WO2022083858A1

WO2022083858A1 - Acacia gum for iron induced microbial dysbiosis

Info

Publication number: WO2022083858A1
Application number: PCT/EP2020/079652
Authority: WO
Inventors: Raphaelle Bourdet-Sicard; Muriel DERRIEN
Original assignee: Compagnie Gervais Danone
Priority date: 2020-10-21
Filing date: 2020-10-21
Publication date: 2022-04-28
Also published as: EP4231851A1

Abstract

The invention relates to non-therapeutic methods and nutritional compositions comprising acacia gum, preferable acacia gum. The invention further relates to methods of improving intestinal microbiota or preventing and treating intestinal microbial dysbiosis and associated conditions, caused by dietary iron.

Description

ACACIA GUM FOR IRON INDUCED MICROBIAL DYSBIOSIS

Field of the invention

The present invention relates to non-therapeutic methods, and uses of nutritional compositions comprising acacia gum for improving intestinal microbiota composition in subjects receiving iron supplementation or iron fortified products, in particular infants, young children and women

Background of the invention

Iron plays several vital roles in the body, as it is present in hemoglobin, cytochromes in the electron transport chain, and some enzymes. Iron deficiency is one of the most common nutritional deficiencies, in particular in infants, toddlers and pregnant women. This is especially a risk in infants that start weaning when the food consumed in the weaning phase is based solely on cereals, and legumes and milk, when heme-iron is not yet part of the diet and human milk consumption (providing lactoferrin-bound iron) is reducing. Iron deficiency can turn into anemia, the most common nutritional disorder in the world, wherein the body's stores of iron have been depleted and the body is unable to maintain levels of hemoglobin in the blood. Especially infants, young children and pregnant women are prone to this disease, as they have increased iron needs. The WHO has estimated that 43% of the world's infants and young children suffer from iron deficiency. Iron deficiency has serious consequences for the health and development of (unborn) infants and young children. A lack of sufficient supply or uptake of iron during the first year of life has for instance been shown to negatively impact neural development and that this negative impact can be irreversible. Iron deficiency anemia is part of the five first leading causes worldwide of years lived with disability (YLDs) in 2016, contributing 34 7 million of total YLDs, respectively (GBD 2016 Disease and Injury Incidence and Prevalence Collaborators, Lancet 2017; 390: 1211-59).

A way to overcome iron deficiency is to increase iron intake, for example by providing iron supplements or by providing nutritional compositions that are rich in iron. WO 03/013283 discloses beverages fortified with ferric EDTA to prevent or treat iron-deficiency anemia. Another approach is to improve the bioavailability of the iron. WO 14/148911 provides partly fermented formula with non-digestible oligosaccharides that improves iron absorption.

However, when solving the problem of iron deficiency by enriching the diet with iron, a disadvantageous side effect occurs in the intestinal microbiota: The beneficial bifidobacteria and lactobacilli are reduced in levels and pathogenic bacteria such as enterobacteria and Clostridia are increased. Iron-induced dysbiosis increases the risk for gastro-intestinal disorders such as diarrhea or intestinal infection, and intestinal inflammation. This is especially the case in Asia and Africa where there are typically much more pathogenic bacteria present in the gut microbiota than for example in Europe or the USA. Naturally, young children and infants are most at risk since their intestinal microbiota is not stable yet and more prone to be disturbed and they are also more exposed to pathogens due to lack of hygiene. Zimmerman et al, 2010, Am J Clin Nutr 92:1406-1415 disclosed the effect of iron fortification on the gut microbiota of African school-age children and found an increase in enterobacteria, an increase in intestinal inflammatory markers and a decrease in lactobacilli. Jaeggi et al, 2015, Gut 64:731-742 demonstrated that iron fortification in African young children resulted in an increase of enterobacteria, particularly Escherichia/Shigella, the enterobacteria/bifidobacteria ratio and Clostridium. Also markers of intestinal inflammation were increased. The iron-supplemented group required more treatment of diarrhea. In Pakistani young children, daily supplementation of micronutrient powder containing 12.5 mg of iron was associated with significant increased proportion of days with diarrhea and increased incidence of bloody diarrhea (Soofi et al. Lancet 2013).

Some have tried to develop means to prevent or treat iron-induced intestinal microbial dysbiosis. Paganini et al, 2017, Gut, 66(1 1 ): 1956- 1967 disclosed that supplementation with prebiotic galactooligosaccharides (GOS) mitigated the adverse effect of iron fortification on the gut microbiota of Kenyan infants, while the iron supplementation resulted in decreased anemia. The higher abundances of Bifidobacterium and Lactobacillus and lower abundance of Clostridiales, virulence and toxin genes of pathogens was restored by adding GOS.

Still there is a need for further improved compositions that promote, improve and/or maintain healthy microbiota in subjects undergoing iron supplementation, as well as that prevent and treat iron-induced microbial dysbiosis.

Summary of the Invention

The inventors tested a range of prebiotic fibers for their ability to prevent or reduce iron-induced microbial dysbiosis in both the microbiota of healthy and dysbiotic donors, employing an in vitro fermentation system. A healthy microbiota is highly enriched in lactobacilli and bifidobacteria, whereas a dysbiotic microbiota has low bifidobacteria levels, no detectable levels of lactobacilli and, more importantly, has moderate levels of Enterobacteriaceae.

It was surprisingly found that under conditions of high iron levels and an added pathogenic enterotoxigenic E. coll, acacia gum had the best effect, among all tested fibers, on stimulating growth of bifidobacteria and lactobacilli, while preventing or inhibiting growth of enteropathogenic bacteria. This effect was observed for donors with a well-balanced, healthy microbiota, as well as for donors with an already dysbiotic microbiota. The effect observed on preventing iron-induced dysbiosis was better than that observed with other prebiotic fibers, such as galacto-oligosaccharides, inulin and fructooligosaccharides.

Remarkably, in dysbiotic microbiota, acacia gum has shown to lead to the highest total short-chain fatty acids levels under high iron conditions, as well as highest acetate and butyrate levels, when compared to the other tested prebiotic fibers. Short chain fatty acids (SOFA) are the major metabolic products of fermentation by microbial communities that colonize the mammalian gut, being indicative of a healthy microbiota. Detailed Description of the Invention

In a first aspect, the present invention relates to a non-therapeutic method to promote, improve and/or maintain a healthy intestinal microbiota in a human subject receiving an iron-fortified diet, preferably an iron supplementation diet comprising iron supplements and/or iron-fortified nutrition, by administering a nutritional composition comprising acacia gum to the human subject. The invention may also be worded as (non-medical) use of acacia gum (in the manufacture of a nutritional composition) for promoting, improving and/or maintaining a healthy intestinal microbiota in a human subject receiving an iron-fortified diet, preferably an iron supplementation diet comprising iron supplements and/or iron-fortified nutrition, by administering a nutritional composition comprising acacia gum to the human subject. Preferably, the human subject does not suffer from iron deficiency, or iron deficiency associated disorders, such as anemia.

In a second aspect, the present invention relates to a nutritional composition comprising acacia gum for use in preventing ortreating iron-induced intestinal microbial dysbiosis in a human subject. The invention can also be worded as a method of preventing or treating iron-induced intestinal microbial dysbiosis in a human subject comprising administering a nutritional composition comprising acacia gum to said subject. Alternatively, the invention may also be worded as the use of acacia gum in the manufacture of a nutritional composition to prevent or treat iron-induced intestinal microbial dysbiosis in a human subject. Preferably, the human subject suffers from or is at (imminent or increased) risk of suffering from iron deficiency or iron deficiency associated disorders, such as anemia. The acacia gum is present in therapeutically effective amounts.

In a third aspect, the present invention relates to a nutritional composition comprising acacia gum for use in preventing ortreating iron-induced gastrointestinal disorder, preferably a gastrointestinal disorder selected from diarrhea, gastrointestinal inflammation, and/or gastrointestinal infections in a human subject. The invention can also be worded as a method of preventing or treating iron-induced gastrointestinal disorder, preferably a gastrointestinal disorder selected from diarrhea, gastrointestinal inflammation, and/or gastrointestinal infections in a human subject, comprising administering a nutritional composition comprising acacia gum to said subject. Alternatively, the invention may also be worded as the use of acacia gum in the manufacture of a nutritional composition to prevent or treat iron- induced gastrointestinal disorder, preferably a gastrointestinal disorder selected from diarrhea, gastrointestinal inflammation, and/or gastrointestinal infections in a human subject. Preferably, the human subject suffers from or is at risk of suffering from iron deficiency or iron deficiency associated disorders, such as anemia. The acacia gum is present in therapeutically effective amounts.

The present invention is based on the surprising finding that acacia gum, when administered to a human subject undergoing iron supplementation, may mitigate the adverse effect of iron supplementation on intestinal microbiota. Preferably, intestinal microbiota refers to colon microbiota. As used herein, a “healthy intestinal microbiota” (or balanced intestinal microbiota) relates to a microbiota composition with high levels of beneficial bacteria such as bifidobacteria and lactobacilli, whereas the growth of pathogenic intestinal bacteria, such as Clostridium ssp. and E. coli, is kept at controlled amounts. A healthy intestinal microbiota has been associated with maintenance of overall health in human subjects, e.g. sustaining gut motility and functioning, ensuring nutrients absorption, and improving immune system, reduced intestinal inflammation, in particular in infants and young children. Naturally, when intestinal microbiota balance is critically affected, i.e. the healthy microflora is reduced or absent, pathogenic bacteria will more easily colonize the gut increasing the risk of leading to gastrointestinal disorders, such as diarrhea, infections and inflammation. The present invention relates to both the non-therapeutic and the therapeutic aspects of improved intestinal microbiota.

The non-therapeutic method and nutritional compositions comprising acacia gum for use according to the invention preferably comprise (i) increasing bifidobacteria and/or lactobacilli; and/or (ii) controlling growth of enteropathogenic bacteria; (iii) increasing alpha-diversity; and/or (iv) increasing at least one of total short-chain fatty acids levels, acetate levels, propionate levels, butyrate levels; or combinations thereof. Preferably, bifidobacteria and/or lactobacilli are selected from at least one of B. breve, B. pseudocatenulatum, B. longum and Lactobacillus rhamnosus. Enteropathogenic bacteria, which growth is particularly controlled by the composition of the invention, are preferably selected from Escherichia coli, Enterobacter hormaechei/cloacae, Enterobacter, and Klebsiella pneumoniae and Clostridium XVIII, and mixtures thereof.

Alpha-diversity, as used herein, relates to the stimulation of growth of a wider range of bacterial species in one subject, which is suggested to be involved in limiting pathogenic bacteria (such as ETEC) growth. The nutritional compositions comprising acacia gum and methods using the same according to the invention have shown an effect in increasing alpha-diversity in tested samples of healthy and dysbiotic microbiota samples. Importantly, from all tested fibers, acacia gum has shown high stimulatory effects over beneficial microbiota, particularly B. breve, B. pseudocatenulatum, B. longum, Bocteroidesfragilis, B. thetaiotaomicron, B. cellulosiltycus, and B. vulgatus, whilst it did not stimulate or it inhibited growth of pathogenic bacteria (such as E. coli, Enterobacter, and Klebsiella pneumoniae). In one preferred embodiment, alpha-diversity is increased in infants or toddlers older than 6 months (e.g. 6-12 months, and 12-36 months), children, adolescents and adults. More preferably, alpha-diversity is increased in infants or toddlers older than 6 months, even more preferably, weaning infants or toddlers of 6-36 month of age (i.e., no longer being exclusively with breastmilk or infant milk formula).

Preferably, the nutritional composition comprising acacia gum for uses according to the invention (second and third aspects of the invention) is preferably administered to a human subject who concomitantly undergoes iron supplementation treatment and/or receives iron fortified nutrition. The human subject preferably suffers from or is at risk of suffering from iron deficiency or iron deficiency associated disorders, such as anemia. Iron deficiency (sideropaenia or hypoferraemia) is a stage preceding iron deficiency anaemia. The body has less than adequate iron levels. It can for example be determined by measuring an abnormal value for at least two of the three following indicators, serum ferritin, transferrin saturation, and free erythrocyte protoporphyrin, while still having a haemoglobin content above the threshold for anaemia. Iron deficiency anaemia is abnormal values of 2 out of 3 indicators with anaemia (a haemoglobin content below the threshold for anaemia). In the context of the present invention, 'prevention' of a disease or certain disorder also means 'reduction of the risk' of a disease or certain disorder and also means 'treatment of a human subject at risk' of said disease or said certain disorder.

Anaemia is a decrease in number of red blood cells or less than the normal quantity of hemoglobin in blood. In the present invention anaemia refers in particular to iron deficiency anaemia, i.e. anaemia caused by insufficient iron store . Iron-deficiency anaemia is caused by insufficient dietary intake and absorption of iron and causes approximately half of all anaemia cases in the world. According to the WHO anaemia is defined as a hemoglobin content of less than 6.83 mmol/l blood in infants or young children of 6 months to 5 years, of less than 7.13 mmol/l in children of 5 to 11 years of age, of less than 7.45 mmol/l in teens of 12 to 14 years of age, of less than 7.45 mmol/l in non-pregnant women with age above 15 years, of less than 6.83 mmol/1 in pregnant women, and of less than 8.07 mmol/l in men above 15 years of age. Symptoms are pallor, fatigue, lightheadedness and weakness. Other symptoms can be headaches, trouble sleeping, loss of appetite, paleness, reduced resistance to infection, fragile nails. Iron- deficiency anaemia for infants in their earlier stages of development has greater consequences than it does for adults. An infant made severely iron-deficient during its earlier life cannot recover to normal iron levels even with iron therapy. Iron-deficiency anaemia affects neurological development by decreasing learning ability, negatively altering motor functions and negatively effecting socioemotional functioning as behavior. Additionally, iron-deficiency anaemia has a negative effect on physical growth. In pregnant women, of which it is estimated that 50% suffers from iron deficiency or anaemia, there is an increased need for iron. Anaemia may increase the risk of preterm or small birth weight babies.

The methods and uses according to the invention is preferably directed to a child (human subject from 0 to 10 years), more preferably an infant or young child (6-36 month of age). In this group, iron deficiencies or anemia may have irreparable effects on the growth and development especially effects on brain. Likewise, the adverse effects of iron supplementation on intestinal microbiota may impact the immunity system and cognitive development.

In one embodiment, the methods and uses according to the invention is preferably directed to women in the reproductive age, in particular pregnant women. Women in reproductive age already are more at risk for iron deficiency. Iron deficiency in pregnant women may impact growth and development of the unborn child. In another embodiment, the methods and uses according to the invention is preferably directed to human of any age who is consuming iron supplements or iron fortified nutrition. Iron supplementation is increasingly used in healthy subjects opting for a vegetarian or vegan diet. Iron supplementation may negatively impact the intestinal microbiota composition leading to gastrointestinal discomfort, e.g. increased production of gas, abdominal bloating, etc.

Acacia gum

The present invention relates to methods/uses using compositions comprising acacia gum. Particularly, the methods/uses are preferably directed at subjects receiving iron supplementation or iron fortified nutrition. This gum was found to prevent iron induced microbial dysbiosis.

Acacia gum is a soluble and fermentable fibre. A fibre is defined here as a non-digestible carbohydrate. Non-digestible carbohydrates are carbohydrates that are resistant to digestion and absorption in the human small intestine and enter the colon intact. The term "soluble" as used herein, when having reference to a non-digestible carbohydrate, 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). The high solubility of acacia gum in water is due to the high degree of branching and the small hydrodynamic volume (Williams et al. Food Hydrocolloids 1990, 4:305-311). Moreover, the solubility is stable over a wide range of pH from 3 to 9 (D'Angelo LL. Gums and Stabilisers for the Food Industry, Royal Society of Chemistry, 2010). The term "fermentable" as used herein 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 Am. J. Clin. Nutr. 53, 1418-1424 (1991).

Acacia gums are complex, branched polysaccharides consisting of arabinose and galactose monosaccharides. Acacia gum is a branched complex polysaccharide whose main chain consists of 1 ,3-linked p-D-galactopyranosyl units. Acacia gum is also known as gum arabic, gum Sudani, Senegal gum and Indian gum. Acacia gum is commercially available and used in the food industry, for example as a stabilizer, emulsifier or thickener and has E number E414. Other suitable classifications to characterize acacia gum of the invention is CAS 9000-01-5, EC 232-519-5 and Codex Alimentarius INS number 414. The only two botanical species allowed for food applications are Acacia Senegal and Acacia seyal (cf. FAO specification for Acacia gum). Hence, the acacia gum of the invention is Acacia Senegal or Acacia seyal.

Acacia gum consists mainly of high molecular weight polysaccharides and their calcium, magnesium and potassium salts, which on hydrolysis yield arabinose, galactose, rhamnose and glucuronic acid. The basic structural units of acacia gum are galactose, rhamnose, arabinose, glucuronic acid and 4-0- methyl glucuronic acid (Al-Assaf et al. Gum Arabic, Royal Society of Chemistry, 2012; Kapoor et al. Carbohyd Res 1991 , 221 :289-293). Arabinogalactans from larch gum, larch wood sugar, galactoarabinan, L-arabino-D-galactan, and stractan (e.g. obtained from trees of the genus Larix) or form obtained by microbial origin is not considered acacia gum in the context of the invention. Gum Arabic/acacia and larch arabinogalactan are for example different in molecular complexity, werein the former is more complex and branched (having a higher amount of rhamnose and gluconic acid residues) and known to be metabolized by different intestinal bacteria. In particular, it is known that acacia gum can be metabolized by certain commensal bacteria due to their ability to remove rhamnose cap of acacia gums (Cartmell et al. Nat Microbiol. 2018 November ; 3(11): 1314-1326).

Acacia gum has been described for its bifidogenic effects (Cherbut, et al., "Acacia Gum is a Bifidogenic Dietary Fibre with High Digestive Tolerance in Healthy Humans," Microbial Ecology, in Health and Disease, 15(1):43-50 (2003). However, it is generally less effective as bifidogenic fiber than other prebiotics such as GOS and FOS. The present inventors observed that, as compared to other prebiotic fibers, this gum was found to be surprisingly superior in maintaining healthy microbiota or preventing iron induced microbial dysbiosis and gastrointestinal disorders under high iron conditions.

Preferably, the methods of the invention and nutritional composition for use according to the invention provide acacia gum at a daily dose of 1-40 g day, more preferably 1 to 20 g/day, even more at least 5 g per day, even more preferably 5 to 10 g/day. The methods of the invention and nutritional composition for use according to the invention preferably comprise acacia gum in an amount between 0.2 to 12 g per 100 ml, more preferably 0.4 to 8 g per 100 ml, even more preferably at least 1 g/100 ml, even more preferable, 1 to 2 g per 100 ml, when in ready to consume form. Preferably, the nutritional composition comprising acacia gum is selected from follow on formula, young child formula or a liquid cereal drink.

The nutritional composition comprising acacia gum and methods and uses of the same preferably comprise 1 .0 to 50 g acacia gum per 100 g, more preferably 2.5 to 35 g per 100 g, even more preferably at least 6 g per 100 g , even more preferably 6 to 15 g per 100 g dry weight. Preferably, the compositions comprise acacia gum in 0.5 to 20 g per 100 g of dairy wet product or cereal milk porridges (e.g. yoghurt, dairy drinks, cereal porridges, and the like), more preferably 0.5 to 10 g per 100 g, even more preferably at least 2.5 g/100 g, even more preferable, 2.5 to 5 g per 100 g, when in ready-to-consume form.

Nutritional compositions

The nutritional compositions according to the invention are selected from nutritional supplements or nutrition for infants, children, or adults (in particular, women and/or pregnant or lactating women). Preferably the nutritional composition according to the invention is selected from the group consisting of an infant formula, follow on formula, toddler milk or formula and growing up milk (also known as young child formula), more preferably from the group consisting of follow-on formula and young child formula, even more preferably a young child formula. An infant formula is defined as a starter formula, intended for infants of 0 to 4 to 6 months of age. A follow on formula, intended for infants of 4 to 6 months, preferably 6 months, until 12 months of age. Such infants are no longer solely dependent on the formula but start eating other food. Young child formulae are intended for children of 12 to 36 months of age. Follow on formulae comprise vitamins, minerals, trace elements and other micronutrients according to international directives. Young child formulae preferably comprise vitamins, minerals, trace elements and other micronutrients according to international directives for follow on formulae. Preferably, the nutritional compositions according to the invention are selected from: follow on formula for infants aged 6-12 months, young child formula for young children aged 12- 36 months, or a growing up milk for infants older than 36 months, preferably up to and including 6 years.

In one embodiment, the nutritional composition is not an infant formula.

The nutritional compositions according to the invention may comprise iron or may be supplemented with iron prior to consumption, e.g. wherein the composition is for use in a subject receiving iron supplementation diet. For instance, the nutritional compositions according to the invention include but are not limited to non-iron enriched compositions such as infant formula, follow-on formula, young child formula, liquid cereal drinks, instant baby cereal foods, baby food purees, yogurt, dairy drinks, cereal porridges and the like. Accordingly, in one embodiment, the nutritional compositions according to the invention do not comprise iron. Non limiting examples of iron supplementation are e.g. iron pills, and iron fortified nutrition may include e.g. food preparations (other than the composition according to the invention) fortified with iron. Preferably, the iron-fortified nutrition is selected from follow-on formulae, young child formulae, cereals, porridges, milk, dairy drinks, yoghurt, and the like.

In one embodiment, the nutritional composition does not comprise acacia gum. The compositions according to the invention may be supplemented with acacia gum prior to consumption. The nutritional composition may comprise iron and be supplemented with acacia gum, optionally prior to consumption. In one embodiment the nutritional compositions according to the invention are compositions not comprising iron and/or acacia gum, preferably selected from follow on formula, young child formula, liquid cereal, yogurt type nutrition, dairy beverages and drinks, and the like.

In one embodiment, the nutritional compositions according to the invention comprise both acacia gum and iron, particularly iron at effective amounts to meet daily recommended intake.

In a preferred embodiment, nutritional compositions according to the invention comprise at least acacia gum. More preferably, the compositions are supplemented with acacia gum and iron.

The nutritional compositions according to the invention may comprise additional fibres in the form of non-digestible oligosaccharides, such as galactooligosaccharides, fructooligosaccharides, uronic acid oligosaccharides, glucooligosaccharides, xylooligosaccharides, mannanoligosaccharides, arabinooligosaccharides, glucomannooligosaccharides, galactomannooligosaccharides, soy oligosaccharides, isomaltooligosaccharides, non-digestible dextrin, arabinogalactooligosaccharides, gentiooligosaccharides, nigerooligosaccharides, chitooligosaccharides, fucooligosaccharides, sialyloligosaccharides. If present, preferred additional prebiotics fibers are selected from galactooligosaccharides, fructooligosaccharides, or combinations thereof. In one preferred embodiment, acacia gum is the only fibre added to the composition.

In one embodiment the nutritional composition is in a liquid form. In another embodiment the nutritional composition is a powder suitable for making a liquid nutritional composition after reconstitution with an aqueous solution, preferably with water. Preferably the nutritional composition is a powder, suitable for reconstitution with water to a liquid. Preferably the infant or young child formula is a powder to be reconstituted with water.

Preferably, the iron fortified nutritional composition of the invention comprises 0.2 - 1.8 mg/100 ml iron, more preferably 0.4-1 .7 mg/100 ml iron. The iron fortified nutritional composition according to the invention is preferably selected from follow on formula for infants aged 6-12 months, young child formula for young children aged 12- 36 months, or a growing up milk for children older than 36 months, preferably up to and including 6 years of age. Preferably, the iron fortified nutritional composition of the invention comprises 1.5 - 15 mg iron/100 g dry weight, more preferably 3 to 12 mg/100 g. The iron fortified nutritional composition according to the invention is preferably selected from follow on formula for infants aged 6-12 months, young child formula for young children aged 12- 36 months, or a growing up milk for infants older than 36 months, preferably up to and including 6 years.

It is noted that wherever in the present description wording like "the present nutritional composition" or "nutritional composition according to the (present) invention" is used, this also refers to the methods and uses according to the present invention.

Iron and iron deficiency

In the context of this invention, iron equivalent refers to the equivalent amount of the ions Fe2+ or Fe3+. Wherever in this description an amount or concentration of iron is mentioned, this refers to the amount or concentration of Fe2+ or Fe3+, hence excluding the weight of the counter ion such as sulphate, lactate, gluconate, etc., of the iron source.

In one embodiment, the nutritional compositions according to the invention comprise iron and/or are fortified with iron. When present, the nutritional composition preferably comprises non-haem iron, more preferably one or more iron sources selected from the group consisting of ferrous sulphate, ferrous lactate, ferrous gluconate, ferrous bisglycinate, ferrous citrate, ferrous fumarate, ferric diphosphate (ferric pyrophosphate), and ferric ammonium citrate. Sources of ferrous iron are preferred as sources of ferric iron need to be converted to ferrous iron in the body, the capacity of which may be limited in human subjects with an age of 0 to 36 months, e.g. infants and young children. Preferably, the nutritional compositions comprise ferrous sulfate, ferrous fumarate or ferric pyrophosphate. Preferably, the nutritional composition comprising ferrous sulfate is selected from follow on formula or young child formula. In another preferred embodiment, the nutritional composition comprising ferrous fumarate is selected from baby foods, baby instant cereals, baby purees and the like, preferably baby instant cereals. In yet another preferred embodiment, the nutritional composition comprises ferric pyrophosphate is a dairy wet product or ready-to-drink beverage or liquid composition, such as growing up milks. Preferred dairy wet products include but are not limited to porridges, yoghurts, quark, dairy beverages and the like, preferably yoghurts.

The amount of iron added to the nutritional compositions according to the present invention depend on the recommended nutrient intake (RNI) for the target population. The recommended daily dosages provided herein correspond to 100% RNI.

Preferably, the nutritional composition according to the invention is an follow on formula or growing up milk. These compositions may be provided in dry or liquid form ready for consumption. When provided in dry form, typically 13.5g powder is diluted in 100 ml prior to consumption.

Accordingly, in one embodiment, the nutritional compositions of the invention are follow on formulae (e.g. adapted for the nutrition of infants 6-12months). The iron recommended intake for infants 6-12 months is 2.4-11 mg iron per day, preferably 2.4-7.6 mg iron per day. Infant formulae consumption for infants aged 6-12 months is around 600 ml/day. The follow-on formula according to the invention preferably comprises 0.4-1 .8 mg iron per 100 ml, more preferably 0.4-1 .6 mg per 100 ml. The present nutritional composition preferably comprises 3-14 mg iron per 100 g dry weight, more preferably 3 - 10 mg per 100 g dry weight, even more preferably not more than 6.5 mg iron per 100 g dry weight. A minimal amount is preferred in order to ensure sufficient iron uptake and prevent iron deficiency. However, too much iron can result in poor product quality by peroxidising polyunsaturated acids and can have adverse health effects like promoting intestinal inflammation and growth of intestinal pathogens.

According to the present invention, when iron is provided in a young child formula (e.g. nutritional compositions for infants 12-36 months old), the daily recommended dosage is 1.7-7 mg/day, preferably 1.95-6.75 mg/day. Daily consumption of young child formula or growing up milk by infants aged 12-36 months old ranges from 300 to 500 ml. The young child or growing up milk according to the invention, e.g. for infants of 1 to 3 years old, preferably comprises 0.4-1 .8 mg per 100 ml, even more preferably 0.5-1 .7 mg iron per 100 ml. The present nutritional composition preferably comprises 3 - 13 mg per 100 g dry weight, more preferably 4 - 12 mg iron per 100 g dry weight, even more preferably not more than 10 mg iron per 100 g dry weight.

In another embodiment, the nutritional compositions according to the invention are growing up milk, e.g. for infants older than 3 years. The compositions may be specially adapted to the recommended iron intake of children aged 4-10, preferably 4-6 years old. At this age, about 400 ml of the nutritional composition in ready to drink form is ideally consumed, per day. The daily recommended iron intake is 0.9-7 mg iron day, preferably 1 .95-6.75 mg iron per day. The nutritional composition according to the invention preferably comprise 0.2-1 .8 mg iron per 100 ml, more preferably 0.4-1 .7 mg iron per 100 ml. The present nutritional composition preferably comprises 1.5-15 mg iron per 100 g dry weight, more preferably 3 - 13 mg per 100 g dry weight.

In another embodiment, the nutritional compositions according to the invention are specially adapted to the recommended iron intake of children aged 7-10 years old. At this age, about 400 ml of the nutritional composition in ready to drink form is ideally consumed, per day. The daily recommended iron intake is 1 .2-6.2 mg iron day, preferably 2-6 mg iron per day. Particularly for this target population, the nutritional composition according to the invention preferably comprise 0.3-1 .6 mg iron per 100 ml, more preferably 0.5-1 .5 mg iron per 100 ml. The present nutritional composition preferably comprises 2-15 mg iron per 100 g dry weight, more preferably 3.5 - 12 mg per 100 g dry weight.

In another embodiment, the nutritional compositions according to the invention are specially adapted to the recommended iron intake of pre-adolescent subjects, e.g. aged 11-14 years old. At this age, about 300 ml of the nutritional composition in ready to drink form is ideally consumed, per day. The daily recommended iron intake varies for males and females, overall ranging from 1 .2-22.9 mg iron day, preferably 3-20 mg iron per day. Particularly for this target population, the nutritional composition according to the invention preferably comprise 0.4-7.7 mg iron per 100 ml, more preferably 1-7 mg iron per 100 ml. The present nutritional composition preferably comprises 3-60 mg iron per 100 g dry weight, more preferably 7 - 50 mg per 100 g dry weight.

In another embodiment, the nutritional compositions according to the invention are specially adapted to the recommended iron intake of young adults, or subjects in puberty, e.g. aged 15-17 years old. At this age, about 300 ml of the nutritional composition in ready to drink form is ideally consumed, per day. The daily recommended iron intake varies between males and females, overall ranging from 1 .6-22 mg iron day, preferably 2-20 mg iron per day. The nutritional composition according to the invention preferably comprise 0.5-7.3 mg iron per 100 ml, more preferably 0.6-7 mg iron per 100 ml. The present nutritional composition preferably comprises 3.5-55 mg iron per 100 g dry weight, more preferably 4 - 52 mg per 100 g dry weight.

In another embodiment, the nutritional compositions according to the invention are specially adapted to the recommended iron intake in adulthood (e.g. >18 years old). At this age, about 300 ml of the nutritional composition in ready to drink form is ideally consumed, per day. The daily recommended iron intake varies between males and females, overall ranging from 1.2-20.6 mg iron day, preferably 2-18 mg iron per day. The nutritional composition according to the invention preferably comprise 0.4-6.9 mg iron per 100 ml, more preferably 0.5-6 mg iron per 100 ml. The present nutritional composition preferably comprises 3-52 mg iron per 100 g dry weight, more preferably 3.5-4 mg per 100 g dry weight. Iron supplements are recommended to pregnant women whose blood tests indicate iron deficiency. Iron deficiency anemia may lead to fatigue and severe anemia can lead to complications in pregnancy, including weakening the mother’s immune system and increasing risk of infections. It also increases the risk of the baby weighing too little at birth (low birth weight) therefore indirectly affecting the baby’s health. In another embodiment, the nutritional compositions according to the invention are specially adapted to the recommended iron intake in pregnancy. About 300 ml of the nutritional composition in ready to drink form is ideally consumed, per day. The daily recommended iron intake is 4.0-20.6 mg iron day, preferably 4.5-18 mg iron per day. The nutritional composition according to the invention preferably comprise 1 .2-6.9 mg iron per 100 ml, more preferably 1 .5-6.9 mg iron per 100 ml. The present nutritional composition preferably comprises 8.5-52 mg iron per 100 g dry weight, more preferably 10-52 mg per 100 g dry weight.

EXAMPLES

Example 1: Selecting microbiota donors and in vitro fermentation conditions.

Stool samples of 10 babies between 6-12 months old were collected in anaerobic containers. These samples were aliquoted, flash frozen in liquid nitrogen and preserved at -80°C in presence of a cryoprotectant. DNA was extracted from the fecal inocula of the ten baby donors A to J and qPCR was performed to assess the abundances of three bacterial populations, i.e. bifidobacteria, lactobacilli and enterobacteriaceae. The methods were known from the literature: for lactobacilli, the qPCR method used is as described in Furet et al, FEMS MicrobiolEcol, 2009, 68: 351-62; for Bifidobacteria, the qPCR method used is as described in Rinttila et al, JAppIMicrobiol, 2004, 97: 1166-77; for enterobacteriaceae, the qPCR method used is as described in Nakato et al, JFoodProt, 2003, 66: 1798-804; and for E. coli, the qPCR method used is as described in Taniuchi et al, Diagn Microbiol Infect Dis, 2012, 73(2): 121 - 128.

The aim of the pre-screening was to select donors with high background levels of lactobacilli and bifidobacteria on one hand, and to identify donors with lower lactic acid bacteria levels on the other hand. Donors with high bifidobacteria and lactobacilli levels are more likely to allow for the observation of antipathogenic effects by prebiotic compounds. Donors with low bifidobacteria and lactobacilli levels are likely to be more vulnerable to the colonization of pathogens.

Bifidobacteria were detected in the fecal inocula of all donors. The highest levels were obtained for 2 donors F and J; lowest levels for 3 donors B, G and H. Donors B, D and J contained the highest lactobacilli levels. Lactobacilli were not detected in the fecal inocula of donors C, F, G and H. Enterobacteriaceae were detected in the fecal inocula of all donors, in abundances approximately 1 log lower than bifidobacteria. The highest levels were obtained for donors D and F; lowest levels were detected in donors H and I. High levels of Enterobacteriaceae are indicative of dysbiosis. Based on these considerations, the microbiota of 5 donors were selected for further experiments: Donors B, D and J were selected as healthy microbiota donors, since their gut microbiota was highly enriched with lactobacilli and bifidobacteria.

Donors G and H were selected as dysbiosis-like microbiota donors, given their low bifidobacteria levels and the fact that lactobacilli were not detected, while still having moderate enterobacteriaceae levels.

In the subsequent test the frozen fecal material of these five selected baby donors was used in the final experiments.

As a first step it was tested whether anaerobic conditions were required to promote Enterobacteriaceae growth. For this purpose, oxygen concentrations of 0%, 2.5%, 5%, 7.5% and 10 % by controlled introduction of oxygen, were tested on Enterobacteriaceae growth. 5 g/L galacto-oligosaccharides (GOS) was used as a carbon- and energy source. Iron (fumarate ferrous (C4H2FeO4)) was dosed to obtain a final concentration of 3 mg Fe/L (fumarate ferrous (C4H2FeO4)), corresponding with low iron levels, in the reactors. As a source of the colonic microbiota, a frozen fecal sample of donor A was added. Incubations were performed for 48 h at 37°C, under shaking (90 rpm) conditions. Medium composition was e.g. as described in Van den Abbeele P, Taminiau B, Pinheiro I, Duysburgh C, Jacobs H, Pijls L, et al. Arabinoxylo-oligosaccharides and inulin impact inter-individual variation on microbial metabolism and composition, which immunomodulates human cells. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY. 2018;66(5):1 121-30. The reactors were inoculated with 10% of a fecal suspension.

It was shown that growth of Enterobacteriaceae was not affected by the presence or absence of oxygen and the rate of acetate production was reduced with increasing oxygen concentration (data not shown), that could be explained by bifidobacteria activity reduction (main acetate producers in infant microbiota) known to be vulnerable to oxygen exposure. Therefore, strict anaerobic conditions, achieved by flushing with nitrogen gas, were applied in the subsequent experiments.

Example 2: Effect of iron supplementation on microbiota composition

Subsequently, the effect of two iron concentrations were tested on the growth of Enterobacteriaceae, Bifidobacteria and Lactobacilli and an added pathogenic E. coli strain in the samples of the 5 selected donors as described in Example 1 (Donors B, D, J, G and H). Tested were a high iron level (30 mg Fe/I - fumarate ferrous (C4H2FeO4)) inside the reactors and a low iron level (3 mg Fe/I - fumarate ferrous (C4H2FeO4)) inside the reactors. The amount of high iron as tested corresponds to 100 % RNI (recommended nutrient intake). No prebiotic fibers were added at this stage. As a representative for an enteropathogen, enterotoxigenic Escherichia coli (ETEC) strain LMG 2092 was employed. Three doses of ETEC were tested: 10⁵ CFU per reactor vessel, corresponding with a density of 1 .43*10³ CFU/ml in the reactor at Oh, 10⁷ CFU, corresponding with a density of 1.43*10⁵ CFU/ml in the reactor at Oh, and 10⁹ CFU, corresponding with a density of 1.43*10⁷ CFU/ml in the reactor at Oh. Each incubation was performed in single repetition, resulting in 45 independent incubations (9 per donor). Per donor three blanks were included, containing only the sugar-depleted nutritional medium with a low iron level and absence of ETEC. The abundances of bifidobacteria, lactobacilli, Enterobacteriaceae and ETEC were assessed by qPCR at the start, after 24h and 48h of incubation. Medium composition and qPCR methods are as described in Example 1.

The following observations were made with respect to Bifidobacteria, See also Table 1 : Growth of Bifidobacteria was in many cases stronger in the blank incubations, when compared with the incubations where ETEC was added, indicating a potential inhibitory effect of the ETEC pathogen on growth of the lactic acid bacteria. Bifidobacteria tended to be inhibited by high iron concentrations. Most growth occurred during the first 24 h, and the inhibitory effect of iron and/or ETEC was most observed at this timepoint, which is indicative for reduced growth rate. This effect was observed in most donors, independent whether they had a balanced or a disbalanced microbiota at the start. See Table 1 . Using 10⁷ cfu ETEC/70 ml, a further slight inhibition of bifidobacterial growth rate was observed (data not shown).

Table 1 : Absolute abundance of bifidobacteria (log (copies/ml)) on t=0, 24 and 48 in the absence (bl) or presence of ETEC (10⁵ cfu/70 ml), under high (H) or low (L) iron conditions. Faecal inocula were obtained from 5 selected donors.

Bl=blank, Fe=iron, H=high, L=low

Lactobacilli were not detectable in all donors, and for the donors in which the levels were not detectable at t=0, no outgrowth was observed after 24h. Lactobacilli were slightly inhibited in growth when ETEC was added. The amount of iron had less effect on the outgrowth. A similar effect was observed when 10⁷ cfu ETEC/70 ml was dosed (Data not shown).

Enterobacteriaceae levels were similar between blanks and corresponding treatments (i.e. low iron levels) after 24h for most donors. Only when 10⁹ CFU ETEC was dosed, higher Enterobacteriaceae levels were detected after 24h than the blanks.

The observations with respect to the added ETEC are shown in Table 2. ETEC levels were relatively unaffected by iron levels, which may favor ETEC over indigenous bifidobacteria and other bacterial groups. ETEC levels were higher in high iron conditions only for the donors with a dysbiosis in microbiota composition. Table 2: Absolute abundance of ETEC (log (copies/ml)) on t=0, 24 and 48 in the absence (bl) or presence of ETEC (10⁵ cfu/70 ml), under high (H) or low (L) iron conditions. Faecal inocula were obtained from 5 selected donors.

Nd: not determined, bl=blank, Fe=iron, H=high, L=low

Example 3: Testing the effect of different fibers on microbiota composition under high iron conditions.

The same procedure was used as in example 2, but with the following modifications:

Frozen fecal material of three selected baby donors was used. Donor B, J and H, to keep the number of samples workable. These donors were shown to respond to iron level changes and ETEC presence in example 1 .

Four different fibers were tested at a concentration of 5 g/L., namely Acacia gum, (Fibregum LI, Nexira), native inulin (Frutafit IQ, Sensus), GOS/lcFOS 9 :1 w/w ratio (source GOS VivinalGOS, Friesland Campina Domo, source IcFOS RaftilinHP Orafti), oligofructose (Frutalose L85 Sensus).

All reactors (including the blank) were inoculated with 10⁵ CFU ETEC/70 ml and contained high iron concentrations, corresponding with 30 mg Fe/I (fumarate ferrous (C4H2FeO4)). Each incubation was performed in triplicate to account for biological variation. This resulted in 45 independent incubations (15 per donor). An assessment was made of the abundance of Bifidobacteria, Lactobacilli, Enterobacteriaceae and ETEC by qPCR at t=0, t=24h and t=48h. An in-depth analysis of microbial community composition was performed by means of 16S-targeted Illumina sequencing (coupled with flow cytometry) of samples collected at the start, after 24h and 48h of incubation. 16S-based Illumina sequencing is a molecular technique, which is based on the amplification of the 16S rRNA gene. The methodology applied involves primers that span 2 hypervariable regions (V3-V4) of the 16S rDNA, i.e. 341 F (5'-CCTACGGGNGGCWGCAG-3') and 785R (5'-GACTACHVGGGTATCTAAKCC-3'). Using a pair-end sequencing approach, sequencing of 2x250bp resulted in 424 bp amplicons. Read assembly and cleanup was largely derived from the MiSeq SOP described by the Schloss lab. Briefly, mothur was used to assemble reads into contigs, perform alignment-based quality filtering (alignment to the mothur- reconstructed SILVA SEED alignment), remove chimeras, assign taxonomy using a naive Bayesian classifier and SILVA and cluster contigs into Operational Taxonomic Unit (OTUs) at 97% sequence similarity. All sequences that were classified as Eukaryota, Archaea, Chloroplasts and Mitochondria were removed. Also, if sequences could not be classified at all (even at (super)Kingdom level) they were removed. For each OTU representative sequences were picked as the most abundant sequence within that OTU. Reads with maximum abundances of only 5 in all samples were removed, as they were supposedly artefacts or bacteria that were not having any biological impact. For the most abundant OTUs, the obtained consensus sequences were classified manually through the RDP web interface using the RDP SeqMatch tool. The database search was restricted to isolates (uncultured organisms were not taken into account) with only near-full-length and good quality sequences.

Assessment of the total bacterial levels in the incubations by flow cytometry was done by staining the appropriate dilutions with SYTO 24. Samples were analyzed on a BDFacs verse. The samples were run using the high flow rate. Bacterial cells were separated from medium debris and signal noise by applying a threshold level of 200 on the SYTO channel.

The Mothur software package (v.1.39.5) and guidelines were used for data-processing. An Operational Taxonomic Unit (OTU) was defined as a collection of sequences with a length between 402 and 427 nucleotides that are found to be more than 97 % similar to one another in the V3-V4 region. Taxonomy was assigned using the RDP version 16 and silva. nr_v123 database. The resulting file, containing the number of reads observed for each OTU in each sample and a consensus sequence for each OTU, was loaded into Microsoft® Excel®. Reads occurring with a maximum abundance of 5 in all samples were removed, supposedly artefacts. Consensus sequences of the remaining OTUs were classified manually through the RDP web interface using the RDP SeqMatch tool, restricting the database search to type strains with only near-full-length, good quality sequences. Although identification to the species level based on short 300 bp reads may involve some ambiguity, the most likely species classification of some interesting OTUs is reported in the results sections.

Relative abundances of each OTU were calculated in the different samples. Conversion of relative abundances to absolute abundances was performed by multiplying the total cell count in a given sample (obtained by flow cytometry) with the relative abundance of any taxonomic entity within that sample. Total counts were used, considering that Illumina sequencing does not differentiate between living or dead bacteria. Two-tailed T-tests were performed to assess for any endpoint whether differences between averages in the blank incubations and averages in the treatment incubations were statistically significant at a level of significance a=0.05. This was done for each individual treatment on the different timepoints.

Further, microbial activity was measured at t=Oh, t-6h, t=24h and t-48h in by measuring pH, production of SCFA and lactate. pH, SCFA and lactic acid was measured as known in the art. Lactate concentrations were determined using a commercially available enzymatic assay kit (R-Biopharm, Darmstadt, Germany) according to manufacturer’s instructions. Short-chain fatty acids (SCFA) were measured as described by De Weirdt et al. (2010). The fermentation experiments with the donor microbiota and fibers disclosed that all the fibers were fermented. The pH decreased in all cases, when compared to the blank, indicating that sugar fermentation (producing acids) was higher than proteolytic fermentation (producing ammonium), see Table 3. Metabolites like short chain fatty acids (SCFA) (Table 4), which were for the large part acetic acid (Table 5), were formed. The drop of pH was highest in the first 24 h of fermentation. All fibers decreased pH vs blank. Acacia gum induced a mild pH decrease mainly after 24h. Native inulin and GOS:FOS were rapidly fermented, between 6-24h. Oligofructose induced the strongest pH decrease and was gradually fermented during 48h.

All fibers increased SCFA versus the blank. The highest amounts of total SCFA and acetate (a metabolite formed by amongst others Bacteroides, Bifidobacteria) were with inulin after 24h in all 3 donors. After 48 h acacia gum also showed high total SCFA and acetate level, in particular in Donor H, the “dysbiotic” donor. GOS:FOS (9:1) induced the lowest acetate after 48h under conditions with ETEC and high iron. The amounts of propionate and butyrate production were much lower and were fiber and donor-dependent. Acacia gum stimulated propionate production in all 3 donors, especially in Donor H (Table 6). Butyrate production was mainly observed at the second stage of the fermentation, where it is produced by the microbiota upon consumption of lactic acid and acetate. Only acacia gum was able to stimulate butyrate production in all three donors (Table 7). The other fibers only showed butyrate production in donor J. The production of butyrate is considered beneficial for the intestinal health and having anti-inflammatory properties. Branched short chain fatty acids (which are indicative for a proteolytic fermentation) were only observed in donors J and H, in a very low amount, and not dependent on the fiber. For all cases the amount of branched short chain fatty acids was reduced with fibers (data not shown).

Table 3: pH upon fermentation of fibers by the microbiota of 3 different infants

Bl=blanc, AG = acacia gum, l=inulin, Gf=GOS/lcFOS, Of=oligofructose.

Table 4: Total SCFA formation, in mM, upon fermentation of different fibers by the microbiota of 3 different infants. SCFA at =0 was set at 0.

Bl=blank, AG = acacia gum, l=inulin, Gf=GOS/lcFOS, Of=oligofructose Table 5: Acetic acid formation, in mM, upon fermentation of different fibers by the microbiota of 3 different infants. Acetic acid at t=0 was set at 0.

Bl=blank, AG = acacia gum, l=inulin, Gf=GOS/lcFOS, Of=oligofructose

Table 6: Propionic acid formation, in mM, upon fermentation of different fibers by the microbiota of 3 different infants. Propionic acid at t=0 was set at 0.

Bl=blank, AG = acacia gum, l=inulin, Gf=GOS/lcFOS, Of=oligofructose

Table 7: Butyric acid formation, in mM, upon fermentation of different fibers by the microbiota of 3 different infants. Butyric acid at t=0 was set at 0.

Bl=blank, AG = acacia gum, l=inulin, Gf=GOS/lcFOS, Of=oligofructose

With regard to the effect of these fibers on specific bacteria in the microbiota the following could be observed. All fibers stimulated the growth of Bifidobacteria (Table 8). The strongest effects, under these conditions with high iron and ETEC, was observed for acacia gum and inulin. Also a moderate stimulatory effects on lactobacilli were observed, except in donor H which had no level of detectable lactobacilli (data not shown).

Table 8: Absolute abundance of Bifidobacteria (log (copies/ml)) on t=0, 24 and 48 in the presence of ETEC (10⁵ cfu/70 ml), under high (H) iron conditions.

Bl=blank, AG = acacia gum, l=inulin, Gf=GOS/lcFOS, Of=oligofructose Table 9: Absolute abundance of ETEC (log (copies/ml)) on t=0, 24 and 48 in the presence of ETEC (10⁵ cfu/70 ml), under high (H) iron conditions.

Bl=blank, AG = acacia gum, l=inulin, Gf=GOS/lcFOS, Of=oligofructose

In this experiment with high iron and added ETEC inulin, GOS/lcFOS and FOS stimulated the growth of Enterobacteriaceae to some extent in all 3 donors. Levels observed with acacia gum were the lowest and closest to the blank (data not shown). In this experiment inulin, GOS/lcFOS and FOS, in particular inulin, stimulated the growth of ETEC. Acacia gum did not stimulate the growth of ETEC (Table 9).

The findings with the qPCR method were confirmed with the Illumina sequencing method combined with accurate enumeration of the cell count via flow cytometry, examining the shifts in the microbial community upon the fiber fermentation. Only acacia gum did not result in increased Enterobacteriaceae and ETEC. Acacia gum consistently stimulated Bifidobacteriaceae (phylum Actinobacteria), Eggerthellaceae (phylum Actinobacteria), Tannerellaceae (phylum Bacteroidetes), Lachnospiraceae (phylum Firmicutes) and Veillonellaceae (phylum Firmicutes). Native inulin consistently stimulated Enterobacteriaceae (phylum Proteobacteria) and Bifidobacteriaceae. GOS:FOS consistently stimulated Acidaminococcaceae (phylum Firmicutes), Lachnospiraceae and Enterobacteriaceae and oligofructose consistently stimulated Enterobacteriaceae only (data not shown).

Table 10: Average reciprocal Simpson diversity indices of the microbiota at t=0, 24 and 48 h during fermentation of different fibers in the presence of ETEC (10⁵ cfu/70 ml), under high (H) iron conditions.

Bl=blank, AG = acacia gum, l=inulin, Gf=GOS/lcFOS, Of=oligofructose

The effects on alpha diversity were studied by analyzing the reciprocal Simpson diversity index. Simpson diversity index is calculated by the square of the relative abundances of each OTU in a given sample, followed by the reciprocal sum of the obtained values for that sample. This value represents the reciprocal Simpson Div index. The data are shown in Table 10. Fermentation with inulin resulted in the lowest alpha-diversity (associated with enrichment in ETEC strain). Acacia gum on the other hand resulted in the highest alpha-diversity. This is indicative of a stimulation of growth of a wider range of bacterial species, which may be involved in limiting ETEC growth. The effect on beta-diversity was analysed by PCoA plots and a box polt of Bray Curtis dissimilarities. Consistent product-specific differences were observed amongst the 3 donors. Treatment with acacia gum came closer to the inocula, and inulin was the most distant form the initial inocula. GOS/FOS and oligofructose clustered well with the blank containing high iron and ETEC after 24h and 48h.

It can be concluded that of all the tested fibers the acacia gum fiber is the best performing fiber to prevent microbial dysbiosis induced by high iron levels, in either dysbiotic or healthy microbiota. Although other fibers presented beneficial effects overstimulation of Bifidobacteria growth (e.g. GOS/FOS), acacia gum has surprisingly shown an ability to also suppress growth of Enterobacteriaceae and ETEC under these conditions. Moreover, acacia gum was associated with a high amount of SCFA formation, including butyrate formation and acetate, propionate and butyrate formation particularly in dysbiotic samples, and the shift to the highest alpha diversity, whereas a beta-diversity clustering close to the inoculum.

Example 4: Young child formula

A young child formula is prepared with standard infant formula manufacturing techniques. An young child formula according to the invention comprises:

66 kcal/100 ml energy

2.6 g fat/100 ml (mix of vegetable oils fish oil)

1.3 g protein/100 ml (casein and whey protein form cow’s milk)

8.7 g digestible carbohydrates/ 100 ml )(mainly lactose)

1 .0 g fiber per 100 ml Acacia gum

1.2 mg iron per 100 ml

Other minerals, trace elements, vitamins as known in the art.

Claims

1 . Non-therapeutic method to promote, improve and/or maintain a healthy intestinal microbiota in a human subject receiving an iron-fortified diet, preferably an iron supplementation diet comprising iron supplements and/or iron fortified nutrition, by administering a nutritional composition comprising acacia gum to the human subject.

2. The non-therapeutic method according to claim 1 , wherein the human subject does not suffer from iron deficiency, or iron deficiency associated disorders.

3. The non-therapeutic method according to claim 1 or 2, wherein promoting, improving and/or maintaining a healthy intestinal microbiota is selected from (i) increasing bifidobacteria and/or lactobacilli; and/or (ii) controlling growth of enteropathogenic bacteria; (iii) increasing alpha-diversity; and/or (iv) increasing at least one of total short-chain fatty acids levels, acetate levels, propionate levels, butyrate levels; or combinations thereof.

4. A nutritional composition comprising acacia gum for use in preventing or treating iron-induced intestinal microbial dysbiosis in a human subject.

5. A nutritional composition comprising acacia gum for use in preventing or treating iron-induced gastrointestinal disorder, preferably a gastrointestinal disorder selected from diarrhea, gastrointestinal inflammation, and/or gastrointestinal infections in a human subject.

6. The nutritional composition comprising acacia gum for use according to claim 4 or 5, wherein the human subject suffers from or is at risk of suffering from iron deficiency or iron deficiency associated disorders, such as anemia.

7. The nutritional composition comprising acacia gum for use according to anyone of claims 4-6, wherein the human subject is concomitantly administered iron supplementation and/or iron-fortified nutrition.

8. The nutritional composition comprising gum acacia for use according to anyone of claims 4-7, wherein preventing or treating iron-induced intestinal microbial dysbiosis is selected from (i) increasing bifidobacteria and/or lactobacilli; and/or (ii) controlling growth of enteropathogenic bacteria; (iii) increasing alpha-diversity; and/or (iv) increasing at least one of total short-chain fatty acids levels, acetate levels, propionate levels, butyrate levels; or combinations thereof.

9. The non-therapeutic method according to claims 1-3, or the nutritional composition according to claims 4-8, wherein enteropathogenic bacteria growth is controlled, preferably E. coli, Enterobacter, and Klebsiella pneumoniae is controlled.

10. The non-therapeutic method according to claims 1-3 or 9, or the nutritional composition for use according to claims 4-9, wherein acacia gum is present in the composition in an amount between 2.5 to 35 g per 100 g.

11. The non-therapeutic method according to claims 1-3, 9 or 10, or the nutritional composition for use according to claims 4-10, wherein the human subject is a human subject at the age between 0 to 10 years, preferably an infant or young child 6-36 month of age, or a woman of reproductive age, preferably a pregnant woman.

12. The non-therapeutic method according to claims 1-3 or 9 - 11 , or the nutritional composition for use according to claims 4-11 , wherein the composition comprises 1.5 - 15 mg iron/100 g dry weight, more preferably 3 to 12 mg/100 g.

13. The non-therapeutic method according to claims 1-3 or 9-12, or the nutritional composition for use according to claims 4-12, wherein the nutritional composition comprises an iron source selected from the group consisting of ferrous sulphate, ferrous lactate, ferrous gluconate, ferrous bisglycinate, ferrous citrate, ferrous fumarate, ferric diphosphate, ferric pyrophosphate and ferric ammonium citrate.

14. The non-therapeutic method according to claims 1-3 or 9 - 13, or the nutritional composition for use according to claims 4-13, wherein the nutritional compositions comprise ferrous sulfate, ferrous fumarate or ferric pyrophosphate.

15. The non-therapeutic method according to claims 1-3 or 9 - 14, or the nutritional composition for use according to claims 4-14, wherein the nutritional composition is an follow on formulae, young child formulae or a supplement for pregnant women.