Mixture of inulin oligosaccharides having different degree of polymerisation for improving iron bioavailability

Abstract

The present invention pertains to a combination of 2 types of inulins and nutritional compositions comprising that combination of inulins for improving the bioavailability of iron and preventing or treating iron deficiency, in particular for infants, young children, and subjects in need thereof.

Description

[0001] Mixture of non-digestible oligosaccharides for improving iron bioavailability

[0002] FIELD OF THE INVENTION

[0003] The invention is in the field of nutritional intervention for treating or preventing iron deficiency or anemia.

[0004] BACKGROUND OF THE INVENTION

[0005] Iron is an essential mineral in body functioning, particularly important for red blood cell functioning. Nearly 70 percent of iron in human body is located within the hemoglobin protein. Iron deficiency is a common nutritional deficiency and poses a significant global health issue, particularly affecting infants, children, elderly and pregnant women. Iron deficiency can lead to anemia, a condition wherein the body's stores or iron are insufficient, characterized by reduced hemoglobin levels, which in turn impairs oxygen transport in the body. This condition can cause fatigue, weakened immune function, and developmental delays in infants and children. Anemia affects about 40% of children worldwide and poses particularly in developing countries a health concern. Cognitive and motor skill development can be particularly affected by anemia, which is irreversible and leading to long-term educational and social challenges. Addressing iron deficiency is crucial for ensuring healthy growth, optimal brain development, and overall well-being, making it a critical focus for public health interventions and nutritional strategies.

[0006] Iron fortification is a way to treat iron deficiency anemia but is hampered by several drawbacks 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. 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. Therefore, interventions that are able to improve absorption of dietary iron and thereby improve iron bioavailability without having to increase the quantity of dietary iron are preferred. Iron is taken up from the diet in the gastro -intestinal tract, in particular via absorption by the enterocytes of the duodenal lining and the proximal jejunum and to a minor extent in the colon where it is mainly used by the intestinal microbiota. Iron can be absorbed, or in its ferrous Fe2+form. The low pH of gastric acid in the proximal duodenum allows a ferric reductase enzyme, duodenal cytochrome B (Dcytb), on the brush border of the enterocytes to convert the insoluble ferric (Fe3+) to absorbable ferrous (Fe2+) ions. Iron in its ferric Fe3+form is first converted to its ferrous form to be taken up. Iron is taken up by a membrane transporter DMTl into the enterocyte cell, where it is stored and bound to apoferritin to form ferritin. The body regulates the iron levels by regulating each of these steps.

[0007] Supplementation of nutrition with prebiotic fibers and iron biotics has the potential to increase iron bioavailability. WO2014 / 148887 Al describes that a partly fermented formula with a mixture of the non- digestible oligosaccharides galacto-oligosaccharides (GOS) and long chain fructo-oligosaccharides (IcFOS) iron bioavailability is increased.

[0008] Keenan Derek F et al. 2013 describes sausages with inulins as replacers of the fat fraction. WO2022 / 083858 Al relates to acacia gum for the treatment of iron-induced dysbiosis . US2007 / 0042992 Al teaches that combining easily fermentable inulin (EFI) with hardly fermentable inulin (HFI) enhances mineral absorption, such as the absorption of calcium and magnesium. W02022 / 248900 Al discloses a cereal composition with iron and GOS and IcFOS for improving iron absorption. Synergyl, a prebiotic blend containing inulin and short chain fructo-oligosaccharides (scFOS) in a 1:1 ratio, has been studied for its impact on iron absorption in preclinical setting showing results similar to the use of scFOS alone (Yashuda et al, (2009). J. Nutr., 139(11), 2018-2023. doi.org / 10.3945 / jn.109.110528).

[0009] There remains a need in the art for compositions that aid in improving the bioavailability of iron. The present invention provides in this need.

[0010] SUMMARY OF THE INVENTION

[0011] The invention pertains to nutritional compositions providing improved bioavailability of iron. The inventors of the present invention have surprisingly found that a combination of two types of inulin improved the bioavailability of iron in a synergistic manner, when compared to the single sources of inulin alone. An increase in iron bioavailability was found in in vitro models representative forthe small intestine and colon. Improved iron absorption and thus bioavailability in the small intestine model was also observed when compared with prior art mixtures of scFOS and inulin or GOS and IcFOS. Therefore, nutritional compositions comprising a combination of these two types of inulin can be used to improve iron bioavailability, iron uptake, iron absorption and / or iron status, especially in subjects at risk of iron deficiency.

[0012] The invention thus pertains to a combination of two different types of inulin wherein the combination consists of inulin I having a number average degree of polymerization (DPn) of 8 to 14 and inulin II having a number average degree of polymerization of 20 to 25. The combination and nutritional compositions comprising a combination of these two sources of inulin are for use in improving, preventing, treating and / or reducing the risk of occurrence of iron deficiency associated anemia and / or iron deficiency. In a preferred embodiment the invention is about a combination of these 2 types of inulin, a source of iron and vitamin C, referred to as iron biotics.

[0013] DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention concerns a combination of two different types of inulin wherein the combination consists of inulin I having a number average degree of polymerization (DPn) of 8 to 14, and inulin II having a number average degree of polymerization of 20 to 25 and wherein the weight ratio of inulin I : inulin II ranges from 4:1 to 0.5:1, preferably 3:1 to 1:1, even more preferably 2:1 to 1:1.

[0015] Said combination of two different types of inulin preferably has a DP profile wherein the total content of inulin with a DP of 9 and lower is preferably at most 10 wt%, more preferably at most 8%, even more preferably at most 7 wt% based on total weight of the combination of two types of inulin.

[0016] Said combination of 2 types inulins preferably has a DP profile wherein the total content of inulin with a DP between 6 - 10 is preferably 18 - 26 wt%, more preferably 19 - 25 wt%, even more preferably 20 - 24 wt% based on total weight of the combination of two types of inulin. The combination of 2 types inulins in addition preferably has a DP profile wherein the total content of inulin with a DP of 10 and higher is preferably 60 - 72 wt%, more preferably 62 - 70 wt%, even more preferably 63 - 69 wt% based on total weight of the combination of two types of inulin. The combination of 2 types inulins preferably further has a DP profile wherein the total content of inulin with a DP between 2 and 5 is at most 17 wt%, preferably 8 - 17 wt%, more preferably 10 - 15 wt%, even more preferably 11 - 14 wt% based on total weight of the combination of two types of inulin.

[0017] The invention can also be worded as a nutritional composition comprising a combination of two different types of inulin wherein the combination consists of inulin I having a number average degree of polymerization (DPn) of 8 to 14, and inulin II having a number average degree of polymerization of 20 to 25. Preferably the nutritional composition further comprises at least dietary iron and vitamin C, preferably the nutritional composition is an iron biotics composition.

[0018] The present invention concerns a method for increasing iron absorption, increasing iron bioaccessibility, increasing iron storage and / or increasing iron bioavailability in a human subject, comprising administering to the human subject a combination of two different types of inulin or a nutritional composition comprising said combination of two different types of inulin wherein the combination consists of inulin I having a number average degree of polymerization of 8 to 14, and inulin II having a number average degree of polymerization of 20 to 25.

[0019] The invention may also be worded as the use of a combination of two different types of inulin or a nutritional composition comprising said combination of two different types of inulin wherein the combination consists of inulin I having a number average degree of polymerization of 8 to 14, and inulin II having a number average degree of polymerization of 20 to 25 in the manufacture of a nutritional composition for increasing iron absorption, increasing iron bioaccessibility, increasing iron storage and / or increasing iron bioavailability in a human subject.

[0020] In one embodiment, the method for increasing iron absorption, increasing iron bioaccessibility, increasing iron storage and / or increasing iron bioavailability in a human subject is a non-medical method.

[0021] In one embodiment, increasing iron absorption, increasing iron bioaccessibility, increasing iron storage and / or increasing iron bioavailability is in a human subject with an age of 0 to 36 months. In yet another embodiment, increasing iron absorption, increasing iron bioaccessibility, increasing iron storage and / or increasing iron bioavailability is in a human subject above 36 months of age, preferably a child above 36 months of age, preferably a pregnant woman, lactating woman, menstruating woman, malnourished subjects and elderly subjects.

[0022] The present invention also concerns a method for treating and / or preventing iron-deficiency anemia and / or treating and / or preventing iron deficiency in a human subject comprising administering to the human subject a combination of two different types of inulin or a nutritional composition comprising said combination of two different types of inulin wherein the combination consists of inulin I having a number average degree of polymerization of 8 to 14, and inulin II having a number average degree of polymerization of 20 to 25.

[0023] The invention can also be worded as a combination of two different types of inulin or a nutritional composition comprising said combination of two different types of inulin wherein the combination consists of inulin I having a number average degree of polymerization of 8 to 14, and inulin II having a number average degree of polymerization of 20 to 25 for use in treating and / or preventing anemia, preferably iron deficiency associated anemia, and / or for use in treating and / or preventing iron deficiency in a human subject.

[0024] For some jurisdictions the invention can be worded as the use of a combination of two different types of inulin or a nutritional composition comprising said combination of two different types of inulin wherein the combination consists of inulin I having a number average degree of polymerization of 8 to 14, and inulin II having a number average degree of polymerization of 20 to 25, for the manufacture of a nutritional composition for treating and / or preventing anemia, preferably iron deficiency associated anemia, and / or treating and / or preventing iron deficiency in a human subject.

[0025] In yet even another preferred embodiment, administering a nutritional composition to an infant may be considered non-therapeutic. In those instances, the invention may be worded as defined above by way of a method comprising administering a nutritional composition. For clarity, the method can also be defined as a non-therapeutic method. By definition, the words "non-therapeutic" exclude any therapeutic effect.

[0026] In an embodiment a nutritional supplement is provided comprising the combination of 2 types of inulin, iron and vitamin C. Such a supplement pertains to iron biotics.

[0027] Iron biotics as used relates to the provision of the combination of 2 types of inulin according to the invention, iron sulphate and vitamin C.

[0028] In some embodiments the invention also pertains to a method of preparing a nutritional composition, said method comprising the steps of a) Adding a type of inulin I having a number average degree of polymerization (DPn) of 8 to 14 to the supplement or nutritional composition and subsequently or concomitantly or beforehand b) Adding a type of inulin II having an average DP of 20 to 25 to the supplement or nutritional composition and c) Mixing the two types of inulin with the nutritional composition to obtain a final nutritional composition comprising the combination of two types of inulin according to the invention.

[0029] It is noted that wherever in the present description wording like "the present nutritional composition", "the present combination" or "nutritional composition according to the (present) invention" is used, this also refers to the methods and uses according to the present invention.

[0030] FIGURES

[0031] The present invention will be discussed in more detail below, with reference to the attached figures.

[0032] Figure 1 shows the HPAEC-PAD profile of inulin I, inulin II and the combination thereof in a 1:1 and 1:2 weight ratio.

[0033] LIST OF PREFERRED EMBODIMENTS

[0034] 1. A combination of two types of inulin wherein the combination consists of

[0035] - inulin I having a number average degree of polymerization (DPn) of 8 to 14, preferably 10 to 13, more preferably 8 to 12 and

[0036] - inulin II having a number average degree of polymerization of 20 to 25, wherein the weight ratio of inulin I : inulin II ranges from 3:1 to 1:1, preferably 2:1 to 1:1, and wherein the total content of inulin with DP8 and DP9 is preferably at most 8.0 %, more preferably at most 7.5 % and even more preferably at most 7.3 % as determined by HPAEC-PAD.

[0037] 2. The combination of two types of inulins according to embodiment 1 wherein the number average DPn of the combination of two types of inulin is 14 to 20, more preferably 15.5 to 18.

[0038] 3. The combination of two types of inulin according to embodiments 1 and 2, wherein the total content of inulin with a DP between 6 - 10 is preferably 18 - 26 wt%, more preferably 19 - 25 wt%, even more preferably 20 - 24 wt% based on total weight of the combination of two types of inulin.

[0039] 4. The combination of two types of inulin according to the preceding to embodiments, wherein the total content of inulin with a DP of 2 to 9 is preferably at most 40 wt%, more preferably at most 35 wt%, even more preferably at most 30 wt% based on total weight of the combination of two types of inulin. 5. The combination of two types of inulin according to the preceding to embodiments wherein the combination contains as most abundant molecules DP4, DP5 and DP14 and / or DP15 inulin, preferably wherein the combination of the two types of inulins has a DP profile wherein the total content of inulin with a DP4, DP5, DP14 an DP15 in the combination is preferably 13.5 to 19 wt%, more preferably 14 to 18.5 wt%, even more preferably 14.5 to 18 wt% based on total weight of the combination of the two types of inulins and, wherein preferably said combination of the two types of inulins has a total content of inulin with DP4 and DP5 of preferably 6.5 to 11 wt%, more preferably 7 to 10.5 wt%, even more preferably 7.5 to 10 wt% and a total content of inulin with DP14 and DP15 of preferably 5.5 to 9 wt%, more preferably 6 to 8.5 wt%, even more preferably 6.5 to 8 wt% based on total weight of the combination of the two types of inulins.

[0040] 6. The combination of two types of inulin according to the preceding embodiments, wherein

[0041] - the total content of inulin with a DP between 2 and 5 is preferably 8 - 17 wt%, more preferably 10 - 15 wt%, even more preferably 11 - 14 wt% based on total weight of the combination of two types of inulin and / or

[0042] - the total content of inulin with a DP of 10 and higher is preferably 60 - 72 wt%, more preferably 62 - 70 wt%, even more preferably 63 - 69 wt% based on total weight of the combination of two types of inulin.

[0043] 7. The combination of two types of inulin according to the preceding embodiments, wherein the polydispersity index of the combination of the two types of inulins is about 1.41 to 1.48.

[0044] 8. A nutritional composition comprising the combination of two different types of inulin according to any one of embodiments 1 to 7.

[0045] 9. Nutritional composition according to embodiment 8 wherein the nutritional composition comprises non-digestible oligosaccharides and wherein 90 wt% to 100 wt%, more preferably 95 wt% to 100wt% of the non-digestible oligosaccharides consist of the combination of two types of inulin.

[0046] 10. Nutritional composition according to any one of embodiments 8 and 9 wherein the nutritional composition comprises the combination of two types of inulin in a total amount of

[0047] 0.1 to 2.0 g / 100 ml, preferably 0.2 to 1.8 g / 100 ml, even more preferably 0.4 to 1.6 g / lOOml and / or

[0048] 1.4 to 28 wt%, more preferably 2.8 to 25 wt%, even more preferably 5.6 to 22.5 wt% based on total dry weight of the nutritional composition, and / or

[0049] 0.14 to 3.3 g per 100 kcal, more preferably 0.3 g to 2.7 g per 100 kcal, even more preferably 0.6 to

[0050] 2.4 g per 100 kcal. 11. Nutritional composition according to any one of embodiments 8 to 10 wherein the nutritional composition is selected from an infant formula, follow-on formula, young child formula, fortified milk, sairy or plant based products, cereal composition, a food for special medical purpose and a dietary supplement wherein the composition is in liquid, spoonable or powder format.

[0051] 12. Nutritional composition according to claim any one of embodiments 8 to 11 wherein the nutritional composition comprises iron and vitamin C, preferably 0.3 mg to 7.5 mg iron per 100 kcal and 1.85 mg to 33 mg vitamin C per 100 kcal.

[0052] 13. Nutritional composition according to any one of embodiments 8 to 12 wherein the source of iron is selected from the group consisting of ferrous sulphate, ferrous lactate, ferrous gluconate, ferrous bisglycinate, ferrous citrate, ferrous fumarate, ferric diphosphate, ferric ammonium citrate, ferric pyrophosphate, ferric sodium EDTA, preferably ferrous sulphate, ferrous fumarate, ferric pyrophosphate and ferrous lactate, more preferably ferrous sulphate,.

[0053] 14. Use of the nutritional composition according to any one of embodiments 8 to 13 for increasing and / or improving iron absorption, increasing and / or improving iron storage, increasing and / or improving iron bioaccessibility and / or increasing and / or improving iron bioavailability, in a human subject, comprising administering the nutritional composition to the human subject.

[0054] 15. Use of the nutritional composition according to embodiment 14 wherein the human subject is selected from a human subject with an age of 0 to 36 months, a human subject above 36 months of age, preferably a child aged 3 to 12 years, a pregnant woman, lactating women, women of reproductive age that are menstruating, malnourished subjects and elderly subjects, preferably an infant or young child.

[0055] 16. Combination of two types of inulin according to any one of embodiments 1 to 7 or the nutritional composition according to any one of embodiments 8 to 13 for use in increasing and / or improving iron absorption, increasing and / or improving iron storage, increasing and / or improving iron bioaccessibility and / or increasing and / or improving iron bioavailability.

[0056] 17. Combination of two types of inulin according to any one of embodiments 1 to 7 or the nutritional composition according to any one of embodiments 8 to 13 for use in preventing and / or treating and / or reducing the risk of iron deficiency and / or anemia, preferably iron deficiency anemia.

[0057] 18. Method of preparing a nutritional composition according to any one of embodiments 8 to 13, said method comprising the steps of a) Adding a type of inulin I having a number average degree of polymerization (DPn) of 8 to 14, preferably 10 to 13, more preferably 8 to 12 to the supplement or nutritional composition and subsequently or concomitantly or beforehand b) Adding a type of inulin II having a number average DP of 20 to 25 to the supplement or nutritional composition and c) Mixing the two types of inulin with the nutritional composition.

[0058] 19. Method according to embodiment 18 further comprising the step of d) adding ferrous sulphate and vitamin C, and optionally e) long chain polyunsaturated fatty acids selected from the group consisting of arachidonic acid, docosahexaenoic acid and eicosapentaenoic acid, to obtain a final nutritional composition

[0059] 20. Nutritional composition obtainable by the method according to any one of embodiments 17 to 19.

[0060] 21. Combination of two types of inulin according to any one of embodiments 1 to 7 or the nutritional composition according to any one of embodiments 8 to 13 wherein inulin type I has a number average DP of 8 to 14, preferably above 10, preferably 10 to 13, preferably 8 to 12.

[0061] 22. Combination of two types of inulin according to any one of embodiments 1 to 7 or the nutritional composition according to any one of embodiments 8 to 13 wherein inulin type I has a number average DP of 8 to 12, preferably of about 12 to about 13.

[0062] 23. Nutritional composition according to any one of embodiments 8 to 13 wherein the composition is an iron biotic composition.

[0063] Anemia is a decrease in number of red blood cells or less than the normal quantity of hemoglobin in blood. In the present invention anemia refers in particular to iron deficiency anemia, i.e. anemia caused by insufficient iron bioavailability. Iron-deficiency anemia is caused by insufficient dietary intake and absorption of iron and causes approximately half of all anemia cases in the world. According to the WHO anemia 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 / l 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.

[0064] Iron-deficiency anemia for infants and young children in their earlier stages of development has greater consequences than it does for adults. An infant or young child made severely iron-deficient during its earlier life cannot recover to normal iron levels even with iron therapy. Iron-deficiency anemia affects neurological development by decreasing learning ability, negatively altering motor functions and negatively effecting socioemotional functioning as behavior. Additionally, iron-deficiency anemia has a negative effect on physical growth. In pregnant women, of which it is estimated that 50% suffers from iron deficiency or anemia, there is an increased need for iron. Anemia may increase the risk of preterm or small birth weight babies. It is therefore of utmost importance to prevent anemia in infants and young children and pregnant women.

[0065] Bioavailability as used herein refers to the proportion of ingested iron that is absorbed in the gastrointestinal tract and available to be used by the body.

[0066] Bioaccesibility as used herein refers to is the proportion of ingested dietary iron released from the digestive matrix during digestion and that is available for absorption.

[0067] Iron deficiency (sideropaenia or hypoferraemia) is a stage preceding iron deficiency anemia. 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 anemia. Iron deficiency anemia is abnormal values of 2 out of 3 indicators with anemia (a haemoglobin content below the threshold for anemia).

[0068] The combination, nutritional compositions 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 (0-36 month of age). In this group, iron deficiencies or anemia may have irreparable effects on the growth and development especially effects on brain.

[0069] Non-digestible oligosaccharides as used herein refers to oligosaccharides with an average degree of polymerization ranging from 2 to 100. Non-digestible oligosaccharides are oligosaccharides that are nondigested in the stomach or small intestine and reach the colon intact. Maltodextrin, lactose, and monomers such as galactose, fucose, and sialic acid are not considered non-digestible oligosaccharides, i.e. they are considered digestible carbohydrates. Inulin is a non-digestible oligosaccharide.

[0070] Inulin is a fructan-type carbohydrate, consisting mostly of fructose units, which occurs in many plants as a reserve carbohydrate. Inulin can be produced by certain bacteria and can also be enzymatically produced in vitro from sucrose. Inulin naturally occurs as a polydisperse mixture of carbohydrate molecules which are essentially composed of fructosyl units forming chains in which the fructosyl units are mainly or exclusively linked to one another by a 0(2,1) bound. Inulin molecules from plant origin mostly contain one terminal glucosyl unit. Inulin has a structure of chain-terminating glucosyl moieties and a repetitive fructosyl moiety, which are linked by beta-2,1 linkages. The mainly linear chains are possibly bearing one or more side chains essentially composed of fructosyl units, thus forming branched inulin molecules with a fructosyl-fructosyl linkage at the branching point commonly formed by a fructosyl- fructosyl 0(2,6) bound.

[0071] In the context of the present invention the term "prevention" means "reducing the risk of (occurrence)" or "reducing the severity of".

[0072] An infant is a child under the age of 12 months.

[0073] A young child is a child that is up to 36 months of age, preferably up to 6 years of age.

[0074] As used herein, the term "degree of polymerization" (DP) means the number of monomer units joined together in a poly- or oligomer. The average degree of polymerisation (DP) is commonly determined by the method described by De Leenheer L. et al., Starch / Starke, 46 (5), 193-196, (1994) and Carbohydrates as Organic Raw Materials, Ed. H. Van Bekkum et al. for CRF, Wageningen, The Netherlands, Vol. Ill, 67-74, (1996). The number average DP as used herein is a measure of the average number of monomer units calculated by devising the number-average molecular weight of the polymer by the molecular weight of individual monomer units.

[0075] The term polydispersity as used herein refers to a numerical measure of the distribution of molecular weights within a polymer. In the context of dietary fibers, the PDI quantifies the heterogeneity of chain lengths or molecular sizes. The PDI is calculated as the ratio of the weight-average molecular weight (Mw) to the number-average molecular weight (Mn), expressed as: PDI = Mw / Mn

[0076] A PDI value close to 1 thus indicates a uniform, monodisperse distribution, whereas higher values reflect greater variability in molecular size.

[0077] The degree of polymerization and polydispersity index can be obtained using techniques common in the art such as high performance anion exchange chromatography (HPAEC)-PAD (pulsed amperometric detection). In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0078] Combination of 2 types of inulin

[0079] A combination of two different types of inulin or a nutritional composition comprising said combination of two different types of inulin, wherein the combination consists of inulin I having a number average degree of polymerization of 8 to 14 and inulin II having a number average degree of polymerization of 20 to 25, were found to enhance iron bioavailability. The DP and all percentages thereof are preferably as determined by HPAEC-PAD.

[0080] Inulins comprise a group of naturally occurring polysaccharides (several simple sugars linked together) produced by many types of plants. Inulins belong to a class of dietary fibers known as fructans. Inulin is used by some plants as a means of storing energy and is typically found in the roots or rhizomes of these plants. Sources of inulin include, dandelion (taraxacum officinale), wild yam (dioscorea spp.), artichoke (helianthus tuberosus), chicory (cichorium intybus), jicama (pachyrhizus erosus), burdock (articium lappa), onion (allium cepa), garlic (allium sativum), agave (agave spp.).

[0081] Inulins are non-digestible oligosaccharides having a structure of repetitive fructosyl moiety and are a type of fructooligosaccharides (FOS) wherein at least 75% of the glycosidic linkages are 0(2,1) linkages. Non- digestible oligosaccharides, including inulins, are not digested in the intestine by the action of digestive enzymes present in the human upper digestive tract (small intestine and stomach). The non-digestible oligosaccharides are fermented by the human intestinal microbiota. The degree of polymerization (DP) or number of fructose units in the fructose polymer of standard or native inulin ranges from mainly 2 to 60 or from 2 to 60 with number average DP of approximately 8-12 or 8-13 for example for native chicory. Inulin chain length distribution to determine the degree of polymerization can be done by conventional analytic means known in the art such as a chromatographic method HPAEC-PAD and gas liquid chromatography (GLC). Commercial sources of (standard) inulin include Frutafit ®IQ from Sensus, Raftiline ST or Orafti ST from Beneo, Fibroline instant from Cosucra.

[0082] Long chain inulin or also called long chain FOS is generally obtained by crystallization and removal of impurities from standard inulin. Hereby short chain inulin or FOS with DP 2-9 is removed. Long chain FOS therefore is a fructose polymer made of poly-0-D-(2->l)-fructofuranosyl-a-D-glucopyranose but also of derivatives thereof such as poly-0-D-(2->l) polyfructofuranose and which has preferably a DP ranged from mainly 10 to 60 and a number average DP of such long chain inulin is 20 -25, preferably 22-23. Suitable commercial sources of IcFOS are RaftilinHP or Orafti HP from Beneo, FrutafitTex from Sensus and fibrulineXL from Cosucra.

[0083] Short chain FOS (scFOS) on the other hand have a DP ranging from 2 - 8, preferably from 2 - 7 and have a number average DP of 3 -5. Suitable commercial sources of scFOS are Raftilose from Beneo, Actilight 950P from Meij and frutalose from Cosucra.

[0084] The combination of two types of inulin according to the invention consist of an inulin and a long chain inulin or long chain FOS. The combination of two different types of inulin consists of inulin I having a number average degree of polymerization (DPn) of 8 to 14, preferably 8 to 12, and inulin II having a number average degree of polymerization of 20 to 25, preferably 22 to 23. The weight ratio of inulin I : inulin II in the combination ranges from 4:1 to 0.5:1, preferably 3:1 to 1:1, even more preferably 2:1 to 1:1.

[0085] Inulin I preferably has a DP range from 10 to 60. Inulin II preferably has a DP range from 2 -60. The combination of the DP profiles of inulin I and inulin II beneficially improve iron bioavailability.

[0086] Preferably inulin I has a number average degree of polymerization of 8 to 14, preferably 10 to 13, preferably 8 to 12. In a preferred embodiment the number average DP of inulin I is above 10, preferably 10 to 13, more preferably about 12 to about 13. In a further embodiment inulin type I has a number average DP of 8 to 14, preferably 8 to 12, preferably of about 12 to about 13. Preferably inulin I has a number average degree of polymerization of 11 to 13.

[0087] Said combination of two different types of inulin according to the invention preferably has a DP profile wherein the total content of inulin with a DP of 2 to 9 is preferably at most 40 wt%, more preferably at most 30 wt%, even more preferably at most 20 wt% based on total weight of the combination of two types of inulin.

[0088] In a further preferred embodiment, the combination of two different types of inulin according to the invention preferably have a total content of inulin with a DP of 2 -6 that is preferably at most 25 wt%, more preferably at most 20 wt%, even more preferably at most 10 wt%.

[0089] In an embodiment, the combination of 2 types inulins preferably has a DP profile wherein the total content of inulin with a DP between 6 - 10 is preferably 18 - 26 wt%, more preferably 19 - 25 wt%, even more preferably 20 - 24 wt% based on total weight of the combination of two types of inulin.

[0090] The combination of 2 types inulins preferably has a DP profile wherein the total content of inulin with a DP of 10 and higher is preferably 60 - 72 wt%, more preferably 62 - 70 wt%, even more preferably 63 - 69 wt% based on total weight of the combination of two types of inulin.

[0091] The combination of 2 types inulins preferably further has a DP profile wherein the total content of inulin with a DP between 2 and 5 is preferably 8 - 17 wt%, more preferably 10 - 15 wt%, even more preferably 11 - 14 wt% based on total weight of the combination of two types of inulin.

[0092] In an embodiment the combination of the two types of inulins has a total content of inulin with DP8 and DP9 is preferably at most 8.0 %, more preferably at most 7.5 % and even more preferably at most 7.3 % based on total weight of the combination of the two types of inulins.

[0093] In an embodiment, the combination of two types of inulin preferably has a DP profile wherein the total content of inulin with a DP between 2 and 5 is preferably 8-17 wt%, more preferably 10-15 wt%, even more preferably 11-14 wt%;wherein the total content of inulin with a DP between 6 and 10 is preferably 18-26 wt%, more preferably 19-25 wt%, even more preferably 20-24 wt%; and wherein the total content of inulin with a DP of 10 and higher is preferably 60-72 wt%, more preferably 62-70 wt%, even more preferably 63-69 wt%; all percentages being based on the total weight of the combination of the two types of inulin. In a further embodiment, the combination of two types of inulin preferably has a DP profile wherein the total content of inulin with a

[0094] DP between 2 and 5 is preferably 8-17 wt%, more preferably 10-15 wt%, even more preferably 11-14 wt%; wherein the total content of inulin with a

[0095] DP between 6 and 10 is preferably 18-26 wt%, more preferably 19-25 wt%, even more preferably 20-24 wt%; and wherein the total content of inulin with a

[0096] DP of 10 and higher is preferably 60-72 wt%, more preferably 62-70 wt%, even more preferably 63-69 wt%; and wherein the total content of inulin with DP8 and DP9 is preferably at most 8.0 %, more preferably at most 7.5 % and even more preferably at most 7.3 % based on total weight of the combination of the two types of inulins, all percentages being based on the total weight of the combination of the two types of inulin.

[0097] In an embodiment the number average DP of the combination of two types of inulin according to the invention has at least a DPn > 11, preferably > 12, more preferably > 14, even more preferably > 16 . In a preferred embodiment the number average DPn of the combination of two types of inulin is > 14. In a further preferred embodiment the number average DPn of the combination of two types of inulin is preferably 14 to 20, more preferably 15 to 19, even more preferably 15.5 to 18.

[0098] In a further embodiment, the combination of two types of inulin preferably has a DP profile wherein the total content of inulin with a DP between 2 and 5 is preferably 8-17 wt%, more preferably 10-15 wt%, even more preferably 11-14 wt%;wherein the total content of inulin with a DP between 6 and 10 is preferably 18-26 wt%, more preferably 19-25 wt%, even more preferably 20-24 wt%; wherein the total content of inulin with a DP of 10 and higher is preferably 60-72 wt%, more preferably 62-70 wt%, even more preferably 63-69 wt%, wherein the percentages are based on the total weight of the combination of the two types of inulin and wherein the number average DPn of the combination of two types of inulin is preferably 14 to 20, more preferably 15 to 19, even more preferably 15.5 to 18.

[0099] In an embodiment the number average molecular weight of the combination of two types of inulins is preferably 2400 to 3200 g / mol, more preferably 2500 to 3100 g / mol, even more preferably 2600 to 3000 g / mol. In a further embodiment the weight-average molecular weight of the combination of the two types of inulins is preferably 3750 to 4400 g / mol, more preferably 3850 to 4300 g / mol, even more preferably 3900 to 4250 g / mol.

[0100] In an embodiment the polydispersity index of the combination of the two types of inulins is above 1.3, preferably above 1.35, more preferably above 1.4. In an embodiment the polydispersity index of the combination of the two types of inulins is about 1.41 to 1.48.

[0101] In a further preferred embodiment the most abundant molecules of the combination of the two types of inulins comprise GF4, GF5 and GF14 and / or GF15. The abbreviation GF as used herein represents the degree of polymerization of the glucose-fructose chains. In an embodiment the combination of the two types of inulin has a bimodal distribution pattern. Most abundant as used herein means the absolute number of molecules based on the total amount of molecules present in the combination of the two types of inulins.

[0102] Alternatively worded the combination of the two types of inulins contains as most abundant molecules of the combination of inulins DP4, DP5 and DP14 and / or DP15 molecules. The combination of the two types of inulins preferably has a DP profile wherein the total content of inulin with DP4, DP5, DP14 an DP15 in the combination is preferably 13.5 to 19 wt%, more preferably 14 to 18.5 wt%, even more preferably 14.5 to 18 wt% based on total weight of the combination of the two types of inulins. Said combination of the two types of inulins has a total content of inulin with DP4 and DP5 of preferably 6.5 to 11 wt%, more preferably 7 to 10.5 wt%, even more preferably 7.5 to 10 wt% and a total content of inulin with DP14 and DP15 of preferably 5.5 to 9 wt%, more preferably 6 to 8.5 wt%, even more preferably 6.5 to 8 wt% based on total weight of the combination of the two types of inulins.

[0103] In a preferred embodiment the combination of 2 types of inulin is present in a nutritional composition. In some embodiments a nutritional composition preferably does not comprise other non-digestible oligosaccharides than the combination of two types of inulin according to the invention. Preferably the non-digestible oligosaccharides in a nutritional composition consist of at least 50 wt%, more preferably at least 75 wt%, even more preferably at least 90 wt% of the combination of two types of inulin according to the invention. In a preferred embodiment the non-digestible oligosaccharides in the nutritional composition consist of 90 wt% to 100 wt%, more preferably 95 wt% to 100wt% of the combination of two Y1 types of inulin according to the invention based on total weight of non-digestible oligosaccharides in the nutritional composition. In a most preferred embodiment 100 wt% of the non-digestible oligosaccharides in the nutritional composition consists of the combination of two types of inulin according to the invention.

[0104] Based on 100 ml ready to drink composition the nutritional composition preferably comprises 0.1 to 2.0 g in total of the combination of two types of inulin, more preferably 0.2 to 1.8 g, even more preferably 0.4 to 1.6 g, based on 100 ml of the nutritional composition. The nutritional composition preferably comprises 1.4 to 28.0 wt% of the combination of two types of inulin, more preferably 2.8 to 25 wt%, even more preferably 5.6 to 22.5 wt% based on total dry weight of the nutritional composition. Preferably the amount of the combination of two types of inulin per 100 kcal of the nutritional composition is 0.15 to 3.0 g per 100 kcal, more preferably 0.3g to 2.7 g per 100 kcal, even more preferably 0.60 to 2.4 g per 100 kcal.

[0105] Nutritional compositions

[0106] The present nutritional composition comprising the combination of 2 types of inulin is preferably particularly suitable for providing the complete daily nutritional requirements to a human subject in need of improved iron bioavailability. In a preferred embodiment the nutritional composition comprising the combination of 2 types of inulin further comprises a source of iron and vitamin and preferably is a composition for iron biotics.

[0107] In a preferred embodiment the nutritional composition is suitable for an infant or a child, or in other words to a human subject with an age of 0 months to 12 years, more preferably to an infant or young child, or in other words to a human subject with an age of 0 months to 6 years. In a preferred embodiment the nutritional composition is thus suitable for a child above 36 months of age.

[0108] In another preferred embodiment the nutritional composition is suitable for infant or young children aged 0-36 months, more preferably 6-36 months. In an alternative preferred embodiment, the nutritional composition is suitable for adults, preferably for subjects at risk of iron deficiency including pregnant women, lactating women, women of reproductive age that are menstruating, malnourished subjects, and elderly subjects.

[0109] The present nutritional composition comprises digestible carbohydrate. Preferably the digestible carbohydrate is lactose. Thus herein, lactose is considered to be a digestible carbohydrate. However, also other digestible carbohydrates such as glucose, sucrose, fructose, galactose, maltose, starch and maltodextrin may be present. When in liquid form, e.g. as a ready-to-feed liquid, the nutritional composition preferably comprises 6.0 to 30 g digestible carbohydrate per 100 ml, more preferably 6.0 to 20, even more preferably 7.0 to 10.0 g per 100 ml. Based on dry weight the present nutritional composition preferably comprises 40 to 80 wt.%, more preferably 40 to 65 wt.% digestible carbohydrates. Based on total calories the nutritional composition comprises 9 to 20 g digestible carbohydrates per 100 kcal, more preferably 9 to 15 g.

[0110] The present nutritional composition preferably comprises lipid. The lipid of the present nutritional composition provides 3 to 7 g per 100 kcal of the nutritional composition, preferably the lipid provides 4 to 6 g per 100 kcal. When in liquid form, e.g. as a ready-to-feed liquid, the nutritional composition preferably comprises 2.1 to 6.5 g lipid per 100 ml, more preferably 3.0 to 4.0 g per 100 ml. Based on dry weight the present nutritional composition preferably comprises 12.5 to 40 wt.% lipid, more preferably 19 to 30 wt.%. Preferably the lipid comprises the essential fatty acids alpha-linolenic acid (ALA), linoleic acid (LA) and / or long chain polyunsaturated fatty acids (LC-PUFA). The LC-PUFA, LA and / or ALA may be provided as free fatty acids, in triglyceride form, in diglyceride form, in monoglyceride form, in phospholipid form, or as a mixture of one of more of the above. Preferably the present nutritional composition contains at least one, preferably at least two lipid sources selected from the group consisting of rape seed oil (such as colza oil, low erucic acid rape seed oil and canola oil), high oleic sunflower oil, high oleic safflower oil, olive oil, marine oils, microbial oils, coconut oil, palm kernel oil and milk fat. Preferably the present nutritional composition comprises at least 0.2 wt.%, more preferably at least 0.4 wt.% long chain poly unsaturated fatty acids based on total fatty acids, wherein the long chain poly unsaturated fatty acids are one or more selected from the group consisting of arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid. Suitable sources are fish oil, egg phospholipids and oil form microorganisms. Preferably the present nutritional composition comprises at most 2 wt.% long chain poly unsaturated fatty acids, more preferably at most 1 wt.%, based on total fatty acids of long chain poly unsaturated fatty acids, wherein the long chain poly unsaturated fatty acids are one or more selected from the group consisting of arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid. Herein, the wt.% of long chain poly unsaturated fatty acids refers to the sum of arachidonic acid, docosahexaenoic acid and eicosapentaenoic acid. Especially preferred is docosahexaenoic acid. It is believed that the provision of long chain poly unsaturated fatty acids provides beneficial effects on brain and cognition and thereby assists in improving the consequences of iron deficiency. Preferably the present nutritional composition comprises protein. The protein is preferably selected from the group consisting of plant proteins and milk proteins. Preferably the present nutritional composition comprises one or more selected from the group consisting of whey, whey protein, whey protein hydrolysate, casein and casein hydrolysate. The nutritional composition preferably contains casein, and / or whey protein, more preferably bovine whey proteins and / or bovine casein. The nutritional composition preferably comprises casein and whey proteins in a weight ratio casein:whey protein of 10:90 to 90:10, more preferably 20:80 to 80:20, even more preferably 35:65 to 55:45.

[0111] The nutritional composition of the present invention preferably provides protein in an amount of 1.25 to 9 g per 100 kcal, preferably providing 1.5 to 7.5 g, even more preferably 1.7 to 6 g per 100 kcal. When in liquid form, the nutritional composition preferably comprises 0.5 to 6.0 g, more preferably 1.0 to 5.0 g, even more preferably 1.0 to 4.0 g protein per 100 ml. Based on dry weight the present nutritional composition preferably comprises 5 to 20 wt.% protein, preferably at least 8 wt.%, more preferably 8 to 14 wt.%, protein even more preferably 8 to 9.5 wt.% based on dry weight of the nutritional composition.

[0112] The nutritional composition of the present invention preferably provides lipid in an amount of 0.4 to 7 g per 100 kcal, preferably 0.6 to 6 g per 100 kcal, protein in an amount of 1.25 to 9 g per 100 kcal, preferably 1.5 to 7.5 g per 100 kcal, preferably 1.7 to 6 g per 100 kcal and digestible carbohydrate in an amount of 5 to 20 g per 100 kcal, preferably 8 to 15 g per 100 kcal of the nutritional composition. Preferably the present nutritional composition comprises lipid providing 0.4 to 7 g per 100 kcal, protein providing 1.25 to 9 g per 100 kcal, and digestible carbohydrate providing 8 to 15 g per 100 kcal of the final nutritional composition.

[0113] The amount of total calories is determined by the sum of calories derived from protein, lipids, digestible carbohydrates and non-digestible oligosaccharides. Protein and carbohydrates are considered to have a caloric density of 4 kcal / g, fat of 9 kcal / g and non-digestible oligosaccharides 2 kcal / g.

[0114] The present nutritional composition is not human breast milk. The nutritional composition according to the invention or the nutritional composition used according to the invention preferably comprises other fractions, such as vitamins, minerals, trace elements and other micronutrients in order to make it a complete nutritional composition. In a preferred embodiment the nutritional composition is an iron biotic composition. In a preferred embodiment the nutritional composition is selected from the group consisting of an infant formula, follow on formula, toddler milk or formula, growing up milk, fortified milk, weaning compositions such as cereal based compositions, nutritional supplements and food for special medical purposes. In an embodiment the nutritional compositions may be dairy and / or plant-based compositions. In an embodiment the nutritional composition is in liquid, spoonable or powder form.

[0115] An infant formula is defined as a formula for use in infants and can for example be a starter formula, intended for infants of 0 to 4 to 6 months of age or a follow-on formula, intended for infants of 4 to 6 months until 12 months of age. A toddler milk or growing up milk or formula is intended for children of 12 to 36 months of age. In one embodiment the nutritional composition is an infant formula. Infant formulae comprise vitamins, minerals, trace elements and other micronutrients according to international directives.

[0116] In one embodiment the nutritional composition is in a liquid or spoonable 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 toddler formula is a powder to be reconstituted with water. Preferably the liquid composition has a viscosity below 100 mPa.s, more preferably below 60 mPa.s, more preferably below 35 mPa.s, even more preferably below 6 mPa.s as measured in a Brookfield viscometer at 20°C at a shear rate of 100 s’1. A low viscosity is important for infant or follow on formula, since it mimics the viscosity of breast milk and can then be administered via a teat.

[0117] In order to meet the caloric requirements of an infant or toddler, the nutritional composition preferably comprises 45 to 200 kcal / 100 ml liquid. For infants the nutritional composition has more preferably 60 to 90 kcal / 100 ml liquid, even more preferably 65 to 75 kcal / 100 ml liquid. This caloric density ensures an optimal ratio between water and calorie consumption. For toddlers, human subjects with an age from 12 to 36 months, the nutritional composition more preferably has a caloric density from 45 to 65, even more preferably from 50 to 60 kcal / 100 ml. For children from 3 to 12 years of age, the nutritional composition more preferably has a caloric density from 45 to 65, even more preferably from 50 to 60 kcal / 100 ml. The osmolarity of the present composition is preferably from 150 to 420 mOsmol / l, more preferably from 260 to 320 mOsmol / l. The low osmolarity aims to further reduce the gastrointestinal stress. When the nutritional composition is in a liquid form, the preferred volume administered on a daily basis is in the range of about 80 to 2500 ml, more preferably about 200 to 1200 ml per day. Preferably, the number of feedings per day is from 1 to 10, preferably from 3 to 8. In one embodiment the nutritional composition is administered daily for a period of at least 2 days, preferably for a period of at least 4 weeks, preferably for a period of at least 8 weeks, more preferably for a period of at least 12 weeks, in a liquid form wherein the total volume administered daily is from 200 ml to 1200 ml and wherein the number of feedings per day is from 1 to 10.

[0118] The pH of the present nutritional composition is preferably from 5.0 to 7.5, more preferably from 5.0 to 6.5, most preferably from 5.5 to 6.3.

[0119] The nutritional composition may be cow's milk based drink comprising cows' milk and these two types of inulin of the invention. Preferably the cow's milk drink is fortified with iron as described below. Preferably the cow's milk is fortified with iron and vitamin C drink as described below

[0120] The composition may in some embodiments also be a food for special medical purpose or a nutritional supplement, such as a supplement for pregnant, menstruating or lactating women, a supplement for children or a supplement for elderly or malnourished subjects. These subjects, weaning infants, children, pregnant, menstruating or lactating women, malnourished subjects and elderly have a higher risk of suffering from iron deficiency, and thus benefit the most from the composition according to the invention. Thus, in one aspect, the invention concerns a nutritional supplement comprising the combination of two types of inulin according to the invention. All benefits and possible application defined herein for the combination of two types of inulin according to the invention equally apply to the nutritional compositions and nutritional supplements according to the invention.

[0121] In some embodiments there is further provided a method of preparing a nutritional composition comprising the combination of inulins according to the invention, said method comprising the steps of a) adding a type of inulin I having a number average degree of polymerization (DPn) of 8 to 14, preferably 8 to 12 to the supplement or nutritional composition and subsequently or concomitantly or beforehand, b)Adding a type of inulin II having a number average DP of 20 to 25 to the supplement or nutritional composition and c) mixing the two types of inulin with the nutritional composition. Said method in an embodiment further comprises the step of d) adding ferrous sulphate and vitamin C, and optionally e) long chain polyunsaturated fatty acids selected from the group consisting of arachidonic acid, docosahexaenoic acid and eicosapentaenoic acid, to obtain a final nutritional composition

[0122] Dietary iron

[0123] The present nutritional composition comprising the combination of two types of inulin preferably comprises iron. In the context of this invention, iron means Fe2+or Fe3+. Preferably the nutritional composition 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, and ferric ammonium citrate, ferric pyrophosphate, ferric sodium EDTA more preferably ferrous sulphate and ferrous fumarate and ferric pyrophosphate, even more preferably ferrous sulphate.

[0124] 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. 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, as well as in malnourished subjects, pregnant, lactating or menstruating women and elderly subjects.

[0125] The present nutritional composition preferably comprises at least 0.2 mg iron per 100 ml, more preferably at least 0.4 mg per 100 ml. The present nutritional composition preferably comprises at least 0.015 mg iron per g dry weight, more preferably at least 0.03 mg per g dry weight. The present nutritional composition preferably comprises at least 0.3 mg iron per 100 kcal, more preferably at least 0.6 mg per 100 kcal. A minimal amount is preferred to ensure sufficient iron uptake and prevent iron deficiency.

[0126] The present nutritional compositions preferably comprise not more than 5 mg iron per 100 ml, more preferably not more than 4 mg iron per 100 ml, more preferably not more than 3.5 mg iron per 100 ml, even more preferably not more than 1.1 mg iron per 100 ml. The present nutritional compositions preferably comprise not more than 0.3 mg iron per g dry weight, more preferably not more than 0.26 mg iron per g dry weight, even more preferably not more than 0.23 mg iron per g dry weight. The present nutritional compositions preferably comprise not more than 9 mg iron per 100 kcal, more preferably not more than 8 mg iron per 100 kcal, even more preferably not more than 7.5 mg iron per 100 kcal. Too much iron can result in poor product quality by peroxidizing polyunsaturated acids and can have adverse health effects amongst others by an effect on the gut microbiota.

[0127] Vitamin C

[0128] In a preferred aspect the present nutritional composition comprising a combination of two types of inulin and dietary iron additionally comprises ascorbic acid (vitamin C). The ascorbic acid enhances the absorption of iron into the bloodstream. In a preferred embodiment vitamin C is provided in a molar ratio to iron in a range of 2:1 to 4:1.

[0129] The present nutritional composition preferably comprises at least 1.25 mg vitamin C per 100 ml, more preferably at least 2.5 mg per 100 ml. The present nutritional composition preferably comprises at least 0.095 mg vitamin C per g dry weight, more preferably at least 0.2 mg per g dry weight. The present nutritional composition preferably comprises at least 1.9 mg iron per 100 kcal, more preferably at least 3.8 mg per 100 kcal.

[0130] The present nutritional compositions preferably comprise not more than 22 mg vitamin C per 100 ml, more preferably not more than 18 mg vitamin C per 100 ml, more preferably not more than 15 mg vitamin C per 100 ml, even more preferably not more than 14 mg vitamin C per 100 ml. The present nutritional compositions preferably comprise not more than 1.3 mg vitamin C per g dry weight, more preferably not more than 1.15 mg vitamin C per g dry weight, even more preferably not more than 1.1 mg vitamin C per g dry weight. The present nutritional compositions preferably comprise not more than 40 mg vitamin C per 100 kcal, more preferably not more than 35 mg vitamin C per 100 kcal, even more preferably not more than 32 mg vitamin C per 100 kcal.

[0131] In a preferred embodiment a nutritional supplement is provided comprising the combination of 2 types of inulin, iron and vitamin C.

[0132] APPLICATION

[0133] Preferably the present combination of two different types of inulin and nutritional composition are suitable for, or suitable for administration to, a human subject. In one embodiment, the present combination of two different types of inulin and nutritional composition is suitable for infants and / or children. In one embodiment the present nutritional composition is for use in providing nutrition to human subjects with an age of 0 to 36 months. Young children, or toddlers, are defined as human subjects with an age of 12 to 36 months. Infants are defined as human subjects with an age of below 12 months. So in other words, the present nutritional composition is suitable for human subjects with an age of 0 to 36 months.

[0134] Wherever in this description the term "infants and / or young children" is used, this can be replaced by "human subjects with an age of 0 to 36 months". Healthy full-term infants are born with a supply of iron that usually lasts for 4 to 6 months. Preferably the present nutritional composition is suitable for a human subject with an age of 4 months to 36 months. In one embodiment the present nutritional composition is preferably for use in providing nutrition to a human subject with an age of 4 months to 36 months. These infants or young children have a higher need for iron and are therefore more prone to suffer from iron deficiency or anemia.

[0135] Preterm infants have less iron stores, which are built up in the third trimester of pregnancy. Preterm infants, defined as infants born before week 37 of gestation, preferably before week 32, are in particular at risk of iron deficiency or anemia. In a preferred embodiment, the present nutritional composition is suitable for a preterm infant, preferably for a preterm infant born before week 37 of gestation, more preferably for a preterm infant born before week 32 of gestation.

[0136] In a further embodiment the present combination of two types of inulin and nutritional composition are for use in providing nutrition to human subjects with an age above 36 months. In an embodiment the present nutritional composition is suitable for, or suitable for administration, to children aged 3 to 12 years that are in higher need of iron. In one embodiment, the present nutritional composition is suitable for, or suitable for administration to pregnant or lactating women or women of reproductive age that are menstruating. Pregnant women are in higher need for iron and are therefore more prone to suffer from iron deficiency or anemia. Iron anemia during pregnancy will also adversely affect the development of the fetus. Likewise, women of reproductive age that are menstruating and lactating women are in higher need of iron and at increased risk of iron deficiency or anemia. In a further preferred embodiment, the present combination and nutritional composition are suitable for malnourished and / or elderly subjects. Elderly subjects are at risk of decreased mineral absorption and in higher need for iron and therefor at risk of iron deficiency and / or anemia. The present combination of two types of inulin and nutritional composition are suitable to improve iron uptake, increasing iron absorption, increasing iron bioaccessibility, increasing iron storage and / or iron bioavailability, more preferably iron bioavailability.

[0137] The present combination of two types of inulin and nutritional composition are preferably enterally administered, more preferably orally.

[0138] In one embodiment the present combination of two types of inulin and nutritional composition are preferably for human subjects suffering from iron deficiency or anemia or human subjects that are at risk of iron deficiency or anemia. In one embodiment the present combination of two types of inulin and nutritional composition are for use in treating or preventing anemia, preferably iron deficiency anemia, and / or iron deficiency. In one embodiment the present combination of two types of inulin and nutritional composition is for use in improving iron uptake, increasing iron absorption, iron bioaccessibility and / or iron bioavailability, more preferably iron bioavailability.

[0139] Bioaccessibility is the proportion of an ingested nutrient that is potentially available for absorption and is dependent on digestion and / or release from the food matrix. Bioavailability is the proportion of an ingested nutrient that is absorbed and available for physiological functions and is dependent on digestion and / or release from the food matrix, absorption by intestinal cells and transport to the body cells. Absorption is the uptake of a nutrient into the cell, and is dependent on digestion and / or release from the food matrix

[0140] EXAMPLES

[0141] The present invention is described in more detail by reference to the following examples.

[0142] Example 1

[0143] Mixture of two sources of inulin increases iron bioavailability in intestinal cells.

[0144] A base milk formula contains 1.5 g / 100 ml protein with a casein / whey protein ratio of 80:20, 75 mg / 100 ml calcium, 1.0 mg / 100 ml iron (iron sulphate), 9.8 mg / 100 ml vitamin C, and further vitamins, minerals, trace elements according to EU directive for follow on formula but does not contain NDO. To this formula, different combinations of non-digestible oligosaccharides (NDO) were added, resulting in, after reconstitution, ready-to-drink follow-on formula differing in non-digestible oligosaccharide content, see

[0145] Table 1.

[0146] Table 1: Tested infant formulas with different ratio's and concentrations of non-digestible oligosaccharides. a. Inulin was obtained from Beneo Orafti (Oreye BE, Pemuco CL) as OraftiGR, and has an average DPn of > 10. The DP ranges from 2 to 60. This inulin is representative of inulin source 1. b. Long chain fructo-oligosaccharides (IcFOS) was obtained as FrutafitTex from Sensus, Zwolle, NL and has an average DPn of about 23. The DP ranges from 10-60. This inulin is representative of inulin source 2. c. Short chain fructo-oligosaccharides (scFOS) was obtained from Beneo Orafti (Oreye BE and Pemuco CL) as oligofructose P95 and has an average degree of polymerization (DPn) of about 4. The DP range from 2 to 8. d. Galacto-oligosaccharides (GOS) were obtained from FrieslandCampina, Borculo NL, as VivinalGOS and has an average DP of about 3-4.

[0147] Small intestinal iron bioavailability.

[0148] The infant formulas underwent in vitro digestion as described previously in Abrahamse et al. nutrients 2022, 14, 1512, followed by heat treatment at 105°C for 5 minutes and centrifugation at 2460 x g, 10 minutes at 4°C. The supernatant was mixed v / v 1:1 with cell culture medium and incubated for 2 hours on Caco-2 intestinal cells. The supernatants were then replaced with fresh cell culture medium and incubated for an additional 22 hours to allow for ferritin (iron storage protein) formation. Caco-2 cells were then lysed using a protein extraction buffer (CelLytic M, Sigma), and ferritin levels were quantified with an ELISA kit following the manufacturer's protocol (Human Ferritin ELISA kit, RayBiotech). These ferritin levels served as an indicator of iron absorption and bioavailability. Because a direct effect of the NDOs after incubation with intestinal cells on internal ferritin levels was measured, this is representative for the iron uptake in the small intestine.

[0149] Colon iron bioavailability.

[0150] In short, next to the direct effect of NDOs on ferritin levels in the intestinal cells, also the situation in the colon was mimicked in vitro. Focus was on the inulins. Here the NDOs of 2-8 were fermented by microbiota and the amount and profile of the short chain fatty acids formed was determined (SCFA). Subsequently these SCFA as formed upon fermentation by the NDOS 2-10 were mixed with digested and dialysed IMF and added to the Caco-2 cells to determine the effect on ferritin level.

[0151] In vitro fermentation:

[0152] Under anaerobic conditions, 4 fecal samples from 4 different infant donors were thawed and approximately a 10% (w / v) suspension of the fecal samples were made in age adapted Colonic Microbiota medium containing 25 mM acetate and 12 mM lactate, 25 mg / L bile acids (Sigma), 15 mmol / L ammonium sulphate without carbon source adjusted to toddler stool pH (6.5) to mimic uncompromised intestinal conditions. The diluted fecal samples were homogenized, allowed to sediment for 5 minutes, and subsequently filtered over a Millex 100 pm vacuum filter.

[0153] The filtered fecal samples.

[0154] A Biolector Pro plate (BOH2 round well, M2P-labs) with pH optodes was used. The feeding row A was filled with sterile prebiotics that are to be used during the overnight feeding in a 6 pL / h flow rate. The feeding row B of the plate was filled with sterile 3M NaOH for the pH adjustment. All the wells of the row C were filled with 800 pL of the fecal solution from donor A, all the wells of row D, E and F were filled with 800 pL of the fecal solution from donor B, C and D, respectively. Next, 800 pL of water and the NDO of 2-10 (table 1) were added to all the wells with the fecal solution samples. After this, the plates were sealed with ventilated silicone foil with slits. The plate was incubated (85% moisture, 37 degrees, 600 rpm, anaerobic) in BioLector Pro for 56 hours. Every 4 hours of the prebiotic fermentation, the experiment was paused, the fecal slurry of each well was harvested and shortly centrifuged, under aseptic anaerobic conditions. The supernatant was frozen for SCFA analyses, while the rest of the fecal pellets was resuspended into 800 pL of fresh colonic microbiota medium and pipetted back in the wells of a new BOH2 plate. During the whole experiment, samples were taken at t=Oh, t=4 h, t=8 h, t=24 h (overnight feeding), t=28 h, t=29 h, t=32 h, t=48 h (overnight feeding), t=52 h, t=53 h and t=56 h.

[0155] Follow on formula (Nutristart 2) was digested and dialysed, with the resulting retentate representing the fraction of the follow-on formula that transits from the small intestine to the colon. 20% of dietary iron was bioavailable under small intestinal conditions, hence the retentate contained ~80% of total dietary iron. This retentate was then mixed with acetic, propionic and butyric short-chain fatty acids (SCFAs), replicating the SCFA profiles observed during in vitro fermentation of the non-digestible oligosaccharides. After incubating the retentate-SCFA mixes for 1 hour at 37°C, they were centrifuged at 2460 x g for 10 minutes at 4°C. The resulting supernatant was diluted 1:1 with cell culture medium, IM NaHCO3 was added to neutralize the sample' pHs and the iron content of the colonic digests was measured via Inductively coupled Plasma-Optical emission spectrum (ICP-OES). The colonic digests were than applied to caco-2 intestinal cells for 2 hours. Following the incubation, supernatants were removed, and fresh cell media was added. After 22 hours, iron uptake was assessed by quantifying ferritin, the iron storage protein, using an ELISA kit.

[0156] Statistics

[0157] Ferritin concentrations were assessed under two conditions: small intestinal and colonic.

[0158] Iron Bioavailability experiments direct effect

[0159] Protein concentrations were adjusted to account for variations caused by the plate effect. Estimated Marginal Means (EMM) were calculated and utilized for statistical analysis. One-way ANOVA was used to identify significant differences in iron uptake among groups. Following the ANOVA, multiple comparisons were performed using the Least Significant Difference (LSD) method, without correction for multiple comparisons.

[0160] In vitro fermentation experiments

[0161] SCFA analyses consisted of measuring the concentrations of three different acids: acetic acid, propionic acid, and butyric acid. To account for variations due to differences in microbiota composition (fermentation capacity) among donors, estimated marginal means (EMM) were calculated. This statistical method provides adjusted means for different groups in a study, taking into account other variables in the model. By adjusting for these variables, estimated marginal means offer a clearer comparison between groups. In our case, they help isolate the effect of non-digestible oligosaccharides on SCFA production.

[0162] One-way ANOVA was used to detect significant differences among groups in SCFA production after fermentation with the NDO of interest. Following the ANOVA, multiple comparisons were performed using the Least Significant Difference (LSD) method, without correction for multiple comparisons.

[0163] Results

[0164] Results of direct effect of the NDOs on ferritin levels are shown in table 2.

[0165] The increased ferritin level upon direct contact with the NDO (direct uptake), which is representative for small intestinal uptake, is increased in the presence of NDO compared to control, but only significantly higher with the two mixtures comprising inulin: IcFOS. The ferritin level, hence iron absorption and bioavailability is higher than expected based on IcFOS and inulin alone for both mixtures. Prior art mixtures of GOS:lcFOS and inulimscFOS showed a lower uptake and were not significantly different from the control. Results are shown in Table 2.

[0166] Table 2: Ferritin levels (ng / mg) in cells after exposure to NDO and iron

[0167] *Values with letters in the superscript are not significantly different from each. The uptake iron absorption / bioavailability in the presence of the SCFA as formed upon fermentation, which is indicative of uptake in the colon, is increased significantly with N DO when compared to the blank, though higher with the NDO mixtures. Compared to the single components the mixtures with in ulin :lcFOS showed a significantly higher uptake. The mixture of inulimscFOS on the other hand was not significantly different from the scFOS. Results are shown in Table 3.

[0168] This correlates with the SCFA formed upon fermentation, shown in Table 4. Fermenting the mixes of NDO by microbiota results in a significantly higher SCFA production than expected based on the single components (table 3). Table 3. SCFA mediated uptake in cells after exposure to NDO and iron.

[0169] Table 4. Fermentation profile of different NDO.

[0170] These results are indicative that over the entire Gl tract the iron absorption or bioavailability is increased in an improved and synergistic way when the inulin mixture of the current invention is consumed, when compared to the ingredients alone or when compared to prior art NDO mixtures.

[0171] The mechanism by which mixes of inulin and IcFOS improve small intestinal iron absorption is unknown. It was therefore unexpected and surprising that inulin mixtures of the present invention directly improve the iron uptake in the intestinal cells [when compared to the single ingredients or prior art NDO mixtures].

[0172] For the colon iron uptake is known to increase with lower pH as most iron salts become more soluble and absorbable at lower pH. In this study, the SCFAs indeed lowered the digest's pH (6.5 - 5.5); however, it was found that the addition of SCFA to digest did not increase iron solubility, as FeSO4 has a very high solubility (>89%) at pH 6.5-5.5. As such, the pH effect alone cannot be the explanation of the increased iron bioavailability of FeSO4. A synergistic and further improved effect was found with the mixtures of the invention, and this is indicative for an improved iron uptake along the entire intestinal tract.

[0173] Example 2: Nutritional composition for young children

[0174] Nutritional composition in powder form. After reconstitution of 14.88 g powder with water to 100 ml to a ready to drink formula the iron biotic composition comprises per 100 ml:

[0175] 67.7 kcal

[0176] 1.03 g protein (cow's milk protein)

[0177] 2.7934 g lipids (mainly vegetable lipids)

[0178] 8.6673 g digestible carbohydrates (mainly lactose)

[0179] 0.8 g mixture of non-digestible saccharides, comprising based on total non-digestible saccharides o 50 wt% Inulin (Source OraftiGR, Oreye BE, Pemuco CL) number average DP about 10 o 50 wt % IcFOS (source Raftilin H P), number average DP 23 1.0 mg ferrous sulphate (Iron)

[0180] 9.8 mg Vitamin C

[0181] Further vitamins, minerals as known in the art.

[0182] Example 3: Iron-biotics nutritional supplement for pregnant women

[0183] Nutritional composition in powder form. 12.5 g powder is added to 100 ml of a liquid to provide a ready to drink composition that comprises per 100 ml:

[0184] 90 kcal

[0185] 8 g protein (cow's milk protein)

[0186] 0.4 g lipids (vegetable lipids)

[0187] 14.3 g digestible carbohydrates

[0188] 3.4 mg iron

[0189] 10 mg vitamin C

[0190] 2 g mixture of non-digestible saccharides / 100 kcal o 66 wt% inulin (Source OraftiGR, Oreye BE, Pemuco CL) number average DP about 10 o 33 wt% Ic FOS (source RaftilinHP), number average DP 23

[0191] Supplemented with vitamin A, B2, B12, D3, C, calcium, zinc and iodine

[0192] Example 4: Iron-biotics nutritional composition for young children

[0193] Nutritional composition in powder form. After reconstitution of 14.88 g powder with water to 100 ml to a ready to drink formula the composition comprises per 100 ml:

[0194] 67.7 kcal

[0195] 1.03 g protein (cow's milk protein)

[0196] 2.7934 g lipids (14,7 mg DHA)

[0197] 8.6673 g digestible carbohydrates (mainly lactose)

[0198] 0.8 g mixture of non-digestible saccharides, comprising based on total non-digestible saccharides o 50 wt% Inulin (Source OraftiGR, Oreye BE, Pemuco CL) number average DP about 10 o 50 wt % IcFOS (source Frutafix), number average DP 23

[0199] 1.0 mg ferrous sulphate (Iron)

[0200] 9.8 mg Vitamin C

[0201] Further vitamins, minerals as known in the art. Example 5. Fingerprint of mixture of inulins

[0202] A combination of inulin I (Source OraftiGR, Oreye BE, Pemuco CL) and inulin II (source RaftilinHP) were prepared in a 1 to 1 ratio and in a 2:1 ratio and using High-Performance Anion-Exchange Chromatography (HPAEC) followed by Pulsed Amperometric Detection (PAD), HPAEC-PAD chromatograms were prepared showing the DP distribution of the combinations.

[0203] The inulins were analyzed (Eurofins Food, Feed & Water Testing, Heereveen, The Netherlands) using high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) to determine their average degree of polymerization (DP). Samples were prepared by aqueous extraction and dilution, followed by direct injection into the HPAEC-PAD system under controlled conditions. Chromatographic separation was achieved using a sodium hydroxide and sodium acetate gradient, allowing resolution of mono- and oligosaccharides based on chain length.

[0204] Chromatograms obtained from the samples were compared with reference profiles of a commercial galacto-oligosaccharide (GOS) ingredient and a standard mixture of fructo-oligosaccharides (FOS) and inulin. The reference mixture included short-chain oligomers (Fm and GFn) and long-chain inulin fractions. Peak identities were annotated using known standards, including GF2 (kestose), GF3 (nystose), and F3 (trifructose), among others. GOS quantification was performed using internal specialty testing.

[0205] The degree of polymerization was inferred from the elution order and peak annotation, with higher DP compounds eluting later in the chromatogram.

[0206] It was found that a mixture of two types of inulins with Inulin I having a number average degree of polymerization (DPn) of 8 to 14 and inulin II with a number average DP of 20 to 25 in a ratio of 1 to 1 and in a ratio of 2 to 1 results in a bimodal DP distribution. The fingerprint profile of Inulin I (Figure 1 A) shows it contains predominantly short chains (DP 4-8), while Inulin II (Figure 1 B) is rich in longer chains (DP 15- 20). When combined, the resulting inulin combinations' fingerprint profile exhibits two distinct peaks in DP abundance, specifically at DP 4-5 and DP 14-15 for the 1:1 mixture (Figure 1 C) and at DP4, 5, 6 and 14 for the 2: 1 mixture (Figure ID). These bimodal distributions contrasts with a unimodal distribution centred around DP 16-17, which would be expected from a single inulin population with that average DP. The mixture is therefore compositionally and functionally distinct, as it maintains high abundances of both short and long chains rather than a single peak near the average.

Claims

34CLAIMS1. A combination of two types of inulin wherein the combination consists of- inulin I having a number average degree of polymerization (DPn) of 8 to 14, and- inulin II having a number average degree of polymerization of 20 to 25, wherein the weight ratio of inulin I : inulin II ranges from 3:1 to 1:1, preferably 2:1 to 1:1, and wherein the total content of inulin with DP8 and DP9 is preferably at most 8.0 %, more preferably at most 7.5 % and even more preferably at most 7.3 %.

2. The combination of two types of inulins according to claim 1 wherein the number average DPn of the combination of two types of inulin is 14 to 20, more preferably 15.5 to 18.

3. The combination of two types of inulin according to claims 1 and 2, wherein the total content of inulin with a DP between 6 - 10 is preferably 18 - 26 wt%, more preferably 19 - 25 wt%, even more preferably 20 - 24 wt% based on total weight of the combination of two types of inulin.

4. The combination of two types of inulin according to the preceding claims, wherein the total content of inulin with a DP of 2 to 9 is preferably at most 40 wt%, more preferably at most 35 wt%, even more preferably at most 30 wt% based on total weight of the combination of two types of inulin.

5. The combination of two types of inulin according to the preceding claims wherein the combination contains as most abundant molecules DP4, DP5 and DP14 and / or DP15 inulin, preferably wherein the combination of the two types of inulins has a DP profile wherein the total content of inulin with a DP4, DP5, DP14 an DP15 in the combination is preferably 13.5 to 19 wt%, more preferably 14 to 18.5 wt%, even more preferably 14.5 to 18 wt% based on total weight of the combination of the two types of inulins and, wherein preferably said combination of the two types of inulins has a total content of inulin with DP4 and DP5 of preferably 6.5 to 11 wt%, more preferably 7 to 10.5 wt%, even more preferably 7.5 to 10 wt% and a35 total content of inulin with DP14 and DP15 of preferably 5.5 to 9 wt%, more preferably 6 to 8.5 wt%, even more preferably 6.5 to 8 wt% based on total weight of the combination of the two types of inulins.

6. The combination of two types of inulin according to the preceding claims, wherein- the total content of inulin with a DP between 2 and 5 is preferably 8 - 17 wt%, more preferably 10 - 15 wt%, even more preferably 11 - 14 wt% based on total weight of the combination of two types of inulin and / or- the total content of inulin with a DP of 10 and higher is preferably 60 - 72 wt%, more preferably 62 - 70 wt%, even more preferably 63 - 69 wt% based on total weight of the combination of two types of inulin.

7. The combination of two types of inulin according to the preceding claims, wherein the polydispersity index of the combination of the two types of inulins is about 1.41 to 1.48.

8. A nutritional composition comprising the combination of two different types of inulin according to any one of claims 1 to 7.

9. Nutritional composition according to claim 8 wherein the nutritional composition comprises non- digestible oligosaccharides and wherein 90 wt% to 100 wt%, more preferably 95 wt% to 100wt% of the non-digestible oligosaccharides consist of the combination of two types of inulin.

10. Nutritional composition according to any one of claims 8 and 9 wherein the nutritional composition comprises the combination of two types of inulin in a total amount of0.1 to 2.0 g / 100 ml, preferably 0.2 to 1.8 g / 100 ml, even more preferably 0.4 to 1.6 g / 100ml and / or1.4 to 28 wt%, more preferably 2.8 to 25 wt%, even more preferably 5.6 to 22.5 wt% based on total dry weight of the nutritional composition, and / or0.14 to 3.3 g per 100 kcal, more preferably 0.3 g to 2.7 g per 100 kcal, even more preferably 0.6 to2.4 g per 100 kcal.

11. Nutritional composition according to any one of claims 8 to 10 wherein the nutritional composition is selected from an infant formula, young child formula, fortified milk, cereal composition, a food for special medical purpose and a dietary supplement.

12. Nutritional composition according to claim any one of claims 8 to 11 wherein the nutritional composition comprises iron and vitamin C, preferably 0.3 mg to 7.5 mg iron per 100 kcal and 1.85 mg to 33 mg vitamin C per 100 kcal.

13. Nutritional composition according to any one of claims 8 to 12 wherein the source of iron is selected from the group consisting of ferrous sulphate, ferrous lactate, ferrous gluconate, ferrous bisglycinate, ferrous citrate, ferrous fumarate, ferric diphosphate, ferric ammonium citrate, ferric pyrophosphate, ferric sodium EDTA, preferably ferrous sulphate, ferrous fumarate, ferric pyrophosphate and ferrous lactate, more preferably ferrous sulphate,.

14. Use of the nutritional composition according to any one of claims 8 to 13 for increasing and / or improving iron absorption, increasing and / or improving iron storage, increasing and / or improving iron bioaccessibility and / or increasing and / or improving iron bioavailability, in a human subject, comprising administering the nutritional composition to the human subject.

15. Use of the nutritional composition according to claim 14 wherein the human subject is selected from a human subject with an age of 0 to 36 months, a human subject above 36 months of age, preferably a child aged 3 to 12 years, a pregnant woman, lactating women, women of reproductive age that are menstruating, malnourished subjects and elderly subjects, preferably an infant or young child.

16. Combination of two types of inulin according to any one of claims 1 to 7 or the nutritional composition according to any one of claims 8 to 13 for use in increasing and / or improving iron absorption, increasing and / or improving iron storage, increasing and / or improving iron bioaccessibility and / or increasing and / or improving iron bioavailability.

17. Combination of two types of inulin according to any one of claims 1 to 7 or the nutritional composition according to any one of claims 8 to 13 for use in preventing and / or treating and / or reducing the risk of iron deficiency and / or anemia, preferably iron deficiency anemia.

18. Method of preparing a nutritional composition according to any one of claims 8 to 13, said method comprising the steps of d) Adding a type of inulin I having a number average degree of polymerization (DPn) of 8 to 14 to the supplement or nutritional composition and subsequently or concomitantly or beforehand e) Adding a type of inulin II having a number average DP of 20 to 25 to the supplement or nutritional composition and f) Mixing the two types of inulin with the nutritional composition.

19. Method according to claim 18 further comprising the step of d) adding ferrous sulphate and vitamin C, and optionally e) long chain polyunsaturated fatty acids selected from the group consisting of arachidonic acid, docosahexaenoic acid and eicosapentaenoic acid, to obtain a final nutritional composition20. Nutritional composition obtainable by the method according to any one of claims 17 to 19.

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