WO2025229094A1

WO2025229094A1 - Mixture of non-digestible saccharides stimulates gut barier

Info

Publication number: WO2025229094A1
Application number: PCT/EP2025/061899
Authority: WO
Inventors: Justyna Urszula POLKA; Ana María GIL RODRÍGUEZ; Harm Johannes WOPEREIS
Original assignee: Nutricia NV
Current assignee: Nutricia NV
Priority date: 2024-04-30
Filing date: 2025-04-30
Publication date: 2025-11-06
Anticipated expiration: 2026-10-30
Also published as: WO2025229092A1; WO2025229098A1; WO2025229102A1

Abstract

The invention concerns the specific mixture of non-digestible saccharides 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 improved gut barrier function.

Description

MIXTURE OF NON-DIGESTIBLE SACCHARIDES STIMULATES GUT BARIER

FIELD OF THE INVENTION

The present invention is in the field of nutritional compositions for young children comprising a mixture of dietary fibres that has a beneficial effect on gut barrier.

BACKGROUND OF THE INVENTION

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.

However, research on the effect of fibres 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.

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. Together the diet and microbiota shape the development of the gut barrier. While in young children the gastro-intestinal tract is still developing, establishing and stimulating gut barrier is extremely important.

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

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

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

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.

However, further improvements can still be made in the design of optimal mixes of fibres for young children in order to enable a smooth transition for infant type to adult type gut barrier, 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 to be applied in nutritional composition for young children.

SUMMARY OF THE INVENTION

Based on preclinical data obtained with a faecal slurry fermentation model using faecal material from young children the inventors found after extensive testing that a specific NDS mixture comprising galactooligosaccharides, inulin, oat fibre comprising beta-glucan and resistant starch significantly increased trans-epithelial electrical resistance. To an extent it was observed this increase was beyond that of a specific NDS mixture developed for infant formula comprising GOS and IcFOS with or without HMOS and the NDS mixture was found to beneficially promote propionate- as well as butyrate-producing bacteria thereby promoting the transformation from a microbiota that is infant-like towards a microbiota that is more adult-like such as that of a young child.

It was found that the metabolites formed upon fermentation by the mixture of the NDS specifically beneficially improved the intestinal barrier function.

Therefore, the specific NDS mixture of galactooligosaccharides, inulin, oat fibre comprising beta-glucan and resistant starch 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 improved gut barrier function. DETAILED DESCRIPTION OF THE INVENTION

The invention thus concerns a mixture of non-digestible saccharides comprising betagalactooligosaccharides, inulin, cereal fibre comprising beta-glucan and resistant starch.

Mixture of non-digestible saccharides

The present invention concerns a mixture of specific non-digestible saccharides (NDS) and supplements and compositions comprising such a mixture. 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 gastrointestinal 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, preferably 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 significantly increased trans-epithelial electrical resistance of the epithelial cell layer compared to the control and outperformed prior art mixtures specially adapted for infants containing short chain galactooligosaccharides and long-chain fructooligosaccharides in a 9:1 ratio and 2’-fucosyllactose and beneficially promoted propionate- as well as butyrate-producing bacteria thereby promoting the transformation from a microbiota that is infant-like towards a microbiota that is more adult-like such as that of a young child and thereby improving gut barrier function.

Beta-galactooligosaccharides

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).

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 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, results in an intestinal microbiota intermediate between infant-type and adult-type and thereby improves gut barrier function.

Inulin

Inulin as used in the present invention refers to carbohydrates composed of more than 50%, preferably more than over 65% fructose units based on total monomeric units of the inulin, in which at least 50%, more preferably at least 75%, even more preferably at least 90%, of the fructose units are linked together via a beta-glycosidic linkage, preferably a beta-2, 1-glycosidic linkage. A glucose unit may be present at the reducing end of the chain of fructose units. Inulin can be analyzed according to AOAC method 997.08. Inulin is a water soluble and fermentable non-digestible polysaccharide. Preferably an inulin is used that has an average DP of at least 10. Preferably an inulin is used that has an average DP of 10 - 60. Suitable sources of inulin are Raftiline GR (Beneo, Orafti), Raftiline HP (Beneo, Orafti) and Fibruline (Cosucra).

A sufficient amount of inulin is advantageous for children, in particular young children, 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 according to the invention, results in an intestinal microbiota intermediate between infant-type and adult-type, which is beneficial for young children and thereby improves gut barrier function. Cereal fibre comprising beta-glucan

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.

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.

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 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 betaglucan replaced by soluble soy fibre, resulted in a microbiota less intermediate between infant-type and adult-type.

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.

Resistant starch

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.

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.

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.

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 according to the invention, results in an intestinal microbiota which is intermediate between infants and adult subjects and thereby improves gut barrier function. A too high amount of resistant starch may result in unfavourable product characteristics such as a high viscosity and precipitations.

NDS mixtures

Preferably the mixture of NDS or the nutritional composition comprising the mixture of NDS does not comprise other sources that provide non-digestible saccharides than the mixture of four NDS according to the invention. Preferably the mixture of NDS 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 mixure of NDS 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 mixture of NDS or the nutritional composition comprising the mixture of NDS 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 and thereby on the gut barrier.

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.

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 and thereby improve gut barrier development and function..

Nutritional composition

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

In the context of the present invention, the nutritional composition according to the invention can also be named a 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.

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.

Preferably the mixture of NDS 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.

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

Furthermore, the nutritional composition according to the invention preferably comprises 0.13 - 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. 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.

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.

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

The amount of saturated fatty acids is preferably below 45 wt% based on total 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). These LC-PUFA were found to improve the intestinal barrier function and including DAH and/or EPA, preferably both, preferably works synergistically with the mixture of NDS according to the invention. Preferably the amount of LC-PUFA is in the range of 0.3-5 wt%, preferably 0.4-3 wt%, more preferably 0.5-2.5 wt% based on total fatty acids. Preferably the nutritional composition comprises DHA and EPA in an amount of 0.3-5 wt%, preferably 0.4-3 wt%, more preferably 0.5-2.5 wt% based on total fatty acids. Preferably the nutritional composition comprises DHA and EPA in a weight ratio of 1 : (0.1-1), preferably in a weight ratio of 1 : (0.1-0.7).

The nutritional composition preferably comprises 0.8 - 2.7 g protein per 100 ml, more preferably 0.9 - 2.1 g per 100 ml, more preferably 1 .1 - 1 .6 g per 100 ml nutritional composition. Based on dry weight, the nutritional composition preferably comprises 6 - 20 g protein per 100 g, more preferably 7 - 16 g per 100 g, more preferably 8 - 12 g protein per 100 g dry weight of the nutritional composition. Based on calories, the nutritional composition preferably comprises 1.2 - 4.0 g protein per 100 kcal, more preferably 1.4 - 3.2 g per 100 kcal, more preferably 1.6 - 2.4 g protein per 100 kcal of the nutritional composition. The protein preferably provides 5 - 15%, more preferably 6 - 13% even more preferably 7 - 10% based on total calories of the composition.

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

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

The nutritional composition preferably comprises 4.7 - 11 .2 g digestible 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.

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

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

Nutritional supplement

In one embodiment of the present invention the mixture of NDS 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.

Application

The mixture of NDS of the present invention, or supplement or nutritional composition comprising this mixture, 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 mixture of NDS of the present invention, or supplement or nutritional composition comprising the mixture of NDS 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.

In particular, the mixture of NDS of the present invention, or supplement or nutritional composition comprising the mixture of NDS 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 mixture of NDS of the present invention, or supplement or nutritional composition comprising the mixture of NDS 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.

In particular, the mixture of NDS of the present invention, or supplement or nutritional composition comprising the NDS 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. It was found that the mixture of NDS of the present invention when fermented by microbiota of young children significantly increased trans-epithelial electrical resistance (TEER) of the epithelial cell layer compared to the TEER in the absence of NDS. The mixture of NDS of the present invention showed a better TEER compared to prior art mixtures specially adapted for infants containing scGOS/lcFOS and 2’-FL. Furthermore, the mixture of NDS of the present invention exhibited a strong bifidogenic effect, increasing or tending to increase seven Bifidobacterium species. The present NDS mixture also especially stimulated potent propionate-producing species, yet also stimulated a series of potent butyrate producers. This facilitates the transformation from a microbiota that is infant-like towards a microbiota that is more adult-like.

Therefore the present mixture of NDS 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. At the same time the present mixture of NDS aids in developing advantageous gut barrier integrity.

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

Butyrate beneficially modulates visceral sensitivity and intestinal motility, which can help in conditions like constipation. Therefore this mixture of NDS of the invention will treat or prevent constipation.

It was found that that upon fermentation of the mixture of NDS of the invention the pro-inflammatory cytokines were reduced. Therefore the mixture of NDS 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 mixture of NDS of the present invention is for use in treating or preventing allergy and/or atopic dermatitis. Propionate helps regulate immune cells, which can reduce intestinal inflammation and maintain the gut barrier. This is crucial for a toddler's developing digestive system.

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

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

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

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

Preferably the use is in 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. 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 mixture of NDS of the present invention, or supplement or nutritional composition comprising this mixture of NDS to the young child.

Alternatively, the invention can be worded as the use of non-digestible saccharides for the manufacture of a 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.

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

The invention can be worded as the use of non-digestible saccharides for the manufacture of a composition for improving intestinal microbiota composition and activity in a young child wherein the non-digestible saccharides is a mixture of beta-galactooligosaccharides, inulin, cereal fibre comprising beta-glucan and resistant starch.

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

EXAMPLES

Material and methods

Two mixtures of non-digestible saccharides, two reference controls, and one blanc (NSC - No Substrate Control) were tested.

NDS mixture FM2 was 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 FM2 is a mixture according to the present invention. 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.

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

⁰ Source of inulin is Orafti® GR (Beneo-Orafti). ^d 2’-Fucosyllactose (2’-FL) and 5 HMOS are obtained from Chr Hansen. 5 HMOS contained 2’-FL, 3-FL, 6’-SL, 3’-SL and LNT in the weight ratio: 52 : 13 : 5 : 4 : 26. ^e Source 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. ^f Source of resistant starch is Novelose®330 (Ingredion UK Ltd.).

Faecal slurry fermentations were performed with samples from six healthy toddlers; two were 1 year of age (12 and 13 months), two were 2 years of age (24 months) and two were 3 years of age (36 months). 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.

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 to be tested, then sealed individually, before being rendered anaerobic. After preparation, bioreactors were incubated under continuous agitation (140 rpm) at 37°C for 24 h (MaxQ 6,000, Thermo Scientific, Thermo Fisher Scientific, Merelbeke, Belgium). Samples were taken at t= 24 h and stored at -80°C.

Analysis of SCFA, BCFA, pH, lactate:

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

Microbial composition 16S rRNA gene profiling:

Upon DNA extraction, library preparation and sequencing were performed on an Illumina MiSeq platform with v3 chemistry. The 16S rRNA gene V3-V4 hypervariable regions were amplified using primers 341 F (50 -CCT ACG GGN GGC WGC AG-30) and 785Rmod (50 -GAC TAC HVG GGT ATC TAA KCC-30 ). Results were 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.

Data analysis:

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

Additional analyses were performed for 0 h (only blanc) and 24 h FM1 and FM2 were tested, along with two 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.

Microbial composition (quantitative shallow shotgun sequencing):

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

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

The LC-MS analysis was carried out using a Thermo Scientific Vanquish LC coupled to Thermo Q Exactive HF MS. An electrospray ionization interface was used as ionization source. Analysis was performed in negative and positive ionization mode. The UPLC was performed using a slightly modified version of the protocol described by Doneanu, C.E. UPLC/MS Monitoring of Water-soluble vitamin Bs in Cell culture mmedia in Mimutes. 7 (2011). Peak areas were extracted using Compound Discoverer 3.1 (Thermo Scientific). In addition to the automatic compound extraction by Compound Discoverer 3.1 , a manual extraction of compounds included in an in-house library was performed using Skyline 21.1 (MacCoss Lab Software). Identification of compounds were performed at three levels; Level 1 : identification by retention times (compared against validated standards), accurate mass (with an accepted deviation of 3ppm), and MS/MS spectra, Level 2a: identification by retention times (compared against validated standards), accurate mass (with an accepted deviation of 3 ppm). Level 2b: identification by accurate mass (with an accepted deviation of 3 ppm), and MS/MS spectra. Level 3: identification by accurate mass alone (with an accepted deviation of 3 ppm).

Results are shown in examples example 1 and 2.

Example 1: Effect of different non-di estible saccharides mixtures on the metabolites formed by the intestinal microbiota of young children

Both FM1 and FM2 provided high SCFA production and FM1 resulted in lower pH (higher pH reduction) compared to FM2. Overall, NDS administration markedly boosted acetate, propionate and butyrate and reduced bCFA in a NDS-specific fashion. The reference products BM1 and BM2 also strongly increased SCFA production. Overall BM1 and BM2 resulted in the highest acetate levels and butyrate levels across all test conditions, being reminiscent to FM1. With FM2, the pH, amounts of propionate and butyrate transitioned from amounts characteristic for infant-like to amounts characteristic for young children and towards those that as characteristic for adult-like.

Table 2: Average butyrate and propionate production

Subsequently, the NDS mixtures FM1/FM2 were subjected to additional in-depth analysis. The test products were compared with 2 reference products (BM1/BM2) and a no substrate control (blanc). The in-depth metabolomic analysis by LC-MS revealed marked effects on metabolites well beyond the traditionally studied SCFA.

The NDS FM1/FM2 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). These results are indicative that the mixture of beta-galactooligosaccharides, inulin, cereal fibre comprising beta-glucan and resistant starch 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.

Example 2: Faecal fermentation of the NDS mixture of the invention results in a microbiota intermediate between infant and adult.

As can be expected, the mixtures containing NDS resulted in higher intestinal bacteria growth. Total bacterial cells were lower with FM1 and BM1/2 compared to fermentation with FM2.

The three key phyla across toddlers were Actinobacteria/Bacteroidota/Firmicutes with lower levels of Fusobacteria/ProteobacteriaA/errucomicrobiota being detected. No significant treatment effects were noted for the less abundant phyla so the focus is on effects on Actinobacteria, Bacteroidota and Firmicutes.

The NDS mixtures significantly increased the phylum to which Bifidobacteriaceae belong, i.e., Actinobacteriota. FM1 , and most markedly FM2, significantly increased Bacteroidota, whole BM1 and BM2 did not. FM2 also significantly increased Firmicutes.

The effect of FM2 on the microbiota that was observed is illustrative for promoting the transformation from a microbiota of an infant towards a microbiota that of an adult.

FM1 and FM2 strongly increased the most prevalent Bifidobacterium species in the toddler’s microbiota, i.e., B. pseudocatenulatum.

To corroborate health benefits of FM1 and FM2 the products after fermentation were subjected to additional in-depth analysis of microbial composition (quantitative shallow shotgun sequencing). The test products were compared with 2 reference products (BM1/BM2) and a no substrate control (blanc).

In-depth analysis of microbial composition using shallow shotgun sequencing was carried out. Strong effects were observed for NDS-containing products. Shotgun sequencing provided high species level resolution and enabled observation of effects on B. adolescentis and B. catenulatum i.e. these Bifidobacterium species were stimulated by the FM1 and FM2 mixtures. Overall FM2 exerted stronger effects on Bacteroides species, most notable for B. 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.

The impact of FM2 on the gut microbiota of toddlers was assessed. Using the ex vivo SIFR® technology, the impact on microbial composition (quantitative shallow shotgun sequencing), metabolite production (untargeted metabolomics) and barrier integrity was assessed (co-culture model of epithelial and immune cells). The various test products were compared with reference products BM1 and BM2 and a no substrate control (NSC). In-depth analysis of microbial composition using shallow shotgun sequencing on revealed marked effects of the test products on a wide range of species.

FM2 exhibited strong bifidogenic effects, increasing or tending to increase 7 Bifidobacterium species. Also FM2 stimulated potent propionate-producing Bacteroides spp., Phocaeicola spp., Alistipes spp., Veillonella spp. species as well as succinate-producing Parabacteroides spp. This would explain a higher propionate production observed for FM2.

Also FM2 stimulated a series of potent butyrate producers, i.e., Anaerobutyricum hallii, Blautia spp., Anaerostipes hadrus, Faecalibacterium prausnitzii, Gemmiger formicilis. Cross-feeding between A. hallii/F. prausnitzii and Bifidobacterium spp. has been shown to boost butyrate levels.

FM2 also significantly increased the levels of health related metabolites such as N-acetylated amino acids and 3-phenyllactic acid, which are related to healthy gut barrier function and indole-3-propionic acid, which is related to gut barrier integrity.

Overall, the beneficial effect of FM2 originated from its selective utilization by specific host microorganisms and resulting in the production of a spectrum of health-related metabolites,

Example 3: Effects on gut barrier function of the fermentation supernatants obtained after fermentation of mixtures of NDS by the microbiota of young children.

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

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

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

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

At the start of the main experiment, culture media in the apical chamber were replaced with samples derived from the SIFR® incubations, diluted in cell medium. Upon measuring TEER, plates were incubated for 24 h after which the TEER was again measured and 500 ng/ml of LPS was added to the basal chamber of the transwells containing the THP-1 cells. Upon a 6 h LPS challenge TEER was measured

Results gut barrier:

After 24 h incubation it was found that 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.

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, see table 3.

Table 3: Trans-epithelial electrical resistance (TEER)

Overall, the beneficial effect of FM2 originated from 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. The FM2-containing product 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.

Example 4: Nutritional composition for young children

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

- 67 kcal

1 .0 g protein (milk protein)

2.79 g lipids (mainly vegetable lipids)

8.66 g digestible carbohydrates (mainly lactose)

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) Vitamins, minerals as known in the art. Example 5: Nutritional composition

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:

59 kcal

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

3.1 g lipids (vegetable lipids)/100 kcal

5.4 g digestible carbohydrates (mainly lactose)/100 kcal

1 .76 g mixture of non-digestible saccharides/100 kcal (2 g/100 ml) o 0.67 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)

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

Example 6: Nutritional composition

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

- 67 kcal

2.7 g fat comprising, based on fatty acids, 0.3 wt% EPA wt% and 0.6 wt% DHA (mix of vegetable oils and fish oil)

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

8.4 g digestible carbohydrates (mainly lactose)

2.0 g non-digestible oligosaccharides per 100 ml: with beta-galactooligosaccharides I inulin I 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.

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 Grey) 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.

Example 7:

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

Sachet comprising per pack:

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). Maltodextrin to make total content 10 grams.

Claims

1 A mixture of non-digestible saccharides comprising beta-galactoohgosacchandes, inulin, cereal fibre comprising beta-glucan and resistant starch.

2 The mixture according to claim 1 , wherein the cereal fibre comprising beta-glucan is cereal fibre comprising at least 30 wt% beta-glucan based on total cereal fibre.

3 The mixture according to claim 1 or 2, wherein the cereal fibre comprising beta-glucan is provided as oat fibre.

4 The mixture according to any one of the preceding claims, wherein the cereal fibre comprising betaglucan is oat fibre comprising at least 30 wt% beta-glucan based on total oat fibre.

5 The mixture according to any one of the preceding claims, wherein the inulin has an average degree of polymerization above 15, preferably above 20.

6 The mixture according to any one of the preceding claims, comprising betagalactooligosaccharides : inulin : cereal fibre comprising beta-glucan : resistant starch in a weight ratio of 1 : 0.1-10 : 0.05-5 : 0.01-1.

7 The mixture according to any one of the preceding claims, comprising, based on weight, 20 - 60 % beta-galactooligosaccharides, 20 - 60 % inulin, 10-30 % cereal fibre comprising beta-glucan, and 2 - 6 % resistant starch.

8 A supplement comprising the mixture of non-digestible saccharides according to any one of claims 1-7.

9 A nutritional composition comprising digestible carbohydrates, lipids and proteins and the mixture of non-digestible saccharides according to any one of claims 1-7.

10 The nutritional composition according to claim 9 comprising 3 to 45 g of the mixture of non- digestible saccharides per 100 g dry weight of the total composition.

11 The nutritional composition according to claim 9 or 10, which is a young child formula.

12 The mixture, or supplement or nutritional composition according to any one of claims 1 to 11 , for use in a method of therapy, wherein the method of therapy is preventing or treating intestinal disorders selected from the group consisting of microbial dysbiosis, constipation, intestinal infections, intestinal inflammation, and diarrhea. The mixture, or supplement of nutritional composition for use according to claim 12, wherein the method of therapy is preventing or treating intestinal inflammation and/or increasing the intestinal barrier function in a subject.