Synbiotic combination for young children

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 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

[0001] SYMBIOTIC COMBINATION FOR YOUNG CHILDREN

[0002] FIELD OF THE INVENTION

[0003] The present invention is in the field of nutritional compositions for young children comprising mixtures of dietary fibres and probiotics that have a beneficial effect on the development of the microbiota.

[0004] BACKGROUND OF THE INVENTION

[0005] Fibres 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 fibres are the human milk oligosaccharides (HMOS) as found in human milk. Infant formula 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 formula with molecules structurally identical to human milk oligosaccharides are also known in the art. In adult nutrition the dietary fibres are mainly derived from plant sources and consist of a mixture of soluble and insoluble fibre, a mixture of indigestible poly- and oligosaccharides, and fermentable and non- fermentable fibres. EP0756828 describes a fibre mix with a composition representing the dietary fibre in a typical adult Western diet.

[0006] 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.

[0007] Ensuring in a young child that the transition from infant to adult microbiota follows the right progression, can provide long lasting health benefits. Furthermore, unhealthy eating habits are quite prevalent in this age category leading to several health and nutritional challenges. Many toddlers do not consume a sufficient amount of fibres. 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 fibres not naturally occurring in food, having their effect mainly in the proximal colon and having no additional benefits for the child. In addition, in 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.

[0008] WO 2022 / 122958 discloses a mixture of beta-galactooligosaccharides, inulin, soluble soy fibre and resistant starch suitable for young children.

[0009] WO 2022 / 103321 and WO 2022 / 103320 disclose a composition for children with B. breve, oat betaglucan, inulin, and resistant starch.

[0010] CN109349618 discloses a medicinal composition with oat beta-glucan, inulin, resistant dextrin and L. acidophilus for use in constipation.

[0011] CN112516265 discloses herbal medicinal compositions for constipation. Mixtures of eleven non- digestible saccharides including oat beta-glucan, inulin, polydextrose, GOS, and 13 strains of probiotics, including L. acidophilus and Bifidobacterium longum, B. breve together with herbal extracts are disclosed.

[0012] However, further improvements can still be made in the design of optimal mixes of fibres and probiotics for young children in order to enable a smooth transition for infant type to adult type gut microbiome and to improve gut health. Especially in optimal mixes that result in increased amounts of intestinal butyrate. 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.

[0013] SUMMARY OF THE INVENTION

[0014] 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 combination of specific LAPB with a specific NDS mixture comprising galactooligosaccharides, inulin, oat fibre comprising beta-glucan and resistant starch was intermediate in intestinal metabolites and microbiota formed, between a combination of the LAPB with a specific NDS mixture developed for infant formula, comprising GOS and IcFOS with or without HMOS, and a combination of the LAPB with a NDS mixture based on the adult diet. 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 Bifidobacterium longum subsp. longum and at least one Lactobacillus selected from the group consisting of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus paracasei and Lactobacillus plantarum, when compared to the mixture of NDS without LAPB or combination with other LAPB. This butyrate production was best increased when Bifidobacterium longum subsp. longum was combined with L. acidophilus. The increased butyrate production was associated with a specific increase of lactate-utilizing and butyrate producing bacterial species. 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 and reduced pro- inflammatory cytokines.

[0015] Therefore, the specific combination of the NDS mixure of galactooligosaccharides, inulin, oat fibre comprising beta-glucan and resistant starch and the LAPB mixture of Bifidobacterium longum subsp. longum and at least one Lactobacillus selected from the group consisting of L. acidophilus, L. helveticus, L. paracasei and L. plantarum is especially suitable for 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, and on top of that, enabling a high level of butyrate formation, resulting in anti-inflammatory effects and improved gut barrier function.

[0016] DETAILED DESCRIPTION OF THE INVENTION

[0017] The invention thus concerns a combination of a mixture of non-digestible saccharides and a mixture of lactic acid producing bacteria comprising

[0018] - as non-digestible saccharides a mixture of beta-galactooligosaccharides, inulin, cereal fibre comprising beta-glucan and resistant starch, and

[0019] - as lactic acid producing bacteria a mixture of Bifidobacterium longum subsp. longum and at least one Lactobacillus selected from the group consisting of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus paracasei and Lactobacillus plantarum.

[0020] Mixtures of non-digestible saccharides

[0021] 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, cereal fibre comprising betaglucan and resistant starch. The present NDS mixture preferably comprises beta- galactooligosaccharides, inulin, cereal fibre comprising beta-glucan provided as oat fibre comprising beta-glucan, preferably oat fibre comprising at least 30 wt% beta-glucan based on total oat fibre, and resistant starch type III. It was found that such a mixture was intermediate with respect to the pattern of metabolites formed between infant-like and adult-like and also that the microbiota established was more like that of a young child.

[0022] Beta-galactooligosaccharides

[0023] 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-galactooligosaccharides (TOS). Beta-galactooligosaccharides can be analyzed according to AOAC method 2001 .02. A suitable source of beta-galactooligosaccharides is VivinalOGOS (commercially available from Borculo Domo Ingredients, Zwolle, Netherlands). Other suitable sources are Oligomate® (Yakult), Cupoligo® (Nissin) and Bi2muno® (Classado).

[0024] 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.

[0025] Inulin

[0026] 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. Suitable sources of inulin are Raftiline GR (Beneo, Orafti), Raftiline HP (Beneo, Orafti) and Fibruline (Cosucra).

[0027] A sufficient amount of inulin is advantageous for children, in particular young children, paediatric patients and / or constipated children since it favourably 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.

[0028] Cereal fibre comprising beta-glucan

[0029] The present mixture of NDS comprises cereal fibre comprising beta-glucan, preferably cereal fibre comprising at least 30 wt% beta-glucan based on total cereal fibre, preferably cereal fibre comprising at least 50 wt% beta-glucan based on total cereal fibre. Beta-glucan is a non-starch polysaccharide composed of beta-D-glucose monomer units holding a glycosidic linkage at beta (1— >3), (1 — >4), and / or (1 — >6), either in a branched or in an unbranched manner. Cereal beta-glucans - including beta-glucan from oat, barley and wheat - are linear polysaccharides joined by 1 ,3 and 1 ,4 carbon linkages. The majority of cereal beta-glucan bonds consist of 3 or 4 beta-1 ,4 glycosidic bonds (trimers and tetramers) interconnected by 1 ,3 linkages. In cereal beta-glucan, these trimers and tetramers are known as cellotriosyl and cellotetraosyl. Oats and barley differ in the ratio of cellotriosyl to cellotetraosyl, and oat has less 1-4 linkages with a degree of polymerization higher than 4. In oat beta-glucan the ratio between cellotriosyl and cellotetraosyl units is between 1 .5 and 2.3 and in barley 1 .8 to 3.5. Oat beta-glucan is water soluble and may reach MW of between 1.1 and 1 .6 MDa.

[0030] According to the present invention the composition preferably comprises oat fibre comprisjng betaglucan or barley fibre comprising beta-glucan. Even more preferably the composition according to the present invention comprises oat fibre comprising beta-glucan. In particular the present mixture of NDS preferably comprises oat fibre comprising at least 30 wt% beta-glucan based on total oat fibre, more preferably oat fibre comprising at least 50 wt% beta-glucan based on total oat fibre.

[0031] A sufficient amount of cereal fibre comprising beta-glucan, preferably oat fibre comprising beta-glucan, is advantageous for children, in particular young children, paediatric patients and / or constipated children since it, 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. It was found that this effect was strongly dependent on the presence of cereal fibre comprising beta-glucan, preferably oat fibre comprising beta-glucan, as the comparative NDS mixture, with the cereal fibre comprising beta-glucan replaced by soluble soy fibre, resulted in a microbiota less intermediate between infant-type and adult-type, and also resulted in less specific effect of the LAPB on butyrate production.

[0032] Suitable sources of cereal fibre comprising at least 30 wt% beta-glucan based on total fibre of the cereal are PromOat from Lantmannen and Oatwell, formerly known as SweOat, from Givaudan. Preferably the cereal fibre comprising beta-glucan comprises at least 40 wt% beta-glucan based on total cereal fibre, more preferably at least 50 wt% beta-glucan based on total fibre of the cereal. Preferably the cereal fibre comprising at least 30 wt% beta-glucan is oat fibre. Preferably the oat fibre comprising beta-glucan comprises at least 40 wt% beta-glucan based on total oat fibre, more preferably at least 50 wt% betaglucan based on total oat fibre.

[0033] Resistant starch

[0034] The NDS mixture according to the present invention comprises resistant starch (RS). Resistant starch is a non-digestible alpha-glucan. Resistant starch relates to non-digestible carbohydrate polymers made up of at least 80% glucose monomers, based on total monomers of the resistant starch, preferably at least 85%, which are bound together for more than 50% via alpha- 1 ,4 glycosidic linkages, and which are resistant to digestion and absorption in the human stomach and small intestine and enter the colon intact.

[0035] Resistant starch can be determined according to the method described by McCleary and Monaghan (2002) J AOAC Int 85, 665-675. Resistant starch includes starch that is physically inaccessible to the digestive enzymes in the stomach and small intestine, starch that is inaccessible to enzymes due to its conformation, e.g. its natural granular form, such as uncooked potato starch, green banana flour and high amylase corn, starch that is formed when starch-containing foods are cooked and cooled (retrograded starch), and starch that is chemically modified to resist digestion.

[0036] Preferably the NDS mixture comprises resistant starch in the form of high amylose starch, preferably from corn, cassava or potato, more preferably from corn. The resistant starch is preferably of type III. Type III RS refers to retrograded starch, also known as RS3. Resistant starch type III is formed when starch-containing foods are cooked and cooled. Retrogradation refers to the collective processes of dissolved starch becoming less soluble after being heated and dissolved in water and then cooled. More preferably the resistant starch is a retrograded, resistant high amylose starch, preferably from corn. A suitable source of resistant starch is Novelose® 330 (Ingredion). Novelose 330 is a retrograded RS3 generated from the hydrolysed products of corn starch. This commercial product contains 28-38 wt% RS3, so about 32 wt% RS. It is composed of a low molecular fraction with a chain length of alpa-1 ,4-D- glucans of between 10 and 40 glucose units and a larger amount of higher-molecular-weight polymers. RS type III is a non-digestible polysaccharide, is fermentable by the intestinal microbiota, and not soluble. The remainder of the ingredient is not a non-digestible saccharide. Other sources of RS3 are C*Actistar form Cargill and Neo-amylose from YMC.

[0037] A sufficient amount of resistant starch is advantageous for children, in particular toddlers, in particular paediatric patients and / or constipated children since it advantageously results in a fermentation at the more distal part of the colon. The use of resistant starch advantageously results in the formation of intestinal butyrate. The use of resistant starch together with the other NDS and in combination with the mixture of LAPB according to the invention, results in an intestinal microbiota which is intermediate between infants and adult subjects. A too high amount of resistant starch may result in unfavourable product characteristics such as a high viscosity and precipitations.

[0038] NDS mixtures

[0039] 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 sources that provide non-digestible saccharides than the mixture of four NDS 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 four NDS according to the invention. Preferably the NDS in the nutritional composition comprising 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 four 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 the four NDS according to the invention. Having a substantial amount of other NDS present besides the four NDS of the present invention may not result in the same effects on the microbiota composition, activity and butyrate formation in young children.

[0040] Preferably the NDS mixture comprises beta-galactooligosaccharides, inulin, cereal fibre comprising beta-glucan, and resistant starch in a weight ratio of 1 : 0.1-10 : 0.05-5 : 0.01-1 , more preferably 1 : 0.2-5 : 0.1-2.5 : 0.02-0.5, more preferably 1 : 0.6-1 .7 : 0.3-0.8 : 0.06-0.2, even more preferably 1 : 0.8-1 .2 : 0.4-0.6 : 0.08- 0.1.

[0041] Preferably, the NDS mixture according to the invention comprises, based on weight, 20 - 60 % beta- galactooligosaccharides, 20 - 60 % inulin, 10-30 % cereal fibre comprising beta-glucan, and 2 - 6 % resistant starch. More preferably the NDS mixture comprises, based on weight, 30 - 50 % beta- galactooligosaccharides, 30 - 50 % inulin, 15-25 % cereal fibre comprising beta-glucan, and 3 - 6 % resistant starch. Even more preferably the NDS mixture comprises, based on weight, 34-42 % beta- galactooligosaccharides, 34-42 % inulin, 18-22 cereal fibre comprising beta-glucan and 3-5 % resistant starch. These ratios of these four NDS ensures a further improved balance and / or interaction between the different types of NDS and their specific beneficial effect, such as intestinal butyrate formation, and development of the microbiota composition and metabolic activity from infant-type to adult-type.

[0042] Lactic acid producing bacteria.

[0043] The combination of NDS and LAPB of the present invention contains at least two types of lactic acid producing bacteria (LAPB). One is a Bifidobacterium longum subsp. longum and one is a Lactobacillus selected from the group consisting of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus paracasei and Lactobacillus plantarum. Preferably the Lactobacillus in combination with the 8. longum subsp. longum is L. acidophilus, even more preferably as a further LAPB B. breve is present.

[0044] The present combination of NDS and LAPB preferably contains 2 x 104to 3 x 1013colony forming units (cfu) LAPB per gram dry weight of the combination, preferably 2 x 105to 3 x 1012cfu, more preferably 2 x 106to 3 x 1010cfu most preferably from 3 x 106to 3 x 109cfu LAPB per gram dry weight.

[0045] Bifidobacterium longum subsp. longum

[0046] The LAPB according to the present invention comprises a strain of Bifdobacterium longum subsp. longum. A syntrophic effect was observed in combinations comprising the mixtures of four NDS of the present invention and mixtures of LAPB comprising B. longum subsp. longum. In particular the formation of butyrate was stimulated in LAPB mixtures where a Lactobacillus and Bifidobacterium longum subsp. longum was present. This result was not, or to a lesser extent, observed when Bifidobacteria belonging to 8. breve or 8. bifidum were used.

[0047] 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 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): IQ- 16. doi:10. 1016 / j.mimet .2011.06.014. PMID 21756944 describe a method to identify 8. longum subsp. longum.

[0048] 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), or R0175 (Lallemand),

[0049] The present combination of NDS and LAPB preferably contains at least 104cfu 8. longum subsp. longum per gram dry weight of the combination of NDS and LAPB, more preferably at least 105cfu, even more preferably at least 106cfu per gram dry weight. The present combination of NDS and LAPB preferably contains 104to 1013colony forming units (cfu) B. longum subsp. longum per gram dry weight of the combination of NDS and LAPB, preferably 105to 1012cfu, more preferably 106to 101° cfu most preferably from 106to 109cfu per gram dry weight.

[0050] 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 8. longum subsp. infantis. Lactobacilli

[0051] The LAPB according to the present invention comprises a strain of Lactobacillus. It was found that the combination of B. longum subsp. longum and a Lactobacillus selected from L. acidophilus, L. helveticus, L. paracasei or L. plantarum 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.

[0052] The present combination of NDS and LAPB preferably contains at least 104cfu of a Lactobacillus selected from L. acidophilus, L. helveticus, L. paracasei or L. plantarum per gram dry weight of the combination of NDS and LAPB, more preferably at least 105cfu, even more preferably at least 106cfu per gram dry weight. The present combination of NDS and LAPB preferably contains 104to 2.1013colony forming units (cfu) Lactobacillus selected from L. acidophilus, L. helveticus, L. paracasei or L. plantarum per gram dry weight of the combination of NDS and LAPB, preferably 105to 2.1012cfu, more preferably 106to 2.1 O10cfu most preferably from 106to 2.109cfu per gram dry weight.

[0053] Lactobacillus acidophilus

[0054] The LAPB according to the present invention preferably comprises a strain of L. acidophilus. This species was special in that it enhanced the formation of butyrate the most when the NDS mixture of the present invention and a B. longum subsp. longum was fermented by the microbiota of young children. 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.

[0055] 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 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.

[0056] 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. Also other strains of L. acidophilus can be used instead.

[0057] The present combination of a mixture of NDS and a mixture of LAPB preferably contains at least 104cfu L. acidophilus per gram dry weight, more preferably at least 105cfu, even more preferably at least 106cfu L. acidophilus per gram dry weight. The present combination preferably contains not more than 1013cfu L. acidophilus per gram dry weight of the combination of a mixture of NDS and mixture of LAPB, more preferably not more than 1012cfu, even more preferably not more than 1 O10cfu L. acidophilus per gram dry weight. The present combination of NDS and LAPB preferably contains 104to 1013colony forming units (cfu) L. acidophilus per gram dry weight, preferably 105to 1012cfu, more preferably 106to 1 O10cfu most preferably from 106to 109cfu L. acidophilus per gram dry weight.

[0058] Lactobacillus helveticus

[0059] The LAPB according to the present invention preferably comprises a strain of L. helveticus. This species enhanced the formation of butyrate when the NDS mixture of the present invention and a B. longum subsp. longum was fermented by the microbiota of young children. B. longum subsp. longum together with L. helveticus in combination with the NDS of the present invention resulted in an higher SCFA and especially butyrate production than in combination with one of the other bifidobacteria.

[0060] L. helveticus is a Gram- positive, anaerobic, branched rod-shaped bacterium. It is a homofermentative microorganism, producing lactate via the EMP pathway. The L. helveticus according to the present invention preferably has at least 95 % identity with the 16S rRNA sequence when compared to the type strain of L. helveticus AT CC 15009, more preferably at least 97% identity (Stackebrandt & Goebel, 1994, Int. J. Syst. Bacteriol. 44:846-849). L. helveticus strains are typically commercially available from producers of lactic acid bacteria, but they can also be directly isolated from fermented products, identified, characterised and produced.

[0061] Suitable L. helveticus strains are available. An example of a suitable L. helveticus strain is R0052 (Lallemand) CNCM 1-1722 or SBT2171 (Mori nag a).

[0062] The present combination of a mixture of NDS and a mixture of LAPB preferably contains at least 104cfu L. helveticus per gram dry weight, more preferably at least 105cfu, even more preferably at least 106cfu L. helveticus per gram dry weight. The present combination preferably contains not more than 1013cfu L. helveticus per gram dry weight of the combination of a mixture of NDS and mixture of LAPB, more preferably not more than 1012cfu, even more preferably not more than 1 O10cfu L. helveticus per gram dry weight. The present combination of NDS and LAPB preferably contains 104to 1013colony forming units (cfu) L. helveticus per gram dry weight, preferably 105to 1012cfu, more preferably 106to 1 O10cfu most preferably from 106to 109cfu L. helveticus per gram dry weight.

[0063] Lactobacillus paracasei

[0064] The LAPB according to the present invention preferably comprises a strain of L. paracasei. This species, recently renamed as Lactocaseibacillus paracasei, enhanced the formation of butyrate when the NDS mixture of the present invention and a B. longum subsp. longum was fermented by the microbiota of young children. B. longum subsp. longum together with L. paracasei in combination with the NDS of the present invention resulted in an higher SCFA and especially butyrate production than in combination with one of the other bifidobacteria.

[0065] L. paracasei is a Gram- positive, anaerobic, branched rod-shaped bacterium. It is a homofermentative microorganism, producing lactate via the EMP pathway. The L. paracasei according to the present invention preferably has at least 95 % identity with the 16S rRNA sequence when compared to the type strain of L. paracasei ATCC 25302, more preferably at least 97% identity (Stackebrandt & Goebel, 1994, Int. J. Syst. Bacteriol. 44:846-849). L. paracasei strains are typically commercially available from producers of lactic acid bacteria, but they can also be directly isolated from the human gastrointestinal tract or fermented products, identified, characterised and produced. Suitable L. paracasei strains are available. An example of a suitable L. paracasei strain is Lactobacillus paracasei Lpp1 , Danone, CNCM 1-1518 or L. paracasei 431 (Novonesis).

[0066] The present combination of a mixture of NDS and a mixture of LAPB preferably contains at least 104cfu L. paracasei per gram dry weight, more preferably at least 105cfu, even more preferably at least 106cfu L. paracasei per gram dry weight. The present combination preferably contains not more than 1013cfu L. paracasei per gram dry weight of the combination of a mixture of NDS and mixture of LAPB, more preferably not more than 1012cfu, even more preferably not more than 1 O10cfu L. paracasei per gram dry weight. The present combination of NDS and LAPB preferably contains 104to 1013colony forming units (cfu) L. paracasei per gram dry weight, preferably 105to 1012cfu, more preferably 106to 1 O10cfu most preferably from 106to 109cfu L. paracasei per gram dry weight.

[0067] Lactobacillus plantarum

[0068] The LAPB according to the present invention preferably comprises a strain of L. plantarum. This species enhanced the formation of butyrate when the NDS mixture of the present invention and a B. longum subsp. longum was fermented by the microbiota of young children. B. longum subsp. longum together with L. plantarum in combination with the NDS of the present invention resulted in an higher SCFA and especially butyrate production than in combination with one of the other bifidobacteria.

[0069] Lactobacillus, plantarum (recently renamed as Lactoplantibacillus plantarum) is a Gram positive, bacilli shaped bacterium. L. plantarum cells are rods with rounded ends, straight, generally 0.9-1 .2 pm wide and 3-8 pm long, occurring singly, in pairs or in short chains. The L. plantarum according to the present invention preferably has at least 95 % identity with the 16S rRNA sequence when compared to the type strain of L. plantarum ATCC 4356, more preferably at least 97% identity (Stackebrandt & Goebel, 1994, Int. J. Syst. Bacteriol. 44:846-849). L. plantarum strains are typically commercially available from producers of lactic acid bacteria, but they can also be directly isolated from the human gastrointestinal tract or fermented products, identified, characterised and produced.

[0070] Suitable L. plantarum strains are available. An example of a suitable L. plantarum strain is L. plantarum 299v (Probi) DSM9843 (can also be referred to as Lactiplantibacillus plantarum 299v) or PPLP-217 (Novonesis).

[0071] The present combination of a mixture of NDS and a mixture of LAPB preferably contains at least 104cfu L. plantarum per gram dry weight, more preferably at least 105cfu, even more preferably at least 106cfu L. plantarum per gram dry weight. The present combination preferably contains not more than 1013cfu L. plantarum per gram dry weight of the combination of a mixture of NDS and mixture of LAPB, more preferably not more than 1012cfu, even more preferably not more than 1 O10cfu L. plantarum per gram dry weight. The present combination of NDS and LAPB preferably contains 104to 1013colony forming units (cfu) L. plantarum per gram dry weight, preferably 105to 1012cfu, more preferably 106to 1 O10cfu most preferably from 106to 109cfu L. plantarum per gram dry weight.

[0072] Bifidobacterium breve

[0073] In a preferred embodiment, a combination of B. longum subsp. longum and B. breve is present together with L. acidophilus. Additional experiments testing the combination of B. longum subsp. longum and B. breve were indicative of an even further improvement in metabolite formation when a mixture of L. acidophilus, B. longum subsp. longum and B. breve was present. The effects were not strain specific. The LAPB according to the present invention preferably comprises a strain of Bifdobacterium breve. A syntrophic effect was observed with B. longum subsp. longum and L. acidophilus and the NDS mixture of the invention, especially with respect to butyrate formation. Especially a combination of B. longum subsp. longum and B. breve with L. acidophilus was able to form the highest amounts of butyrate in combination with the NDS mixture of the present invention.

[0074] 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, characterised and produced.

[0075] 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).

[0076] 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).

[0077] The present combination of NDS and LAPB preferably contains at least 104cfu B. breve per gram dry weight of the combination of NDS and LAPB, more preferably at least 105cfu, even more preferably at least 106cfu B. breve Per gram dry weight. The present combination preferably contains 104to 1013colony forming units (cfu) B. breve per gram dry weight of the combination of NDS and LAPB, preferably 105to 1012cfu, more preferably 106to 101° cfu most preferably from 106to 109cfu B. breve per gram dry weight. The present combination of NDS and LAPB preferably contains at least 104cfu 8. breve and at least 104cfu B. longum subsp. longum per gram dry weight of the combination of NDS and LAPB more preferably at least 105cfu B. breve and at least 105cfu B. longum subsp. longum, even more preferably at least 106cfu B. breve and at least 106cfu B. longum subsp. longum.

[0078] The present combination of NDS and LAPB preferably contains 104to 1013cfu B. breve and 104to 1013cfu 8. longum subsp. longum per gram dry weight of the combination of NDS and LAPB, preferably 105to 1012cfu of each of both, more preferably 106to 1 O10cfu of each of both most preferably from 106to 109cfu of each of both 8. breve and 8. longum subsp. longum per gram dry weight.

[0079] Bifidobacterium bifidum

[0080] The LAPB preferably comprises a strain of Bifidobacterium bifidum. This species combined with B. longum subsp. longum and L. acidophilus showed an enhanced butyrate production. 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 8. 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 B. bifidum R0071 from Lallemand or B. bifidum Bb-06 (Dupont Dansico). Most preferably, the B. bifidum is 8. 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. B. bifidum CNCM 1-4319 has also been disclosed in US 9,402,872.

[0081] The present combination of NDS and LAPB preferably contains at least 104cfu B. bifidum per gram dry weight, more preferably at least 105cfu, even more preferably 106cfu 8. bifidum per gram dry weight. The present combination of NDS and LAPB preferably contains 104to 1013colony forming units (cfu) 8. bifidum per gram dry weight, preferably 105to 1012cfu, more preferably 106to 101° cfu, most preferably 106to 109cfu B. bifidum per gram dry weight.

[0082] Preferably the B. longum subsp. longum and at least one Lactobacillus selected from the group consisting of L. acidophilus, L. helveticus, L. paracasei and L. plantarum, 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

[0083] 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.

[0084] In the context of the present invention, the nutritional composition according to the invention can also be named a young child formula. In order to meet the caloric requirements of the 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.

[0085] The nutritional composition according to the invention comprises lipid, protein, digestible carbohydrate 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 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 3 - 7% of the total calories of the nutritional composition.

[0086] 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 0.4 - 6.0 g of the present NDS mixture per 100 ml, preferably 0.6 - 5.3 g, more preferably 0.9 - 3.3 g and even more preferably 1.1 - 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 3 - 45 g of the present NDS mixture per 100 g dry weight, preferably 4 - 40 g, more preferably 7 - 25 g and even more preferably 8-20 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 0.6 - 9.0 g of the present NDS mixture per 100 kcal, preferably 0.8 - 8.0 g, more preferably 1 .4 - 5.0 g and even more preferably 1.6 - 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 purpose of the present invention the caloric density of the NDS is set at 2 kcal per gram.

[0087] The present nutritional composition comprising the combination of NDS and LAPB preferably contains 2 x 103to 3 x 1012colony forming units (cfu) LAPB per gram dry weight, preferably 2 x 104to 3 x 1011cfu, more preferably 2 x 105to 3 x 109cfu most preferably from 3 x 105to 3 x 108cfu LAPB per gram dry weight of nutritional composition.

[0088] 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.

[0089] Furthermore, the nutritional composition according to the invention preferably comprises 0.13 - 2.7 g inulin per 100 ml, more preferably 0.20 - 2.0 g, even more preferably 0.40 - 1.3 g inulin per 100 ml. Based on dry weight, the nutritional composition according to the invention preferably comprises 1 .0 - 20 g inulin per 100 g, more preferably 1.5 - 15 g, even more preferably 3.0 - 10 g inulin per 100 g. Based on calories, the nutritional composition according to the invention preferably comprises 0.2 - 4.0 g inulin per 100 kcal, more preferably 0.3 - 3.0 g, even more preferably 0.6 - 2.0 g inulin per 100 kcal.

[0090] The nutritional composition according to the invention preferably comprises 0.07 - 1 .33 g cereal fibre comprising beta-glucan per 100 ml, more preferably 0.09 - 1.00 g, even more preferably 0.20 - 0.67 g cereal fibre comprising beta-glucan per 100 ml. Based on dry weight, the nutritional composition according to the invention preferably comprises 0.5 - 10 g cereal fibre comprising beta-glucan per 100 g, more preferably 0.7 - 7.5 g, even more preferably 1 .5 - 5 g cereal fibre comprising beta-glucan per 100 g. Based on calories, the nutritional composition according to the invention preferably comprises 0.10 - 2.0 g cereal fibre comprising beta-glucan per 100 kcal, more preferably 0.14 - 1 .5 g, even more preferably 0.30 - 1.0 g cereal fibre comprising beta-glucan per 100 kcal. Cereal fibre comprising betaglucan preferably is cereal fibre comprising at least 30 wt% beta-glucan based on total cereal fibre, more preferably at least 50 wt% beta-glucan based on total cereal fibre. Cereal fibre comprising beta-glucan preferably is oat fibre.

[0091] Furthermore, the nutritional composition according to the invention preferably comprises 0.01 - 0.27 g resistant starch per 100 ml, more preferably 0.02 - 0.20 g, even more preferably 0.04 - 0.13 g resistant starch per 100 ml. Based on dry weight, the nutritional composition according to the invention preferably comprises 0.10 - 2.0 g resistant starch per 100 g, more preferably 0.14 - 1 .5 g, even more preferably 0.30 - 1.0 g resistant starch per 100 g. Based on calories, the nutritional composition according to the invention preferably comprises 0.02 - 0.4 g resistant starch per 100 kcal, more preferably 0.03 - 0.3 g, even more preferably 0.06 - 0.2 g resistant starch per 100 kcal. The resistant starch is preferably type III resistant starch.

[0092] Furthermore, the present nutritional composition comprising the combination of NDS and LAPB preferably contains 103to 1012colony forming units (cfu) Bifidobacterium longum subsp. longum per gram dry weight, preferably 104to 1011cfu, more preferably 105to 109cfu most preferably from 105to 108cfu Bifidobacterium longum subsp. longum per gram dry weight of the nutritional composition.

[0093] In addition to the Bifidobacterium longum subsp. longum, the present nutritional composition comprising the combination of NDS and LAPB preferably contains 103to 2.1012colony forming units (cfu) of a Lactobacillus selected from the group consisting of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus paracasei and Lactobacillus plantarum per gram dry weight of the nutritional composition comprising the combination of NDS and LAPB, preferably 104to 2.1011cfu, more preferably 105to 2.109cfu most preferably from 105to 2.108cfu per gram dry weight of the nutritional composition.

[0094] In addition to the Bifidobacterium longum subsp. longum, the present nutritional composition comprising the combination of NDS and LAPB preferably contains 103to 1012colony forming units (cfu) Lactobacillus acidophilus per gram dry weight of the nutritional composition comprising the combination of NDS and LAPB, preferably 104to 1011cfu, more preferably 105to 109cfu most preferably from 105to 108cfu per gram dry weight of the nutritional composition.

[0095] In another embodiment, in addition to the Bifidobacterium longum subsp. longum, the present nutritional composition comprising the combination of NDS and LAPB preferably comprises 103to 1012colony forming units (cfu) B. breve per gram dry weight, preferably 104to 1011cfu, more preferably 105to 199cfu most preferably from 105to 108cfu B. breve per gram dry weight of the nutritional composition.

[0096] In yet another embodiment, in addition to the Bifidobacterium longum subsp. longum, the present nutritional composition comprising the combination of NDS and LAPB preferably comprises 103to 1012cfu B. breve and 103to 1012cfu Lactobacillus acidophilus per gram dry weight of the nutritional composition, preferably 104to 1011cfu of each of both, more preferably 105to 109cfu of each of both most preferably from 105to 108cfu of each of both B. breve and Lactobacillus acidophilus per gram dry weight of the nutritional composition.

[0097] In another embodiment, in addition to the Bifidobacterium longum subsp. longum, the present nutritional composition comprising the combination of NDS and LAPB preferably comprises 103to 1012colony forming units (cfu) B. bifidum and 103to 1012cfu Lactobacillus acidophilus per gram dry weight, preferably 104to 1011cfu, more preferably 105to 109cfu, most preferably 105to 108cfu of each of both 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.

[0098] The amount of saturated fatty acids is preferably below 45 wt% based on total lipid 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 more carbon atoms. Preferably the nutritional composition comprises docosahexaenoic acid (DHA) and / or eicosapentaenoic acid (EPA).

[0099] 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.

[0100] 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.

[0101] 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.

[0102] The nutritional composition preferably comprises 4.7 - 11 .2 g digestible carbohydrate 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 carbohydrate per 100 g, more preferably 45 - 80 g per 100 g, more preferably 55 - 70 g digestible carbohydrate per 100 g dry weight of the nutritional composition. Based on calories, the nutritional composition preferably comprises 7 - 17 g digestible carbohydrate per 100 kcal, more preferably 9 - 16 g per 100 kcal, more preferably 11 - 14 g digestible carbohydrate per 100 kcal of the nutritional composition. The digestible carbohydrate preferably provides 30 - 74%, more preferably 40 - 65%, more preferably 45 - 55 % of the total calories of the present nutritional composition.

[0103] 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.

[0104] 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.

[0105] Nutritional supplement

[0106] In one embodiment of the present invention the combination of NDS and LAPB is in the form of a supplement or is comprised in 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.

[0107] Application

[0108] 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 1 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 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.

[0109] 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 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 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.

[0110] 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 fibre intake from the remainder of their diet and may especially benefit.

[0111] 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 B. longum subsp. longum and a Lactobacillus selected from Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus paracasei and Lactobacillus plantarum, enhanced the absolute and relative amounts of butyrate formed. Of these four lactobacilli, combination with Lactobacillus acidophilus resulted in the highest level of butyrate. The metabolites that were formed improved the intestinal barrier function and had an anti-inflammatory effect.

[0112] Therefore the present synbiotic combination is beneficial 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] In another embodiment, the method of therapy is preventing or treating intestinal inflammation and / or increasing the intestinal barrier function in a subject.

[0118] In another embodiment, the method of therapy is improving intestinal microbiota composition and activity.

[0119] Preferably the use is in a young child from 1 up to and including 12 years of age, more preferably in a young child of 1 , 2, or 3 years old.

[0120] 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.

[0121] 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, cereal fibre comprising beta-glucan and resistant starch and the lactic acid producing bacteria is a mixture of Bifidobacterium longum subsp. longum and at least one Lactobacillus selected from the group consisting of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus paracasei and Lactobacillus plantarum.

[0122] 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 betagalactooligosaccharides, inulin, cereal fibre comprising beta-glucan and resistant starch and the lactic acid producing bacteria is a mixture of Bifidobacterium longum subsp. longum and at least one Lactobacillus selected from the group consisting of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus paracasei and Lactobacillus plantarum.

[0123] 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.

[0124] BRIEF DESCRIPTION OF THE FIGURES

[0125] Figure 1 shows a principal component analysis (PCA) summarizing the impact on the gut microbial activity tested via the SIFR® technology platform, for 4 fibre mixtures (FM1 / FM2 / FM3 / FM4), 12 probiotic mixes (circles) and combinations thereof compared to reference products (BM1 / BM2) and a no substrate control (NSC).

[0126] Figure 2 shows the impact on butyrate levels tested via the SIFR® technology platform, for 4 fibre mixtures (FM1 / FM2 / FM3 / FM4), 12 probiotic mixes (1-12) and combinations thereof compared to reference products (BM1 / BM2) and a no substrate control (NSC).

[0127] Figure 3 shows a principal component analysis (PCA) summarizing the impact on microbial composition (at OTU level) as tested via the SIFR®technology for4 fibre mixtures (FM1 / FM2 / FM3 / FM4), 12 probiotic mixes (circles) and combinations thereof compared to reference products (BM1 / BM2) and a no substrate control (NSC). The ellipses represent the 95% confidence intervals for each of the fibre groups.

[0128] EXAMPLES

[0129] Material and methods

[0130] Four different mixtures of non-digestible saccharides 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 67 different combinations of non-digestible saccharides (NDS) and lactic acid producing bacteria (LAPB) that were tested.

[0131] NDS mixture FM2 and FM3 were designed and adapted to the specific dietary needs of young children, taking into account cereals, grains, vegetables and fruit as main dietary sources of NDS. NDS mixture FM3 is a mixture of the prior art. NDS mixture FM2 is a mixture according to the present invention. NDS mixture FM4 is a NDS mixture adapted for adults and the mixture is based on a standard dietary intake of adults with a normal healthy diet. NDS mixture FM1 of beta-galactooligosaccharides (scGOS) and long chain fructooligosaccharides (IcFOS) and five different human milk oligosaccharides was 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.

[0132] Table 1 : Non-digestible saccharides mixtures (weight % based on total NDS) aSource of scGOS is Vivinal® GOS (FrieslandCampina, Domo-Borculo NL).bSource of IcFOS is Raftiline HP (Beneo-Orafti).

[0133] 0Source of inulin is Orafti® GR (Beneo-Orafti).d2’-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.eSource of oat fibre comprising beta-glucan is OatWell BG28 (Givaudan) - contains about 28 wt% beta-glucan based on dry weight, and about 56 wt% beta-glucan based on fibre.fSource of oat fibre comprising beta-glucan for FM4 is PromOat (Lantmannen) - contains about 29 w% beta-glucan based on dry weight and about 96 wt% based on total fibre.

[0134] 9Source of resistant starch is Novelose®330 (Ingredion UK Ltd.).hSource of arabinoxylan PureFiber AX is used (supplier PureFiber). This is corn derived glutenfree and had about 89 wt% fibre and 79 wt% arabinoxylan.

[0135] ' Source of pectin rich soy fibre is Soyafibre-S-Ca100® (Fuji Oil Co, Ltd).

[0136] The following strains of LAPB were used:

[0137] Lactobacillus acidophilus, strain NCFM (Dupont / IFF) - also known as ATCC 700396.

[0138] Lactobacillus plantarum, 299v (Probi) DSM9843 (can also be referred to as Lactiplantibacillus plantarum 299v)

[0139] Lactobacillus paracasei Lpp1 , Danone, CNCM 1-1518

[0140] Lactobacillus helveticus R0052 (Lallemand) CNCM 1-1722

[0141] Bifidobacterium longum subspecies longum, BB536 (Morinaga) ATCC BAA-999

[0142] Bifidobacterium bifidum, (Danone) CNCM 1-4319

[0143] Bifidobacterium breve, BbC50, (Danone) CNCM 1-2219

[0144] 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 108CFU / 5 mL, i.e. 5 x 107

[0145] CFU / mL. Table 2 shows the combinations that were tested.

[0146] Table 2: List of strains, fiber mixtures and their combinations tested.

[0147] Faecal slurry fermentations were performed with samples from six healthy toddlers (1 to 3 years of age).

[0148] No breastfeeding or 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 107cfu / ml.

[0149] 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.

[0150] Analysis of SCFA, BCFA, pH, lactate:

[0151] 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).

[0152] Microbial composition 16S rRNA gene profiling:

[0153] 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 analysed 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.

[0154] Data analysis:

[0155] 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:

[0156] 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 / FM2 / FM3 / FM4) are indicated with “$ / $$ / $$$”, while differences with the respective LAPB (1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12) are indicated with “& / && / &&&”.

[0157] In addition, to estimate the independent effect of the treatment groups (i.e., NDS mixes 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)).

[0158] 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.

[0159] Additional analyses were performed for 16 selected study arms for 0 h (only blanc) and 24 h (all 16 study arms). 15 Test products (selected out of the 66 originally tested test products) were evaluated, i.e., 2 NDS products (FM1 / FM2), 3 LAPB mixes (2 / 6 / 10) as such, the 6 synbiotic combinations, along with 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 107CFU / mL.

[0160] Microbial composition (quantitative shallow shotgun sequencing):

[0161] 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.

[0162] Metabolomics (untargeted LC-MS semi-polar analysis):

[0163] 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).

[0164] 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 1 accepted deviation of 3 ppm), and MS / MS spectra. Level 3: identification by accurate mass alone (with an accepted deviation of 3 ppm).

[0165] Results are shown in examples example 1 and 2.

[0166] Example 1: Effect of different synbiotic combinations on the metabolites formed by the intestinal microbiota of young children

[0167] A principal component analysis (PCA) based on fundamental fermentation parameters, provided comprehensive insight in overall treatment effects since the first two components explained 97.1 % of variation of the dataset. All parameters (acetate, propionate, butyrate, gas), but not pH and bCFA, correlated positively with PC1 (~ X-axis), indicating that samples with enhanced microbial activity positioned to the right. There was a marked differential clustering of 0 h and 24 h samples. As the incubation progressed, samples moved from the left bottom to the upper and right upper side of the PCA, suggesting enhanced production of acetate, propionate, butyrate, bCFA and gasses.

[0168] PCAs were made to visualize product-specific effects, averaged across all donors, thus allowing to zoom in on consistent product effects (Figure 1). The following treatment effects were observed.

[0169] The blanc (No Substrate Control - NSC), and samples of the treatment with LAPB without NDS were positioned closest to 0 h samples along PC1 , suggesting that particularly the NDS mixtures and synbiotic combinations thereof boosted microbial metabolite production compared to the blanc or LAPB alone. Mixtures comprising NDS mixture FM1 , FM2, FM3 and FM4 tended to be positioned as four separate clusters, suggesting product-specific treatment effects. The reference products BM1-2 were positioned in the FM1 cluster, suggesting similar effects on microbial metabolite production.

[0170] 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 were positioned most to the right suggesting strongest overall effects. FM1 , BM1-2 were positioned most downwards relating with butyrate. FM4 was positioned most upwards relating with propionate. FM2 and FM3 showed intermediate effects on these metabolites, with FM2 being the most intermediate in the 2-dimensional space between FM1 and FM4. The label "intermediate" is based on the positioning in the PCA (that considers all 'fundamental fermentation parameters'). The position of FM2 (but also FM3) in the 2-dimensional space indicated to be in between BM / FM1 (infant-like) and FM4 (adult-like).

[0171] Looking at the NDS mixtures without LAPB, FM2 and FM1 resulted in highest SCFA production and FM1 in the lowest pH (highest pH reduction). On the other hand, FM4 resulted in lowest total SCFA and highest pH. FM2 and FM3 were intermediate in pH reduction. Overall, NDS administration markedly boosted acetate, propionate and butyrate and reduced bCFA in a NDS-specific fashion. In particular with FM2, the pH, amounts of acetate and in particular butyrate were intermediate between FM1 and FM4. The reference products BM1-2 also strongly increased SCFA production. Overall BM1-2 resulted in the highest acetate levels and butyrate levels across all test conditions, being reminiscent to FM1. LAPB without added NDS showed a small effect. Less SCFA were formed as less substrate to be fermented is present.

[0172] Subsequently the effect of specific combinations of the NDS mixtures with LAPB was examined. It was found that the addition of various LAPB further impacted SCFA formation, mostly stimulating butyrate, in a specific synbiotic combination-dependent way.

[0173] Within each NDS cluster, 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. Lactate accumulation was not observed, except for low levels in FM1 combinations with some of the LAPB and BM1-2 (data not shown). Lactate is formed by the LAPB, but this is converted by the lactate utilizing bacteria in the microbiota into propionate and butyrate.

[0174] A positive 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. When looking for combinations suitable for young children that are especially butyrogenic, the focus was on selecting the best synbiotic combinations containing the intermediate NDS mixtures FM2 or FM3. The most remarkable butyrogenic effect was observed in FM2 in combinations of B. longum subsp. longum and a Lactobacillus selected from L. acidophilus, L. helveticus, L. paracasei and L. plantarum, see Figure 2. In these combinations acetate was lowered, suggesting that a cross feeding from acetate to butyrate took place.

[0175] The highest butyrate formation was unexpectedly found with the combination of FM2 with B. longum subsp. longum and L. acidophilus.

[0176] Also, in FM3 such effects on butyrate production of the B. longum subsp. longum in combination with lactobacilli in particular L. acidophilus were observed, but to a lesser extent than with FM2. Table 3 lists the average butyrate production for the combinations of the NDS mixtures with LAPB. It shows that for FM2, the combination with B. longum subsp. longum and any of Lactobacillus acidophilus, B. bifidum and B. breve resulted in an unexpected remarkable increase compared to the FM2 mixture. Table 3 also shows that for FM2, the combination with B. longum subsp. longum and any of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus paracasei and Lactobacillus plantarum resulted in an unexpected remarkable increase compared to the FM2 mixture.

[0177] 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

[0178] Subsequently, the NDS mixtures FM1 / FM2, 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.

[0179] The NDS FM1 / FM2 as such significantly increased the levels of metabolites related to diverse health benefits, i.e., N-acetylated amino acids, indole-3-propionic acid, 3-phenyllactic acid, N8- acetylspermidine, pipecolinic acid, 7-methylguanine and nicotinic acid (vitamin B7). Several metabolites were more strongly / specifically elevated by FM2, compared to FM1 , i.e. phenylpyruvic acid (intermediate of 3-phenyllactic acid), indole-3-acetic acid (IAA), 4-guanidinobutyric acid, acetylagmatine, pyridoxic acid (= vitamin B6), trigonelline (= vitamin B3).

[0180] Addition of the LAPB to FM1 / FM2 further stimulated following health-related metabolites, vs. FM1 / FM2 as such, resulting in potent synbiotic effects:

[0181] The combination of FM2 / L. acidophilus / B. longum subsp. longum showed an increase in hydroxyphenyllactic acid, playing a role in immune development and gut barrier function, and 5- methylcytosine. The combination of FM2 / L acidophilus / B. longum subsp. longum showed an increase in indole-3- propionic acid, which is associated with gut barrier integrity and neuroprotection, 4-guanidinobytyric acid (an intermediate of gamma-aminobutyric acid; GABA), serotonin, 3-methylxanthine and hydroxyphenyllactic acid.

[0182] These results are indicative that the synbiotic combination of beta-galactooligosaccharides, inulin, cereal fibre comprising beta-glucan and resistant starch, with Bifidobacterium longum subsp. longum and a Lactobacillus selected from L. acidophilus, L. helveticus, L. paracasei and L. plantarum, in particular Lactobacillus acidophilus is most suitable for young children to improve the activity of the microbiome and the transition from an infant to an adult type microbiome. In particular, the observed effects on elevated butyrate production are indicative for increased intestinal health.

[0183] 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.

[0184] Effect of the NDS

[0185] 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 mixtures and the synbiotic combinations. For the NDS mixtures, total bacterial cells were highest with FM4, lowest with FM1 and the BM1-2, whereas especially fermentation with FM2 resulted in an intermediate bacterial density.

[0186] A PCA was made based on the average results across donors at 24 h. The PCA was made at highest phylogenetic resolution (OTU level), with OTUs being narrowed down to those that were significantly (FDR = 0.20) or consistently affected by any of the treatments. The PCA was based on standardized values (AVG = 0; SD = 1) so that each OTU equally contributed to the PCA (independent of their abundance), allowing to relate more OTUs to treatments. PCAs were made across all samples. The following treatment effects were established based on the PCA (Figure 3):

[0187] FM1 positioned to the left, FM2 and FM3 positioned between FM1 and FM4, though being closer to the FM1 cluster. Positioning to the left related with, amongst others, a series of Bifidobacterium subsp. (B. pseudocatenulatum, B. longum, B. breve and B. bifidum). Further, these treatments 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 also positioned to the left.

[0188] FM4-containing products positioned to the right upper side relating with a series of Bacteroides spp. (B. vulgatus / dorei and 8. xylanisolvens), potent propionate producers. Further, FM4 specifically related with Blautia spp. (8. obeum / wexlerae and 8. fusiformis), and, amongst others, Eisenbergiella tayi, Ruminococcus bromii (keystone species for starch degradation), Waltera intestinalis and Hominilimicola fabiformis (keystone species for xanthan degradation), altogether revealing the effect of FM4 on the toddlers’ microbiota, resulting in a microbiota that is much more adult like.

[0189] FM3 / FM2, and particularly FM2, positioned in the direction of FM4, compared to FM1 and the benchmarks and this indicates that the effects on the microbiota composition of FM2 / 3, and in particular FM2, were intermediate between FM1 and FM4.

[0190] This 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 / Proteobacteria / Verrucomicrobiota being detected. No significant treatment effects were noted for the less abundant phyla so the focus is on effects on Actinobacteria, Bacteroidota and Firmicutes.

[0191] First, all NDS-containing products significantly increased the phylum to which Bifidobacteriaceae belong, i.e., Actinobacteriota. All test products (except BM1 / 2 and FM3) significantly increased Bacteroidota. This increase was most marked for FM4 and also FM2.

[0192] All test products (except BM1 / 2 and specific synbiotic combinations with FM1) also significantly increased Firmicutes. Among the NDS, the increase was most marked for FM4, while FM2 showed an intermediate effect. The effect of FM2 and FM3 on the microbiota that was observed is intermediate between that of an infant and that of an adult, with the FM2 mix being the most intermediate.

[0193] While all NDS mixtures significantly boosted Bifidobacteriaceae, FM1 / 2 / 3 most strongly increased the most prevalent Bifidobacterium species in the toddler’s microbiota, i.e., B. pseudocatenulatum.

[0194] Specific synbiotic interactions

[0195] Within the specific NDS mixtures 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 along with FM2. Another remarkable finding related to the FM2-based synbiotics, was the prominent stimulation of Lactobacillus acidophilus for LAPB mixtures containing L. acidophilus strain. The extent of the L. acidophilus increase was very pronounced reaching abundances as high as almost 1 .5 x 109cells / mL. 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.

[0196] FM1-4 all increased OTUs related to each added probiotic Bifidobacterium strain: The extent of increasing effects on B. longum subsp. longum respectively, was highest for FM1 and lowest for FM4, with FM2 / 3 being intermediate.

[0197] In general effects on the microbiota composition were in line with the effects observed on the amounts of lactate, acetate, propionate and butyrate.

[0198] 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 (e.g. +37% butyrate production when combining L. acidophilus + 8. longum subsp. longum with FM2 compared to FM2 alone).

[0199] To corroborate health benefits of specific products of interest, i.e. FM1 / FM2, 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).

[0200] 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 / 2 mixtures. Again, overall FM2 exerted stronger effects on Bacteroides species, most notable for 8. caccae, B. xylanisolvens, B. stercoris, B. uniformis, compared to FM1. Ruminococcaceae, Faecalibacterium prausnitzii and Gemmiger formicilis increased upon FM1 , and to a higher extent upon FM2 treatment. Strong stimulatory effects of the NDS on potent butyrate producers in the phylum Firmicutes were also observed.

[0201] Further, potent synbiotic effects were noted. First, Lactobacillus acidophilus (part of all 3 synbiotic combinations that were further analyzed) was strongly further promoted by FM2 but not by FM1 . Given that L. acidophilus is a potent lactate producer, this L. acidophilus increase likely boosted butyrate production for L. acidophilus-based synbiotics with FM2. For the combination of FM2 and L. acidophilus and 8. longum subsp. longum a remarkable increase of the lactate consuming, butyrate producing Anaerostipes hadrus was noted, explaining the previously observed remarkable increase of butyrate for this condition. While FM 1 / 2-based synbiotics significantly increased Bifidobacterium spp. related to the three probiotic strains (8. 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 8. bifidum was more pronounced compared to FM2, suggesting a stronger potential of FM1 to boost Bifidobacterium probiotics than FM2.

[0202] 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.

[0203] Host-microbiota interaction assay (Caco2 / THP-1 co-culture): 24 h.

[0204] 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.

[0205] 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 105Caco-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.cm2were selected for the main experiment.

[0206] 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 105cells / ml and split once density had reached 1 x 106cells / 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 105cells) 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.

[0207] 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).

[0208] 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.

[0209] The anti-inflammatory index (Al index) was calculated as follows:

[0210] 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 ; FM2; 2.2; 6.2; 10.2.

[0211] Results gut barrier:

[0212] 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 and FM2 fermentation improved the TEER significantly compared to blanc. Incubation with supernatants from FM1 / FM2 resulted in a higher TEER than from BM1 / 2. FM2 performed better than FM1 . The synbiotic combinations performed similar as the NDS alone.

[0213] 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. But compared to FM2 alone, the TEER remained higher, and hence the protection from barrier disruption was higher, for combinations of FM2 with L. acidophilus with a B. longum subp longum or with a B. breve (data not shown).

[0214] Immune modulation:

[0215] 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 basolaterally. 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.

[0216] Overall, all treatments exhibited positive Al index values, significantly different from the blanc, suggesting strong anti-inflammatory properties for all test products.

[0217] FM1 and FM2 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. But overall the Al Index was increased. Incubation with the supernatant obtained from fermentation with FM2 resulted in a higher Al index, than those of FM1 and BM1-2.

[0218] 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.

[0219] The combination of FM2 and L. acidophilus and B. longum subsp. longum showed a high Al index, in particular a stronger decrease in pro-inflammatory TNF-a, IL-8, CXCL-10, CCL2, when compared to FM2 alone or the specific LAPB combination alone. The Al Index was also higher compared to the other two combinations with LAPB was observed compared to the other synbiotic combination with FM2. Overall, the beneficial effect of FM1 / FM2 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 + B. breve, and in particular + 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-inflmaatory response.

[0220] Example 4: Mixture with beta-galactooligosaccharides, inulin, two different oat fibres comprising betaglucan and resistant starch and single, two or three LAPB strains. Effect on metabolites formed.

[0221] Part of the experiment of examples 1-2 was repeated, but this time with the faecal samples of 6 other young children. Again, two donors were of 1 year of age (+ / - 1 month), 2 donors of 2 years of age (+ / - 1 month) and 2 donors of 3 years of age (+ / - 1 month). No breastfeeding or antibiotics were used in the 90 days before sample collection.

[0222] As from the previous examples, the combination of the NDS mixture FM2 with B. longum subsp. longum and L. acidophilus was found to show the most desired results when fermented by the microbiota of young children, and this combination was explored further.

[0223] First, two variants of FM2 were tested. One contained the same oat fibre as in example 1-2. The other one contained the oat fibre as was present in FM4, namely PromOat. It was tested if the same effects were observed when using different sources of oat fibre containing beta-glucan. The result is shown in Table 4. It appeared that for both sources of oat fibre a specific stimulation of butyrate production, both in absolute and relative amounts, was observed when to the NDS FM2 mixture the LAPB mixture of L. acidophilus and B. longum subsp. longum was added.

[0224] Second, the effect of the LAPB combination L. acidophilus and B. longum subsp. longum was compared to the single strains. OatWell was used as oat fibre source. The result is shown in Table 4. It is shown that the L. acidophilus alone is more butyrogenic than the B. longum subsp. longum alone, but the combination of the two LAPB results in the highest absolute and relative amounts of butyrate.

[0225] Third, in addition to the strains of the above examples 1-3 alternative strains of the same species were tested. For B. breve as an alternative of the BbC50 strain B. breve M-16V (Morinaga) was used. As an alternative for L. acidophilus NCFM, the type strain L. acidophilus La1 CNCM I-2273 was used. The results are shown in Table 4. In all cases the butyrogenic effect was observed in the synbiotic combinations, in absolute and relative amount of butyrate formed, being higher for the combinations with B. longum subsp. longum than B. breve. Fourth, in the most favorable synbiotic combination of two LAPB the further addition of 8. breve was tested. As a B. breve the strain M-16V from Morinaga was used. The results are shown in Table 4. The combination of the three LAPB species with FM2 resulted in a higher absolute and relative amount of butyrate production, when compared to two strains.

[0226] Table 4: The impact of the test products on the key fermentation parameters, represented as % change from blanc, averaged across six toddlers 1-3 years of age: Comparison between Oat fibre source, and LAPB strains These results are indicative for an improved effect on the intestinal microbiota composition and activity of the synbiotic combination of the invention of a non-digestible saccharides mixture with betagalactooligosaccharides, inulin, cereal fibre comprising beta-glucan and resistant starch combined with Lactobacillus acidophilus and a Bifidobacterium selected from B. breve, B. longum subsp. longum, or 8. bifidum, preferably 8. longum subsp. longum. These results indicate an unexpected butyrogenic effect, which was species but not strain dependent. The effect on the metabolism of the microbiota resulted in improved effects on the gut barrier function by improving the barrier function and on the immune function having an ant-inflammatory effect. Example 5: Fermentation of the NDS mixture FM2 by single strain and combinations of strains: effects on acidification, metabolites formed and effect of the fermentation metabolites on the gut barrier function.

[0227] In vitro fermentation with the single strains and NDS mixture FM2 was performed in the absence of faecal microbiota. These experiments were performed in the DasGip fermentation system (pH controlled, anaerobic, 37 °C). Reichardt medium adjusted to bacterial growth was used (per L containing 10 g Tryptone, 1 g yeast extract, 1 g NH4SO4, 2 g K2HPO4, 3.2 g NaHCO3, 4.5 g NaCI, 0.5 g MgSO4.7H2O, 0.5 g L-Cystine hydrochloride, 0.5 g lactose, Menadione, trace elements, vitamins, haemin, pH adjusted to 6.5 with CaCI.H2O). A basal amount of lactose (0.05w / v%) was added to the fermentation medium to allow the propagation of one generation of bacterial cells, thus favouring their growth and the start of the fermentation of more complex carbohydrates. To ensure anaerobic conditions, a nitrogen line was connected to each reactor and the reactors were flushed with nitrogen overnight to ensure the medium was reduced prior to the inoculation of the bifidobacterial strains. For the pre-cultures, lactose at 0.5 w% final concentration was used as carbon source.

[0228] The strains were inoculated from glycerol stock cultures onto agar plates, TOS-mup for Bifidobacterium strains and MRS for Lactobacillus strains, to check for purity. A broth culture was inoculated for each strain from the colonies obtained on the plates and incubated at 37 °C under anaerobic conditions for 24 h. The precultures to inoculate the fermentation were performed in DasGip reactors with pH control at 6.5, each containing 200 mL of Reichardt medium with 0.5% lactose.

[0229] From these pre-cultures, the fermenters were inoculated at a final GD600 of 0.15. For the mixes of two strains, each strain was added at a final OD of 0.08, to add up to 0.15. For the mixes of three strains, each strain was added at a final OD of 0.05, to add up to 0.15. To calculate the amount of each strain’s pre-culture needed to be added to the fermenters, a sample was taken from the pre-culture and the OD measured. The inoculum volume for each strain was calculated as follows:

[0230] Inoculation volume (mL) = Final fermenter volume (mL) I (preculture OD / desired OD)

[0231] In experiment 1 the following strains and their combinations were tested. The same strains were used as in examples 1-2, unless mentioned otherwise.

[0232] L. acidophilus, B. bifidum, B. longum subsp. longum, B. breve M-16V,

[0233] L. acidophilus + B. bifidum, L. acidophilus + B. breve M-16V, L. acidophilus + B. longum subsp. longum. In experiment 2 the NCFM strain of L. acidophilus was replaced with the type strain ATCC 4356 and in addition the mixture L. acidophilus + B breve M-16V + B. longum subsp. longum was tested.

[0234] It was found that, based on NaOH consumption, all strains produce acids. The slowest initial acidification was observed with L. acidophilus alone, the type strain being slower than the L. acidophilus NCFM. The amount and rate of NaOH consumed when L. acidophilus was combined with a Bifidobacterium strain was higher than what was found for a single strain of Bifidobacterium or L. acidophilus. This is indicative for the mixture of LAPB being more effective that the single strains in the synbiotic combination.

[0235] Results of SCFA and lactate formed are shown in Table 5 and 6 and show a similar pattern. Interestingly the lactate / SCFA ratio was high with L. acidophilus or combinations with L. acidophilus, especially B. bifidum or B. longum subsp. longum. This is indicative for the potential to form butyrate and propionate when microbiota is added that contains lactate utilizing bacteria producing butyrate and / or propionate. The combination of L. acidophilus type strain, and two bifidobacteria, B. longum subsp. longum and B. breve M-16V resulted in a further higher acid production than the single strains or combination of L. acidophilus with the single Bifidobacterium strains, see Table 6.

[0236] Table 5: L-lactate, acetate and total acids formed in mmol / l and at t=6 and t=24 h upon fermentation of FM2 by LAPB. Table 6: L-lactate, acetate and total acids formed in mmol / l and at t=6 and t=24 h upon fermentation of

[0237] FM2 by LAPB.

[0238] Agar plating was performed, for experiment 1 , on an agar media specific for Lactobacilli and for Bifidobacteria to follow the growth in time in cfu’s. It was observed that at 24 h the amount of Lactobacilli was highest when 8. breve M-16V was present alone or in combination with 8. longum subsp. longum.

[0239] No cfu stimulatory effect on the amount of Lactobacilli was observed with 8. longum subsp. longum alone. The growth of the Bifidobacteria in cfu was very comparable at all the circumstances (data not shown).

[0240] This is indicative that the addition of B. breve to the combination of L. acidophilus, and B. longum subsp. longum results in a further improved effect of the synbiotic combination.

[0241] Supernatants were obtained by centrifugation samples at 14.000 rpm and storage at -80 °C. Subsequently, the supernatants obtained from the 24 h fermentation time points of NDS mixture FM2 as such or in combination with LAPB were tested for their effect on the gut barrier function in an in vitro model employing Caco2 cells. The organoplateRtechnique (Mimetas) is known in the art (Hagiwara et al. J Pharm Sci 2022; 111 (1):214-224). After a stable cell tubule was formed 4 days after seeing the cells in the tubule TEER was measured (Ohm / cm2)), and the ten times diluted unfiltered supernatants were added in the apical tube, containing the Caco-2 cells. After 16 h the TEER was measured again. Three repetitions with 5 technical replicates were performed. As a blanc the sample from the fermentor before inoculation with LAPB was taken, and also a medium (DMEM + glutaMAX without FCS 1 % sodium pyruvate, 1 % non-essential amino acids, 1 % pen / strep) control was taken.

[0242] The results are shown in Table 7. The TEER obtained with supernatants of the single strains are higher, but not significantly, compared to the blanc. The combination of LAPB however significantly increased the TEER compared to the blanc and compared to the supernatants of the single strains.

[0243] Table 7: Increase in TEER after 16 h incubation with supernatants from specific fermentations

[0244] Statistics: One way anova, post hoc LSD, compared to NCFM + BB536; ** p = <0.01 ; *** p <0.001

[0245] Example 6: Nutritional composition for young children

[0246] 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:

[0247] - 67 kcal

[0248] 1 .0 g protein (milk protein)

[0249] 2.79 g lipids (mainly vegetable lipids)

[0250] 8.66 g digestible carbohydrates (mainly lactose)

[0251] 1 .60 g mixture of non-digestible saccharides, comprising based on total non-digestible saccharides o 38.wt % galactooligosaccharides (source VivinalGOS) o 38 wt% inulin (source RaftilinHP) o 20 wt% oat fibre of which about 60% beta-glucan (source OatWell) o 4 wt% g resistant starch (source Novelose 330)

[0252] About 108cfu Lactobacillus acidophilus (NCFM)

[0253] About 108cfu Bifidobacterium longum subsp. longum BB536

[0254] About 108cfu Bifidobacterium breve M16-V

[0255] Vitamins, minerals as known in the art.

[0256] Example 7: Nutritional composition

[0257] Nutritional composition in powder form, when after reconstitution with water, 12.4 g powder, reconstituted with water to 100 ml ready to drink composition comprises per 100 ml:

[0258] 59 kcal

[0259] 1 .4 g protein (milk protein) / 100kcal

[0260] 3.1 g lipids (vegetable lipids) / 100 kcal

[0261] 5.4 g digestible carbohydrates (mainly lactose) / 100 kcal

[0262] 1 .76 g mixture of non-digestible saccharides / 100 kcal (2 g / 100 ml) o 0.67 g galactooligosaccharides (source VivinalGOS) o 0.67 g Ic FOS (source Raftiline ST) o 0.35 g oat fibre rich in beta-glucan (source PromOat) o 0.07 g resistant starch (source C*Actistar), Cargill)

[0263] About 108cfu Lactobacillus helveticus R0052 (Lallemand)

[0264] About 108cfu Bifidobacterium longum subsp \ongum (BAA-999)

[0265] Supplemented with vitamin A, B2, B12, D3, C, calcium, iron, zinc and iodine

[0266] Example 8: Nutritional composition

[0267] 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:

[0268] - 67 kcal

[0269] 2.7 g fat (mix of vegetable oils and fish oil)

[0270] 1 .3 g protein (casein and whey protein from cow’s milk)

[0271] 8.4 g digestible carbohydrates (mainly lactose)

[0272] 2.0 g non-digestible oligosaccharides per 100 ml: with beta-galactooligosaccharides I inulin I resistant starch I oat fibre high in beta-glucan in wt / wt ratio of about 38 / 38 / 4 / 20 wt / wt minerals, trace elements, vitamins and other micro-nutrients as known and in line with regulations.

[0273] About 107cfu Lactobacillus acidophilus NCFM,

[0274] About 108cfu Bifidobacterium longum subsp. longum BB536

[0275] About 108cfu B. bifidum R0071

[0276] 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 resistant starch Novelose®330 (Ingredion UK Ltd.) is used, and as a source of oat fibre comprising beta-glucan PromOat is used.

[0277] Example 9: Supplement to be added to milk, yoghurt, porridge and the like.

[0278] Sachet comprising per pack:

[0279] 4 g fibre: 1 g galacto-oligosaccharides (VivinalGOS powder), 1 g inulin (raftiline HP), 1 g resistant starch (Novelose330), 1 g Oat bran fibre (Fibercare, 21% beta-glucan based on fibre) and about 109cfu of each L. acidophilus La5 and B. longum subsp. longum BAA999. Maltodextrin to make total content 10 grams.

Claims

CLAIMS1 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, cereal fibre comprising beta-glucan and resistant starch, and- as lactic acid producing bacteria a mixture of Bifidobacterium longum subsp. longum and at least one Lactobacillus selected from the group consisting of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus paracasei and Lactobacillus plantarum.2 The combination according to claim 1 , wherein the mixture of lactic acid producing bacteria is a mixture of Bifidobacterium longum subsp. longum and Lactobacillus acidophilus.3 The combination according to claim 2, wherein the mixture of lactic acid producing bacteria further comprises B. breve.4 The combination according to any one of the preceding claims, wherein the cereal fibre comprising beta-glucan is cereal fibre comprising at least 30 wt% beta-glucan based on total cereal fibre.5 The combination according to any one of the preceding claims, wherein the cereal fibre comprising beta-glucan is provided as oat fibre, preferably oat fibre comprising at least 30 wt% beta-glucan based on total oat fibre.6 The combination according to any one of the preceding claims, wherein the mixture of non- digestible saccharides comprises beta-galactooligosaccharides : inulin : cereal fibre comprising beta-glucan : resistant starch in a weight ratio of 1 : 0.1-10 : 0.05-5 : 0.01-1 .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 3 to 45 g of the mixture of non- digestible saccharides per 100 g dry weight of the total composition and / or at least 103cfu per g dry weight B. longum subsp. longum and at least 103cfu per g dry weight Lactobacillus selected from the group consisting of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus paracasei and Lactobacillus plantarum.The nutritional composition according to claim 8 or 9 which is a young child formula. The nutritional composition according to any one of claims 8-10 which is for use in providing nutrition to a young child, wherein the 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. The combination, or supplement, or nutritional composition according to any one of claims 1-10, for use in preventing or treating intestinal disorders selected from the group consisting of microbial dysbiosis, constipation, intestinal infections, and diarrhea. The combination, or supplement, or nutritional composition according to any one of claims 1-10, for use in preventing or treating intestinal inflammation and / or increasing the intestinal barrier function in a subject. The combination, or supplement, or nutritional composition according to any one of claims 1-10 for use in improving intestinal microbiota composition and activity.

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