, cell counts, microbial diversity (4 indices) and microbial composition (phylum level) across 6 different donors, a repeated measures ANOVA analysis was performed (~ based on paired t-testing, thus accounting for fact that values are compared between samples of a given donor). The statistical significance of the potential treatment effects was determined via Benjamini-Hochberg post hoc testing. The latter involves that a correction for multiple comparisons was implemented where p-values were adjusted by multiplying them with the total amount of comparisons divided by the rank of each original p-value (across all p- values). In practice, this means that while the largest obtained p-value remained uncorrected (i.e., multiplied 1), the lowest p-value was multiplied with the number of conditions assessed, thus strongly decreasing the chance of type 1 errors (i.e., false positives). Statistical differences between treatments and the blanc are indicated with * (0.1 < p adjusted < 0.2), ** (0.05 < p adjusted < 0.1) or *** (p adjusted < 0.05). Further, differences between a synbiotic and the respective fibre (FM1) are indicated with “$”, while differences with the respective LAPB (1/2/3/4/5/6/7/8/9/10/11/12) are indicated with

In addition, to estimate the independent effect of the treatment groups (i.e., NDS mix and LAPB), linear mixed models, an extension of simple linear models allowing both fixed and random effects to compensate for non-independence in the data (i.e., values are compared between samples of a given donor) were used. Here, significance is indicated by * (0.05 < p), ** (p < 0.01) or *** (p < 0.001)).

For the statistical evaluation of the treatment effects on microbial composition (family and OTU level), the Benjamini-Hochberg correction was applied within each comparison, given the large number of features analysed.

Additional analyses were performed for 16 selected study arms for 0 h (only blanc) and 24 h (all 16 study arms). 9 test products (selected out of the 66 originally tested test products) were evaluated, i.e., 1 NDS product (FM1), 3 LAPB mixes (2/6/10) as such, the 3 synbiotic combinations, along with the 2 reference products (BM1/BM2) and compared to a no substrate control (blanc). Again, NDS products were tested at a dose equivalent to 5 g/d, while the LAPB were dosed at a final concentration of 2 x 10⁷ CFU/mL.

Microbial composition (quantitative shallow shotgun sequencing):

Upon DNA extraction, standardized Illumina library preparation was performed followed by 3M total DNA sequencing. Results were analysed at different taxonomic levels (species, family and phylum level). For taxonomic analysis, the proportional data derived from sequencing (%) were corrected for the total amount of cells present in each sample (detected via flow cytometry), allowing to obtain more representative insights in the impact of interventions on the gut microbiota.

Metabolomics (untargeted LC-MS semi-polar analysis):

The LC-MS analysis was carried out using a Thermo Scientific Vanquish LC coupled to Thermo Q Exactive HF MS. An electrospray ionization interface was used as ionization source. Analysis was performed in negative and positive ionization mode. The UPLC was performed using a slightly modified version of the protocol described by Doneanu, C.E. UPLC/MS Monitoring of Water-soluble vitamin Bs in Cell culture mmedia in Mimutes. 7 (2011). Peak areas were extracted using Compound Discoverer 3.1 (Thermo Scientific). In addition to the automatic compound extraction by Compound Discoverer 3.1 , a manual extraction of compounds included in an in-house library was performed using Skyline 21.1 (MacCoss Lab Software).

Identification of compounds were performed at three levels; Level 1 : identification by retention times (compared against validated standards), accurate mass (with an accepted deviation of 3ppm), and MS/MS spectra, Level 2a: identification by retention times (compared against validated standards), accurate mass (with an accepted deviation of 3 ppm). Level 2b: identification by accurate mass (with an accepted deviation of 3 ppm), and MS/MS spectra. Level 3: identification by accurate mass alone (with an accepted deviation of 3 ppm).

Results are shown in examples 1 and 2.

Example 1: Effect of different synbiotic combinations on the metabolites formed by the intestinal microbiota of young children

A principal component analysis (PCA) based on fundamental fermentation parameters, provided comprehensive insight in overall treatment effects since the first two components explained over 50 % of variation of the dataset. All parameters (acetate, propionate, butyrate), but not pH and BCFA, correlated positively with PC1 (~ X-axis), indicating that samples with enhanced microbial activity positioned to the right (Fig 1A).

PCAs were made to visualize product-specific effects, averaged across all donors, thus allowing to zoom in on consistent product effects The following treatment effects were observed.

Mixtures comprising NDS mixture FM1 , positioned as four separate clusters, suggesting productspecific treatment effects. The reference products BM1-2 positioned near but below in the FM1 cluster, suggesting somewhat similar effects on microbial metabolite production.

While blanc samples were related with higher pH and bCFA levels, suggesting a higher proteolytic activity, the NDS clusters were positioned to the right relating with NDS-specific increase of acetate, propionate and butyrate: FM1 , BM1-2 positioned to the right suggesting strongest overall effects on those metabolites. FM1 , BM1-2 were positioned downwards relating with butyrate.

Looking at the NDS mixture without LAPB, FM1 resulted in elevated SCFA production and lowering of the pH. FM1 resulted in significantly elevated butyrate levels at the expense of lower propionate levels illustrated by the downward positioning along the PC1 axis.

When the effect of specific combinations of the NDS mixture FM1 with LAPB was examined, it was found that the addition of various LAPB impacted SCFA formation, mostly stimulating butyrate, in a specific synbiotic combination-dependent way (Fig 1 B - numbers in the graph correspond to the code as in table 2).

Additional administration of LAPB resulted in shifts along PC1/2 axis suggesting additional effects of specific LAPB on the metabolite formation and especially on butyrate.

Said observations are shown in more detail in figures 1 C and 1 D wherein the effect on the metabolites are shown per condition. A beneficial effect on reducing branched chain fatty acid formation was found (Fig 1 C). Furthermore, a beneficial effect on SCFA stimulation and in particular butyrate stimulation was observed for the LAPB mixtures that contained B. longum subsp. longum when compared to the other Bifidobacterium species (Fig 1 D). Taking this together, the highest butyrate formation was unexpectedly found with the combination of FM1 with L. acidophilus and B. longum subsp. longum. Table 3 provides the average butyrate production for the combinations of the NDS mixture FM1 with and without LAPB as well as the calculated percent increase compared to the FM1 blend. It shows that for FM1 , the combination with Lactobacillus acidophilus and any of B. longum subsp. longum, B. bifidum and B. breve resulted in an unexpected increase in butyrate compared to the FM1 mixture. Table 3 also shows that for FM1 , the combinations with B. longum subsp. Longum and any one of Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus paracasei and Lactobacillus plantarum resulted in an unexpected high increase in butyrate production compared to the FM1 mixture.

The LAPB according to the present invention preferably comprises a strain of Bifdobacterium breve or Bifidobacterium longum subsp. longum. A syntrophic effect was observed with L. acidophilus and the

NDS mixture of the invention, especially with respect to reducing branched chain fatty acid formation related to proteolytic

Such consistent stimulation of butyrate is highly remarkable as butyrate cannot be produced by the LAPB themselves, but is produced by indigenous gut microbes, which presence is highly variable among human subjects.

Table 3: Increase of average butyrate production for each condition and percentage increase in butyrate production over the FM1 blend.

Subsequently, the NDS mixture FM1 , and the LAPB mixtures comprising L. acidophilus were subjected to additional in-depth analysis. The various test products were compared with 2 reference products (BM1/BM2) and a no substrate control (blanc). The in-depth metabolomic analysis by LC-MS revealed marked effects on metabolites well beyond the traditionally studied SCFA.

The NDS FM1 as such significantly increased the levels of metabolites related to diverse health benefits, i.e., N-acetylated amino acids, indole-3-lactic acid, indole-3-propionic acid, 2-hydroxyisocaproic acid, 3- phenyllactic acid, N8-acetylspermidine, pipecolinic acid, 7-methylguanine and nicotinic acid (vitamin B7). Addition of the LAPB to FM1 further stimulated following health-related metabolites, vs. FM1 as such, resulting in potent synbiotic effects: The combination of FM1 with L. acidophilus with B. longum subsp longum or B. Bifidum (2.1 and 10.1) showed a significant increase in 2/3-hydroxybutyric acid, said metabolite is formed as a product of fatty acid oxidation and can be used as energy source in the absence of glucose. 3-HBA is an important signaling molecule that can influence gene expression, lipid metabolism, neuronal function, and the overall metabolic rate having clinical relevance amongst others in cognitive functioning. These results are indicative that the synbiotic combination according to the invention is most suitable for young children to improve the activity of the microbiome and the transition from an infant to an adult type of microbiome. In particular, the observed effects on elevated butyrate production are indicative for increased intestinal health.

Example 2: Effect of different synbiotic combinations on the intestinal microbiota composition of young children: Faecal fermentation of the NDS mixture of the invention results in a microbiota intermediate between infant and adult.

Effect of the NDS

As can be expected, all tested mixtures containing NDS resulted in higher intestinal bacteria growth. While the LAPB alone generally also increased bacterial cell density, the extent of this effect was milder compared to the NDS mixture and the synbiotic combinations.

The FM1 mixture influences, amongst others, a series of Bifidobacterium subsp. (8. pseudocatenulatum, B. longum, B. breve and B. bifidum). Further, is mixture related with several butyrate-producing species such as Anaerobutyricum hallii and Faecalibacterium prausnitzii. Several early (non-Bifidobacterium) colonizers of the infant gut such as Ruminococcus gnavus and Clostridium ramosum decreased.

The effect was observed on phylum level as well as on lower phylogenetic levels. The three key phyla across toddlers were Actinobacteria/Bacteroidota/Firmicutes with lower levels of Fusobacteria/ProteobacteriaA/errucomicrobiota being detected. No significant treatment effects were noted for the less abundant phyla, so the focus was on effects on Actinobacteria, Bacteroidota and Firmicutes.

First, the NDS-containing products significantly increased the phylum to which Bifidobacteriaceae belong, i.e., Actinobacteriota. FMI but not BM1/2) significantly increased Bacteroidota. FM1 also specifically boosted B. fragilis. FM1 was further found to strongly stimulate the butyrate-producing species Anaerobutyricum hallii.

FM1 additionally most strongly increased the most prevalent Bifidobacterium species in the toddler’s microbiota, i.e., 8. pseudocatenulatum.

Specific synbiotic interactions

Within the specific NDS mixture FM1 different effects of the combination with LAPB were found. Significant synbiotic effects included the stimulation of Firmicutes for test products containing L. acidophilus and a Bifidobacterium.

Interestingly, these specific synbiotic treatments with L. acidophilus tended to result in lower levels of a series of Lachnospiraceae species, yet higher levels of lactate utilizing butyrate producing Anaerobutyricum hallii and Anaerostipes hadrus. 1

FM1 increased OTUs related to each added probiotic Bifidobacterium strain: A pronounced increase in Actinobacteriota was observed for the combination of FM1 with L. acidophilus and B. longum subsp. longum. In general effects on the microbiota composition were in line with the effects observed on the amounts of lactate, acetate, propionate and butyrate.

Altogether, marked NDS and LAPB-specific effects on microbial metabolite production and microbial composition were noted. The finding that the administration of a single bacterium can alter the overall metabolic output of a gut microbial community consisting of 100’s of species was highly remarkable.

To corroborate health benefits of specific products of interest, i.e. FM1 , LAPB duos with L. acidophilus and combinations thereof, were subjected to additional in-depth analysis of microbial composition (quantitative shallow shotgun sequencing). The various test products were compared with 2 reference products (BM1/BM2) and a no substrate control (blanc).

In-depth analysis of microbial composition using shallow shotgun sequencing was carried out. Strong effects were observed for NDS-containing products. Shotgun sequencing provided high species level resolution and enabled observation of effects on B. adolescentis and B. catenulatum i.e. these Bifidobacterium species were also stimulated by the FM1 mixture. Ruminococcaceae, Faecalibacterium prausnitzii and Gemmiger formicilis increased upon FM1. Strong stimulatory effects of the NDS on potent butyrate producers in the phylum Firmicutes were also observed.

Potent synbiotic effects were noted. FM1 based synbiotics significantly increased Bifidobacterium spp. related to the three probiotic strains (B. longum subsp. longum, B. breve and B. bifidum that were part of the synbiotic combinations), the extent of the stimulatory effect of FM1 on B. longum subsp. longum, B. breve and B. bifidum was pronounced, suggesting a strong potential of FM1 to boost Bifidobacterium probiotics. While levels of B. pseudocatenulatum, B. catenulatum, F. prausnitzii did not increase upon treatment with FM1 alone, their levels were elevated when FM1 was combined with the probiotics L. acidphilus with B. longum subsp. longum, B. breve and B. bifidum (2.1/6.1/10.1). Further, a strong stimulatory effect of the symbiotic FM1 based combination on potent butyrate producers in the phylum Firmicutes, inter alia on Veillonella species was observed.

Example 3: Effects on gut barrier function and inflammation of the fermentation supernatants obtained after fermentation of specific synbiotic combinations by the microbiota of young children.

Host-microbiota interaction assay (Caco2/THP-1 co-culture): 24 h.

The experiment consisted of (i) a 24 h treatment period during which test products were applied on the apical side of the epithelial cells allowing to evaluate the impact on gut barrier integrity and (ii) a subsequent 6 h LPS challenge of THP-1 cells at the basal side to evaluate the impact of the test products on immune functioning.

Caco-2 cell lines obtained from the ATCC were cultured in MEM media supplemented with 1x NEAA and 1 mM Sodium Pyruvate with 10% FBS. 24-well trans-well inserts were coated with Collagen I Rat Tail Protein and 1 x 10⁵ Caco-2 cells seeded onto the apical chambers. The basal chambers were filled with culture media and plates incubated in a 5% CO2 humidified incubator for 14 days. During the differentiation process, media were changed every other day. The TEER was measured to ensure that only transwells with a TEER of more than 300 Q.cm² were selected for the main experiment.

THP-1 cells were cultured in RPMI-1640 supplemented with 10% FBS, 1 mM sodium pyruvate and 10 mM HEPES at 37°C with 5% CO2. Cultures were initially inoculated at a density of 3 x 10⁵ cells/ml and split once density had reached 1 x 10⁶ cells/ml. To differentiate THP-1 cells into macrophages, THP-1 cells were centrifuged and resuspended in cell culture medium containing 100 ng/ml PMA. The PMA- treated THP-1 cells were seeded (5 x 10⁵ cells) on transwell-suitable 24-well plates and incubated at 37°C 5% CO2 to induce differentiation. After 48 hours, Caco-2 bearing inserts were moved to the transwell-suitable 24-well plates containing the PMA differentiated THP-1 cells.

At the start of the main experiment, culture media in the apical chamber were replaced with samples derived from the SIFR® incubations, diluted in cell medium. Upon measuring TEER, plates were incubated for 24 h after which the TEER was again measured and 500 ng/ml of LPS was added to the basal chamber of the transwells containing the THP-1 cells. Upon a 6 h LPS challenge to boost cytokine/chemokine production, TEER was measured and samples from both apical and basal compartments were collected and subjected to cytokine/chemokine analysis using Multiplex Luminex® Assay kit on the MAGPix® analyser (IL-6, CXCL10, IL-10, IL-1 p, TNF-a, CCL2 (=MCP-1), IL-12p70) or ELISA (IL-8 and IL-12p703).

Upon 24 h of interaction between the colonic samples and the co-culture of epithelial and immune cells, macrophages were triggered with LPS to boost their cytokine/chemokine response. LPS was not administered apically of the epithelial cells but basolaterally (in the compartment where immune cells are present), so that immune cells were equally stimulated. The response of immune cells thus reflects a potential differential priming of immune cells in presence of the treatments. Besides presenting the individual cytokines, an anti-inflammatory index (Al) was calculated in which effects on anti-inflammatory markers (IL-10) were accounted as a positive value, while effects on pro-inflammatory markers (IL-1 p, CXCL10, IL-8, TNF-a and CCL2) were accounted as a negative value, so that anti-inflammatory test products increased index values.

The anti-inflammatory index (Al index) was calculated as follows:

To attribute an equal weight to each inflammatory marker as part of the index, results were first normalized by dividing the levels within a marker by those of the corresponding blanc (of a given donor). Subsequently, within each inflammatory marker, values were converted by subtracting the average value of that marker across all samples and dividing by the range that values of a certain marker covered (e.g. 0.75 in case the normalized changes vs. the blanc ranged from 0.80 up to 1.55). The values of anti-inflammatory markers were multiplied by +1 and those of pro-inflammatory markers were multiplied with -1 . Finally, the obtained values of the blanc were subtracted from the values (within a given donor) so the blanc results in an anti-inflammatory index of 0, while test products with anti- and pro- inflammatory effects respectively increase and decrease index values. IL-6 (IL-6) is a pleiotropic cytokine with complex roles in inflammation and there have been conflicting findings on pro-inflammatory and anti-inflammatory effects of IL-6 and hence will not be considered when calculating the antiinflammatory index.

The following supernatants corresponding to the code as in table 2 were tested: blanc, BM1 , BM2, 2, 6, 10, FM1 , 2.1 , 6.1 , 10.1 .

Results gut barrier:

After 24 h incubation it was found that incubation with supernatants from LAPB showed no significant improvement of the TEER, but incubation with supernatants from BM1/2, FM1 fermentation improved the TEER significantly compared to blanc. Incubation with supernatants from FM1 resulted in a higher TEER than from BM1/2. The synbiotic combinations of L. acidophilus with B. longum supsp longum (2.1) and B. bifidum (10.1) had higher, albeit not statistically significant, average TEER values compared to the FM1 mixture alone (data not shown).

After stimulating of THP-1 differentiated macrophages with LPS for 6 h, the effects of the test products on TEER values were largely maintained and showed the same pattern.

Immune modulation:

After 24 h of interaction between the supernatants and the co-culture of epithelial and immune cells, macrophages were triggered with LPS to boost their cytokine/chemokine response. LPS was administered basolateraly. Doing so, immune cells are equally stimulated. The response of immune cells thus reflects a potential differential priming of immune cells in presence of the treatments. Besides presenting the individual cytokines, an anti-inflammatory index (Al) was calculated in which effects on anti-inflammatory markers (IL-10) were accounted as a positive value, while effects on pro-inflammatory markers (IL-1 p, CXCL10, IL-8, TNF-a and CCL2) were accounted as a negative value, so that antiinflammatory test products increased the index values.

Overall, all treatments exhibited positive Al index values, significantly different from the blanc, suggesting strong anti-inflammatory properties for all test products.

FM1 as such reduced the levels of pro-inflammatory TNF-a, CXCL-10, IL1-p, CCL2 and antiinflammatory IL-10, while increasing pro-inflammatory IL-8. Overall, the Al Index was increased. In the synbiotic combinations the combination of FM1 , L. acidophilus and B. bifidum (10.1) had an unexpected high anti-inflammatory effect, when compared to FM1 alone, or the LAPB mix of the two species alone.

For the combination of FM1 with L. acidophilus with a B. bifidum a strong decrease in pro-inflammatory cytokines TNF-a, CXCL-10, IL-1 p and CCL2 was observed and a significant increase in the antiinflammatory index, an effect that was not observed for that LAPB combination alone. For the combination of FM1 with L. acidophilus with a B. longum subsp. Longum or a B. Breve a particular strong effect was observed on an increase in IL-6 and I L1 -p (data not shown). IL-6 is known to promote barrier function, survival of intestinal epithelial cells and antimicrobial defense, while IL1 -p promotes anti-pathogenic T cell responses such as Th17 cell differentiation and IFN production. Regarding the LAPB effect alone, all lowered the pro-inflammatory cytokines. But this was most pronounced for the combination with a B. longum subsp. longum with L. acidophilus, which thus had a higher Al index when compared with the other probiotic combinations.

Overall, the beneficial effect of FM1 originated from their selective utilization by specific microorganisms present in the microbiota of the young children and resulted in the production of a spectrum of health- related metabolites, many of which were further enhanced by the synbiotic combination with LAPB, and the health benefits could extend well beyond those derived from SCFA, but could also be due to other specific metabolites. The NDS-containing products, especially the ones with L. acidophilus + B. longum subsp. longum or L. acidophilus + B. breve, and in particular L. acidophilus + B. longum subsp. longum, also enhanced gut barrier integrity, which is of great interest as a compromised barrier integrity (‘leaky gut’) is considered to contribute to many pathological conditions. In addition to the effects on the gut barrier also effects on the immune response were observed, resulting on an anti-inflammatory response.

Example 4: Nutritional composition

Nutritional composition in powder form. After reconstitution of 14.8 g powder with water to 100 ml to a ready to drink formula, the composition comprises per 100 ml:

- 67 kcal

1 .3 g protein (milk protein)

3.4 g lipids (mainly vegetable lipids)

7.3 g digestible carbohydrates (mainly lactose)

1.15 g mixture of non-digestible saccharides, comprising based on total non-digestible saccharides o 52wt % galactooligosaccharides (source VivinalGOS) o 12 wt% inulin (source RaftilinHP) o 36 wt% of a combination of 5 HMOs consisting of 52 wt% 2’-FL, 13 wt% 3-FL, 26 wt% o LNT, 4 wt% 3’-SL and 5 wt% 6’-SL

About 10⁹ cfu Lactobacillus acidophilus (NCFM)

About 10⁸ cfu Bifidobacterium breve M16-V Vitamins, minerals as known in the art.

Example 5: Nutritional composition for young children

Nutritional composition in powder form, when after reconstitution with water, 1 scoop of 12.4 g powder, reconstituted with water to 100 ml ready to drink composition comprises per 100 ml:

59 kcal

1 .4 g protein (milk protein)/100 kcal

3.1 g lipids (vegetable lipids)/100 kcal

5.4 g digestible carbohydrates (mainly lactose)/100 kcal

1 .76 g mixture of non-digestible saccharides/100 kcal (2 g/100 ml) o 0.92 g galactooligosaccharides (source VivinalGOS) o 0.21 g inulin (source Raftiline HP) o 0.0.64 g of a combination of 5 HMOs consisting of 52 wt% 2’-FL, 13 wt% 3-FL, 26 wt% LNT, 4 wt% 3’-SL and 5 wt% 6’-SL (source Chr Hansen)

About 10⁸ cfu Lactobacillus acidophilus (NCFM)

About 10⁷ cfu Bifidobacterium longum sp \ongum (BAA-999)

Supplemented with vitamin A B2 B12 D3 C, calcium, iron, zinc and iodine.

Example 6: Nutritional composition

A packed powder with on the package instructions to reconstitute 14.4 g of the powder with 90 ml of water to form a ready to drink formula. After reconstitution the drink has per 100 ml the following composition per 100 ml:

- 67 kcal

2.7 g fat (mix of vegetable oils and fish oil)

1 .3 g protein (casein and whey protein from cow’s milk)

8.4 g digestible carbohydrates (mainly lactose)

2.0 g non-digestible oligosaccharides per 100 ml: with beta-galactooligosaccharides I inulin I 5 HMOs in wt/wt/wt ratio of about 521 12/36 wt/wt minerals, trace elements, vitamins and other micro-nutrients as known and in line with regulations. About 10⁹ cfu Lactobacillus acidophilus NCFM, About 10⁷ cfu B. bifidum R0071

The formulation of such young child formula is known to the skilled person. The product is labelled to be for young children with an age of 1 to 3 years. As a source of beta-galactooligosaccharides Vivinal® GOS is used (FrieslandCampina, Domo-Borculo NL), as a source of inulin Orafti® HP (Beneo-Orafti Orey) Beneo BE) is used, as a source of 5 HMOs mix of Chr Hansen is used.

Example 7:

Supplement to be added to milk, yoghurt, porridge and the like.

Sachet comprising per pack:

4 g fibre, 2.1 g Galacto-oligosaccharides (VivinalGOS powder), 0.5 g inulin (raftiline HP), 1 .4 g 5 HMOs (Chr Hansen) and about 10⁹ cfu of each L. acidophilus La5 and B. longum supsp longum BAA999.

Claims

1 . A combination of a mixture of non-digestible saccharides and a mixture of lactic acid producing bacteria comprising

- as non-digestible saccharides a mixture of beta-galactooligosaccharides, inulin, and a mix of 5 human milk oligosaccharides consisting of 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), lacto- N-tetraose (LNT), 3’-sialyllactose (3’-SL) and 6’-sialyllactose (6’-SL), and

2. The combination according to claim 1 , wherein the mixture of lactic acid producing bacteria is a mixture of Lactobacillus acidophilus and B. longum subsp. longum.

3. The combination according to claim 1 , wherein the mixture of lactic acid producing bacteria a mixture of Lactobacillus acidophilus and B. breve.

4. The combination according to claim 1 , wherein the mixture of lactic acid producing bacteria is a mixture of Lactobacillus acidophilus and B. bifidum.

5. The combination according to any one of the preceding claims, wherein the mixture of non- digestible saccharides comprises beta-galactooligosaccharides : inulin : 2’-FL : 3-FL : LNT : 3’-SL : 6’-SL in a weight ratio of 1 : 0.2 - 0.26 : 0.3 - 0.4 : 0.07 - 0.12 : 0.16 - 0.2 : 0.01 - 0.05 : 0.02 - 0.06.

6. The combination according to any one of the preceding claims, wherein the 5 HMOs of the mixture of NDS comprises 42 - 63 wt% 2’-FL, 10 to 16 wt% 3-FL, 21 to 31 wt% LNT, 3 to 5 wt% 3’-SL, 4 to 6 wt% 6’-SL based on weight of the HMOs.

7. A supplement comprising the combination of a mixture of non-digestible saccharides and a mixture of lactic acid producing bacteria according to any one of claims 1-6.

8. A nutritional composition comprising digestible carbohydrates, lipids and proteins and the combination of a mixture of non-digestible saccharides and a mixture of lactic acid producing bacteria according to any one of claims 1-6.

9. The nutritional composition according to claim 8 comprising 1 .5 to 35 g of the mixture of non- digestible saccharides per 100 g dry weight of the total composition.

10. The nutritional composition according to claim 8 or 9, that comprises at least 10³ cfu per g dry weight L. acidophilus and at least 10³ cfu per g dry weight Bifidobacterium selected from the group consisting of B. breve, B. bifidum, and B. longum subsp. longum.

11 . The nutritional composition according to any one of claims 8-10 which is a follow-on or young child formula, preferably a young child formula.

12. The nutritional composition according to any one of claims 8-11 which is for use in providing nutrition to a an infant of 6 months and older and/or a young child, preferably a young child, wherein the infant of 6 months and older and/or young child is treated with or has been treated with antibiotics, is at risk of or suffering from atopic dermatitis, or is a picky eater, preferably is treated with or has been treated with antibiotics.

13 The combination, or supplement of nutritional composition for use according to any one of claims 1 to 12, for use in a method of therapy, wherein the method of therapy is preventing or treating intestinal disorders selected from the group consisting of microbial dysbiosis, constipation, intestinal infections, intestinal inflammation, and diarrhea.

14 The combination, or supplement of nutritional composition for use according to claim 13, wherein the method of therapy is preventing or treating intestinal inflammation and/or increasing the intestinal barrier function in a subject.