The invention relates to nutritional compositions comprising oligosaccharides and to the uses thereof. In particular, it relates to

compositions comprising complex human milk oligosaccharides (HMO) and use thereof as infant food or dietary supplement.

Exclusive breast feeding during the first six months after birth is officially recommended by the WHO and efforts are made to support and promote breast feeding amongst mothers worldwide. Important components of breast milk are human milk oligosaccharides (HMOs), present to 3-19 g/L. They are composed of a core of galactose, glucose, N-acetyl-glucosamine and decorated with fucose and sialic acid to different extents. Numerous studies have pointed out the biological importance of HMOs, e.g. their role in inhibiting the adhesion of pathogenic bacteria to the epithelial surface or the establishment of gut microbiota. The HMO-composition of breast milk cannot be generalized though, as it is genetically determined. The mother's Secretor- and Lewis type determine the fucosylation pattern and thus the set of HMOs present in breast milk. Breast milks from Le(a-b+)-secretors, Le(a+b-)-non- secretors and Le(a-b-)-secretors/-non-secretors can be distinguished and have been thoroughly studied in view of their structural composition as well as their development during different stages of lactation. Differences in HMO-profiles may have an influence on the biological functioning of breast milk.

HMOs are synthesized in the mammary glands and can be further decorated by glycosyltransferases with fucose and sialic acid. Oligosaccharides in human milk can thus be divided into a neutral and an acidic fraction, with acidic oligosaccharides being present in a tenfold lower concentration than neutral oligosaccharides. The structural composition of HMOs has been extensively studied, leading to a well-defined picture on the glycobiology of oligosaccharides in human milk. Table 1 gives an overview on the structural composition of fucosylated and sialylated HMOs up to a core structure of six sugar units, as reviewed by Urashima et al. (Comprehensive glycoscience. From chemistry to system biology; vol.4; Kamerling, J.P., Boons, G.J., Lee, Y.C., Suzuki, A., Taniguchi, N.,Voragen, A.G.J, Eds; Elsevier: Oxford, UK, 2007; pp 695-722)

Table 1. Fucosylation- and sialidation of HMO-core structures and their

concentration in Le(a-b+)-, Le(a+b-)- and Le(a-b-)-breast milk, [lactose (L), lacto-N- tetraose (LNT), lacto-N-neo-tetraose (LNnT), [para] -lacto-N-hexaose (\para-]LNH) and [p r -]lacto-N-neo-hexaose ([para-]LNnH)]. Fuc: fucose; Neu5Ac: sialic acid, nd: not determined, low concentration is expected.

The presence and abundance of HMOs in breast milk is genetically

determined. See Thurl et al. Glycocorijugate J., 1997, 14, 795-799; Oriol et al. Glycobiology, 1999, 9, 323-334.; Oriol et al. Vox Sang., 1986, 51, 161-171.; Erney et al. J. Pediatr. Gastr. Nutr., 2000, 30, 181-192. The (ctl,2)- fucosyltransferase (FUT2) and (al,3/4)-fucosyltransferases (FUT3, 4, 5, 6, 7, 9), which are responsible for the fucosylation of the HMOs in the mammary glands, correspond to the fucosyltransferases responsible for fucosylation of other body glycoproteins (e.g. on erythrocytes and mucins). The expression of FUT2 and FUT3 depends on the maternal Secretor- and Lewis type, respectively. Accordingly, human milk can be classified into four groups:

1) Human milk from Le(a-b+)-secretors contains (al,2)-, (al,3)- and (al,4)- fucosylated oligosaccharides, which accounts for 70% - 80% of the European population.

2) Human milk from Le(a+b-)-non-secretors contains (al, 3)- and (al,4)- fucosylated oligosaccharides, but lacks (al,2)-fucosylated

oligosaccharides. This is found for approximately 20% of the European population.

3) Le(a-b-)-secretor-milk contains (al,2)- and (al, 3)-fucosylated

oligosaccharides and does not contain (al,4)-fucosylated

oligosaccharides. The occurrence of the Le(a-b-)-secretor status is only approximately 10%, as was determined for the French population.

4) Le(a-b-)-non-secretor-milk is composed of (al,3)-fucosylated

oligosaccharides, but lacks (al,2)- as well as (al,4)-fucosylated oligosaccharides. The occurrence of this milk type was estimated as rather low (1%, as was determined for the French population). The concentration of HMOs in breast milk of Le(a-b+)- Le(a+b-)- and Le(a-b-)- mothers is indicated in Table 1.

Breast-feeding is connected to a lowered risk of inflammatory bowel diseases and gastrointestinal, respiratory and urinary infections in the postnatal period. The health-beneficial characteristics of breast milk are mainly connected to the complex HMO-structures, which were also proposed to be involved in the development of the immune system (Klein et al. In Short and long term effects of breast feeding on child health; Koletzko, B., Michaelsen, K.F.,Hernell, 0., Eds; Kluwer Academic / Plenum Publ: New York, 2000; pp 251-259). Even an influence on brain development has been proposed (Wang et al. Am. J. Clin. Nutr., 2003, 78, 1024-1029) Direct pathogen inhibition and bifidogenicity are well-studied examples of health-related structure-function relationships of HMOs.

The intestinal mucosa is the largest surface of the human body and it is among the most heavily glycosylated tissues. The mucosa of the intestine is covered with complex glycans, including glycoproteins, glycolipids, mucins, and others. The principal function of these glycans is thought to be the mediation of communication with the extracellular environment, including cell-cell communication, molecular discrimination, barrier functions, and diverse signalling actions. To overcome this barrier, the first step of bacterial and viral infection is to recognize and bind specific cell surface glycans of the intestinal mucosa, where sialylated and fucosylated oligosaccharides are the primary targets. Because many milk oligosaccharides contain structural units that are homologous to these carbohydrate structures, it has been suggested that they act as soluble receptor analogs that inhibit the adhesion of pathogens, thus preventing infection. In fact, HMO are synthesized by the same glycosyl- and fucosyltranferases, enzymes responsible for the formation of glycans present on different cell types. Fucosylated and sialylated milk oligosaccharides inhibit the binding of pathogenic bacteria by blocking bacteria from attaching to target oligosaccharides on the intestinal mucosal surface. Milk

oligosaccharides, especially those containing alphal-2-linked fucose, have adhesion-inhibiting activity for both Gram-negative and Gram-positive bacteria. HMOs can act as receptor analogs for preventing the adhesion of pathogenic bacteria to the mucosal surface. This ligand-receptor mechanism is based on the structural characteristics of HMOs, which are complementary to the structures of the carbohydrate epitopes in the mucosa. In, vitro

experiments showed that sialylated HMOs prevent the adhesion of S- fimbriated enteropathogenic E.coli and Influenza A virus. Special attention has to be paid to (al,2)-fucosylated HMOs, which showed inhibition of

Campylobacter, norovirus and toxin-producing E-coli in, vitro and a lower incidence of diarrhea in breast-fed infants in, vivo. Babies who consumed milk rich in (al,2)-fucosylated HMOs showed less incidence for diarrhea than babies, who received milk with low (al,2)-fucosylated HMO-levels. There is still insufficient knowledge in order to judge different types of breast milks on their functional value.

The microbiota of breast-fed infants is described as "simple", with a considerable contribution of Bifidobacteria. Bifidobacteria are known to inhibit the growth of pathogens by short chain fatty acid production and to stimulate the immune response, cholesterol assimilation and the synthesis of vitamins. Infant-specific Bifidobacterium species exhibit cellular transporter systems for intact HMOs or exhibit extracellular enzymes necessary to degrade HMOs, such as sialidase, (al,2)-/(al, 3/4)-fucosidase and lacto-N-biosidase- phosphorylase.

Prebiotic oligosaccharide-supplementation to infant formula aims at simulating the health-promoting effect of HMOs. Due to the complexity of HMOs, their synthesis or extraction cannot be performed on a production scale to date. In view of the high galactose content in human milk and the lactose- based structural composition of HMOs, galactooligosaccharides (GOS) are frequently used for the supplementation of infant formula. In addition, long- chain fructooligosaccharides (FOS) are nowadays added to infant formula in order to simulate the higher molecular weight oligosaccharide fraction of human milk.

In vitro- and in vivo studies with prebiotic GOS and FOS demonstrated a specific stimulation of health-beneficial gut bacteria, although structurally different oligosaccharide mixtures were used in the respective studies.

Correspondingly, the presence and expression of saccharidases (6- galactosidases and β-fructofuranosidases) from health-beneficial bacteria and their mode of action has been described. Similar to HMOs, anti-adhesive functions towards an enteropathogenic E.coli-stra. were found for GOS in vitro. For the application in infant formula, a mixture of GOS : FOS (9: 1) has been used (Boehm et al., Arch. Dis. Child., 2002, 86, 178-181). This proportioning emulates the molecular size distribution of HMOs in breast milk. Furthermore, a synergistic effect of GOS and FOS on the stimulation of health-beneficial colonic bacteria was proposed. The FOS added to the infant formula have a size of DP > 10. Feeding infant formula supplemented with a combination of GOS and FOS resulted in the stimulation of bifidobacterial growth and was accompanied by increased stool frequency, soft stools and decreased fecal pH. The health-promoting effects observed were dose- dependent, with 0.8 g/dL supplementation being considered as optimal and approved as safe by the EFSA authority.

The supplementation of infant formula with GOS and/or FOS neglects the presence of acidic oligosaccharides in human milk. These may contribute to the gastrointestinal defense of pathogenic bacteria and to the systemic effects proposed for HMOs. So far, synthetically produced, sialylated oligosaccharides are not suitable for the application in food. Instead, acidic oligosaccharides from pectin (AOS) are being considered for their potential use in infant formula. Pectin, which is a plant cell wall polysaccharide contains acidic polygalacturonan-chains. AOS are obtained by enzymatic hydrolysis and are renowned for their curative functioning for diarrhea, which may be related to the anti-adhesive effects found for these oligosaccharides towards E. coli. The use of non-digestible oligosaccharides in infant foods is for example disclosed in WO2007/067053, WO2005/039597, WO01/642255, US 6,576,251, WO 99/11773, WO2005/055944, WO2007/105945, EP1629850 and

WO2011/008086.

The ample amount of available literature underscores the ongoing efforts made to further improve nutritional oligosaccharide compositions, especially with the aim to enhance infant health and development. The present inventors surprisingly found that a combination of complex HMOs with less complex oligosaccharides leads to unexpected health effects. More in particular, it was observed that the admixture of specific additives, such as prebiotic oligosaccharides, maltodextrin or lactoferrin, delays gastrointestinal metabolization of ocl,2-fucosylated HMOs. Herewith, the efficacy of these beneficial milk saccharides is enhanced. Interestingly, delayed gastrointestinal metabolization of ocl,2-fucosylated HMOs stimulated the body weight gain in preterm infants. Furthermore, the presence of HMOs was found to enhance the prebiotic action of admixed prebiotics. These findings open up new applications of HMOs and known prebiotics. For example, the protective effect of the additive allows for the supplementation with lower concentrations of (synthetic) complex HMOs while still achieving an effect similar to breast milk.

Accordingly, in one embodiment the invention provides a nutritional composition comprising (i) at least one ocl,2-fucosylated human milk

oligosaccharide (HMO) comprising at least four monosaccharide-units and (ii) at least one protective compound selected from the group consisting of prebiotics, maltodextrin and lactoferrin. Typically, prebiotics are

carbohydrates (such as oligosaccharides), but the definition may include non- carbohydrates. As used herein, the term prebiotic refers to a non-digestible food ingredient that beneficially affects the host, by stimulating the growth and/or activity of bacteria in the digestive system e.g. by selectively

stimulating the growth and/or activity of one or a limited number of bacteria in the colon. Prebiotics are well known in the art. Prebiotics suitable for use in infant formula are preferred. Preferably, the prebiotic is a (mixture of) oligosaccharides which are essentially not digested in the upper intestinal tract and thus reach the colon intact to stimulate the growth of beneficial bacteria in the colon, providing several health benefits. The prebiotic definition does not emphasize a specific bacterial group. Generally, however, it is assumed that a prebiotic should increase the number and/or activity of bifidobacteria and lactic acid bacteria. The importance of the bifidobacteria and the lactic acid bacteria is that these groups of bacteria have several beneficial effects on the host, especially in terms of improving digestion (including enhancing mineral absorption) and the effectiveness and intrinsic strength of the immune system.

Exemplary prebiotic oligosaccharides for use in a composition of the invention include galacto-oligosaccharides (GOS), fructose-oligosaccharides (FOS) and GOS/FOS mixtures. GOS prepared by trans-galactosylation, also referred to as trans- galacto-oligosaccharides or TOS, is particularly preferred. Preferably, GOS has a polymerization degree (DP) of 2 to 10. Suitable GOS preparations are commercially available. For example, Vivinal GOS made by the company FrieslandCampina DOMO is a prebiotic ingredient rich in trans- galacto-oligosaccharides. Suitable FOS preparations are those having a polymerization degree (DP) of 2 to 60, preferably 10 to 60. Mixtures of FOS and GOS, preferably in a 1:9 weight ratio, are also envisaged.

Another additive found to have beneficial effects in combination with a ocl,2-fucosylated HMO is maltodextrin. Maltodextrin is an oligosaccharide mixture that is used as a food additive. It is produced from starch by partial hydrolysis and digested and absorbed in the small gastrointestinal tract, although it is not clear whether if digestion is complete in the immature gastrointestinal tract of an (pre)term infant. As shown herein below, a mixed diet of HMOs and maltodextrin yielded increased counts of beneficial lactobacilli in preterm infants as compared to maltodextrin-based infant formula alone. As yet another example, the protective compound is a non-carbohydrate component like lactoferrin. Lactoferrin (LF), also known as lactotransferrin (LTF), is a multifunctional protein of the transferrin family. Lactoferrin is a globular glycoprotein with a molecular mass of about 80 kDa. Besides its antibacterial, antifungal and antiviral effects, whey-derived lactoferrin was shown to support the growth of bifidobacteria and lactobacillus, thus also indicating prebiotic properties (Harper WJ. Biological properties of whey components: a review. Chicago, IL: The American Dairy Products Institute; 2000).

A nutritional composition of the invention is not disclosed or suggested in the art. WO2011/008086 only relates to simple fucosylated oligosaccharide 2'FL and 3'FL, and is silent about more complex HMOs comprising more than three monosaccharide units. US 6, 576,251 discloses carbohydrate mixtures comprising a complex fucosylated structure and GOS, yet fails to teach a preference for oc-l, 2-fucosyl-containing HMOs.

Suitable ocl,2-fucosylated HMOs for use in a nutritional composition of the invention include those found in human breast milk such as

difucosyllactose (DF-L), lacto-N-fucopentaose I (LNFP I), lacto-N- difucohexaose I (LNDFH I), fucosyllacto-N-hexaose I (F-LNH I), difucosyllacto- N-hexaose I (DF-LNH I), trifucosyllacto-N-hexaose I (TF-LNH I), fucosyl-lacto- N-neohexaose I or II (F-LNnH II II) and trifucosyl-para-lacto-N-hexaose I (TF- para-LN I). The more predominant HMOs in milk such as LNFP I, LNDFH I and DF-LNH I (see Table 1, last column) are of particular interest.

In one embodiment, the ocl,2-fucosylated HMO has four monosaccharide units, like DF-L. In another embodiment, the structure has five or more monosaccharide units, like LNFP I. In another embodiment, the ocl,2- fucosylated HMO has six or more monosaccharide units like LNDFH I. In yet another embodiment, the HMO has seven monosaccharide units, such as F- LNH I or F-LNnH I or II. In still a further embodiment, the HMO has eight or more monosaccharide units, for example DF-LNH I. An exemplary structure consisting of nine monosaccharide units is TF-para-LNH I.

In one aspect, the HMO is a monofucosylated compound, preferably selected from the group consisting of LNFP I, F-LNH I and F-LNnH I/II. In another aspect, the HMO is a difucosylated compound, preferably selected from the group consisting of DF-L, LNDFH I and DF-LNH I. In yet another aspect, the HMO is a trifucosylated compound, preferably TF-LNH or TF-para- LNH I.

As is clear from Table 1, the HMO can be based on different core structures, for example lactose, LNT, LNnT, LNH, LNnH, para-LNH or para- LNnH. Lacto-N-biose is considered as bifidogenic component of breast milk . Therefore, the ocl,2-fucosylated complex HMO preferably comprises at least one lacto-N-biose unit, preferably as present in lacto-N-tetraose (LNT).

Preferred LNT -containing ccl,2-fucosylated HMOs are LNFP I and LNDFH I. Interestingly, LNDFH I was also found to be associated with enhanced body weight gain in preterm infants. Also preferred is F-LNH I.

An oc-l,2-fucosylated HMO comprising at least four monosaccharide-units can be obtained from various sources. Commercial suppliers include Dextra, Reading, UK. In one embodiment, it is prepared (semi)-synthetically.

HMOs can be derived using any number of sources and methods known to those of skill in the art. For example, HMOs can be purified from human milk using methods known in the art. One such method for extraction of

oligosaccharides from pooled mother's milk entails the centrifugation of milk at 5,000*g for 30 minutes at 4°C and fat removal. Ethanol may then be added to precipitate proteins. After centrifugation to sediment precipitated protein, the resulting solvent is collected and dried by rotary evaporation. The resulting material may be adjusted to the appropriate pH of 6.8 with phosphate buffer and [beta]-galactosidase is added. After incubation, the solution is extracted with chloroform-methanol, and the aqueous layer is collected. Monosaccharides and disaccharides are removed by selective adsorption of HMOs using solid phase extraction with graphitized nonporous carbon cartridges. The retained oligosaccharides can be eluted with water- acetonitrile (60:40) with 0.01% trifluoroacetic acid. Individual HMOs can be further separated using methods known in the art such as capillary

electrophoresis, HPLC (e.g., high-performance anion-exchange

chromatography with pulsed amperometric detection; HPAEC-PAD), and thin layer chromatography. See, e.g., U.S. Patent Application No. 2009/0098240.

Alternately, enzymatic methods can be used to synthesize HMOs. See for example EP 1637611 and WO2010/070104. In general, any oligosaccharide biosynthetic enzyme or catabolic enzyme (with the reaction running in reverse) that converts a substrate into any of the HMO structures (or their

intermediates) may be used to prepare the HMOs. Another approach to the synthesis of the HMOs entails the chemical or enzymatic synthesis of or isolation of oligosaccharide backbones containing Lacto-N-biose, or Lacto-N- tretrose from non-human mammalian milk sources (e.g., cows, sheep, buffalo, goat, etc.) and enzymatically adding Lacto-N-biose, Fucose and Sialic Acid units as necessary to arrive at HMO structures. Examples of such

oligosaccharide modifying enzymes include sialidases, silate O-Acetylesterases, N-Acetylneuraminate lyases, N-acetyl-beta-hexosaminidase, beta- galactosidases, N-acetylmannosamine-6-phosphate 2-epimerases, alpha-L- fucosidases, and fucose dissimilation pathway proteins, among others, which may be used to catalyze a biosynthetic reaction under the appropriate conditions. Alternatively, conventional chemical methods may be used for the de novo organic synthesis of or conversion of pre-existing oligosaccharides into HMO structures. These techniques for synthesizing HMOs are described in more detail in U.S. Patent Application No. 2009/0098240.

In one embodiment, HMOs for use in the present invention, or precursors thereof, are obtained from non-human mammalian milk. Fucose-al, 2-lactose may be produced in large scale quantities by recombinant E. coli which utilize enzymes FKP (fucokinase pyrophosphorylase) and WbsJ (al, 2- fucoslytransferase) to enzymatically synthesize fucose-al, 2-lactose. FKP is a bifunctional enzyme which catalyzes the reaction between fucose, ATP, and GTP to produce the expensive and hard to obtain GDP-fucose, meanwhile WbsJ is a known al, 2-FucT which demonstrates promiscuous acceptor substrate specificity. This method is cost effective by regenerating the sugar nucleotides required for the synthesis and require only inexpensive,

commercially available, fucose and lactose.

Whey streams have the potential to be commercially viable sources of complex oligosaccharides that have the structural resemblance and diversity of the bioactive oligosaccharides in human milk.

In a specific aspect, a nutritional composition of the invention solely contains ocl,2-fucosylated HMO's obtained from human milk. For example, it is human breast milk obtained from a Le(a-b+) or Le(a-b-)-secretor-type donor subject and supplemented with at least one HMO -protective compound selected from the group consisting of prebiotic oligosaccharides, maltodextrin and lactoferrin.

The relative mixing ratio between (i) ocl,2-fucosyl HMO and (ii) protective compound(s) can vary, depending on type of HMO and/or protective compound. For example, the ratio (i) to (ii) is between about 10: 1 and 1:200 by weight, for example between 10: 1 and 1:200. In one embodiment, the protective compound is present in excess of the HMO. For instance, 4 g/L GOS or maltodextrin was found to delay the metabolisation of 1.2 g/L ocl,2-fucosyl HMO. In another embodiment, the protective compound is present in about equal amounts as the HMO. It is also possible to achieve protection with lower amounts of protective compounds. For example, 1 g/L lactoferrin was found to significantly protect >1 g/L ocl,2-fucosyl HMO.

The absolute concentration of ocl,2-fucosyl HMO and protective compound can vary likewise. In one embodiment, the composition comprises 0.02 to 10 wt% oc- 1,2-fucosylated HMO having at least four monosaccharide-units and/or 0.25 wt% to 15wt% protective compound based on the dry weight of the

composition. In one embodiment, the ocl,2-fucosylated HMO concentration mimics the concentration found in breast milk. The total concentration of oc- 1,2-fucosylated HMOs in breast milk is about 6-9 g/L.

Typical compositions comprising GOS and/or FOS as protective compound contain 0.4 to 0.8 g/dL of GOS and/or FOS in total. Maltodextrin is also suitably used as protective compound at 0.4 to 0.8 g/dL. Lactoferrin can be used at 0.05 to 0.2 g/dL, preferably about 0.1 g/dL. The skilled person will be able to determine, e.g. by HMO profiling of feces, whether a given type and amount of protective compound exerts a desired effect.

A nutritional composition may comprise one or more further

nutritionally useful additives. In one embodiment, the additive(s) is a compound found in human milk, preferably a HMO, more preferably a HMO selected from the group consisting of sialyllactose, fucosyllactose, di-sialylated oligosaccharides, lacto-N-neo-tetraose (LNnT), lacto-N-tetraose (LNT), lacto-N- fucopentaose (LNFP) -isomers, lacto-N-difucohexaose (LNDFH) -isomers, fucosyllacto-N-hexaose (F-LNH) -isomers, difucosyllacto-N-hexaose (DF-LNH)- isomers and trifucosyllacto-N-hexaose (TF-LNH) -isomers. The sialyllactose may originate from bovine milk.

In one embodiment, the nutritional composition further comprises a protein source, a lipid source and a carbohydrate source, preferably in amounts suitable to provide nutrition to an infant. Many different sources and types of carbohydrates, lipids, proteins, minerals and vitamins are known and can be used in the nutritional formulas of the present disclosure, provided that such nutrients are compatible with the added ingredients in the selected formula, are safe for their intended use, and do not otherwise unduly impair product performance.

Carbohydrates suitable for use in the nutritional formulas of the present disclosure can be simple, complex, or variations or combinations thereof. Non- limiting examples of suitable carbohydrates include hydrolyzed, intact, naturally and/or chemically modified cornstarch, glucose polymers, sucrose, corn syrup, corn syrup solids, rice or potato derived carbohydrate, glucose, fructose, lactose, high fructose corn syrup and combinations thereof. Preferably carbohydrate sources contribute between 35 and 65% of the total energy of the formula.

Non-limiting examples of lipids suitable for use in the nutritional formulas include coconut oil, soy oil, corn oil, olive oil, safflower oil, high oleic safflower oil, MCT oil (medium chain triglycerides), sunflower oil, high oleic sunflower oil, palm and palm kernel oils, palm olein, canola oil, marine oils, cottonseed oils, long-chain polyunsaturated fatty acids such as arachidonic acid (ARA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), and combinations thereof.

The lipid source is preferably a lipid or fat which is suitable for use in infant formulas. Preferred fat sources include palm olein, high oleic sunflower oil and high oleic safflower oil. In total, the fat content is preferably such as to contribute between 30 to 55% of the total energy of the composition. The fat source preferably has a ratio of n-6 to n-3 fatty acids of about 5: 1 to about 15: 1; for example about 8: 1 to about 10: 1.

In addition to these food grade oils, structured lipids may be

incorporated into the nutritional formulas if desired. Structured lipids are known in the art, descriptions of which can be found in INFORM, Vol. 8, no. 10, page 1004, Structured lipids allow fat tailoring (October 1997); and US 4,871, 768. Structured lipids are predominantly triacylglycerols containing mixtures of medium and long chain fatty acids on the same glycerol nucleus. Structured lipids are also described in US 6, 160,007.

Non-limiting examples of proteins suitable for use in the nutritional formulas include extensively hydrolyzed, partially hydrolyzed or non- hydrolyzed proteins or protein sources, and can be derived from any known or otherwise suitable source such as milk (e.g., casein, whey), animal (e.g., meat, fish), cereal (e.g., rice, corn), vegetable (e.g., soy), or combinations thereof. It may be desirable to supply partially hydrolyzed proteins (degree of hydrolysis between 2 and 20%), for example for infants believed to be at risk of developing cows' milk allergy. If hydrolyzed proteins are required, the hydrolysis process may be carried out as desired and as is known in the art. For example, a whey protein hydrolysate may be prepared by enzymatically hydrolyzing the whey fraction in one or more steps. The proteins for use herein can also include, or be entirely or partially replaced by, free amino acids known for use in nutritional formulas, non-limiting examples of which include tryptophan, glutamine, tyrosine, methionine, cysteine, arginine, and combinations thereof. Other (nonprotein) amino acids typically added to nutritional formulas include carnitine and taurine. In some cases, the D-forms of the amino acids are considered as nutritionally equivalent to the L-forms, and isomer mixtures are used to lower cost (for example, D,L-methionine).

The nutritional composition of the present disclosure may further comprise any of a variety of vitamins in addition to the components described above. Non-limiting examples of vitamins include vitamin A, vitamin D, vitamin E, vitamin K, thiamine, riboflavin, pyridoxine, vitamin B 12, niacin, folic acid, pantothenic acid, biotin, vitamin C, choline, chromium, carnitine, inositol, salts and derivatives thereof, and combinations thereof. The nutritional composition may further comprise any of a variety of minerals, non-limiting examples of which include calcium, phosphorus, magnesium, iron, zinc, manganese, copper, iodine, sodium, potassium, chloride, and combinations thereof.

The infant formula embodiments of the present disclosure preferably comprise nutrients in accordance with the relevant infant formula guidelines for the targeted consumer or user population. The composition can be a children's food, liquid, semi-liquid or solid, especially for children between 0 and 7 years, or between 0 and 3 years. The composition of the invention can also be targeted to adolescent and adults suffering from particular physio-pathological conditions, especially those in need and/or having compromised gastrointestinal systems and/or compromised immune / defense systems.

The nutritional composition may further comprise other optional components that may modify the physical, chemical, aesthetic or processing characteristics of the formulas or serve as pharmaceutical or additional nutritional components when used in the targeted population. Many such optional ingredients are known or other suitable for use in food and nutritional products, including infant formulas, and may also be used in the nutritional formulas of the present disclosure, provided that such optional materials are compatible with the essential materials described herein, are safe for their intended use, and do not otherwise unduly impair product performance.

Non-limiting examples of such optional ingredients include preservatives, anti- oxidants, emulsifying agents, buffers, colorants, flavors, nucleotides, and nucleosides, probiotics, additional prebiotics, and related derivatives, thickening agents and stabilizers, and so forth.

The probiotic can be present in the composition in an amount equivalent to between 10 ³ and 10 ¹² cfu/g of dry composition. Preferably the probiotic is present in an amount equivalent to between 10 ⁷ to 10 ¹² cfu/ g of dry

composition. The bacteria may be used live, inactivated or dead or even be present as fragments such as DNA or cell wall materials. In other words, the quantity of bacteria which the formula contains is expressed in terms of the equivalent colony forming units of bacteria irrespective of whether they are, all or partly, live, inactivated, dead or fragmented

The probiotic bacterial strain may be any lactic acid bacteria or

Bifidobacteria with established probiotic characteristics. The probiotic of the invention may be any probiotic bacterium or probiotic microorganism

("probiotics"), especially of human origin, in particular probiotics that have been or can be originated from, found in, extracted or isolated from milk upon excretion, preferably in human breast milk. Suitable probiotic lactic acid bacteria include Lactobacillus rhamnosus ATCC 53103 obtainable inter alia from Valio Oy of Finland under the trademark LGG , Lactobacillus rhamnosus CGMCC 1 .3724, Lactobacillus reuteri ATCC 55730 and Lactobacillus reuteri DSM 17938 obtainable from Biogaia, Lactobacillus fermentum VRI 003 and Lactobacillus paracasei CNCM 1-21 16, Lactobacillus johnsonii CNCM I- 1225, Lactobacillus helveticus CNCM 1-4095, Bifidobacterium breve CNCM 1-3865, Bifidobacterium longum CNCM 1-2618

Suitable probiotic Bifidobacteria strains include Bifidobacterium longum ATCC BAA- 999 sold by Morinaga Milk Industry Co. Ltd . of Japan under the trade mark BB536, the strain of Bifidobacterium breve sold by Danisco under the trade mark Bb-03, the strain of Bifidobacterium breve sold by Morinaga under the trade mark M-16V and the strain of Bifidobacterium breve sold by Institut Rosell (Lallemand) under the trade mark R0070. A particularly preferred Bifidobacterium strain is Bifidobacterium lactis CNCM 1-3446 which may be obtained from the Christian Hansen Company of

Denmark under the trade mark Bbl2. A mixture of suitable probiotic lactic acid bacteria and Bifidobacteria may be used. The nutritional composition of the present invention may be prepared as any product form suitable for use in humans, including liquid or powdered complete nutritionals, liquid or powdered supplements (such as a supplement that can be mixed with a drink), reconstitutable powders, ready-to-feed liquids, bars, and dilutable liquid concentrates, which product forms are all well known in the nutritional formula arts.

The nutritional composition may have any caloric density suitable for the targeted or intended population, or provide such a density upon

reconstitution of a powder embodiment or upon dilution of a liquid concentrate embodiment. Most common caloric densities for the infant formula

embodiments of the present disclosure are generally at least about 660 kcal/liter, more typically from about 675-680 kcal/liter to about 820 kcal/liter, even more typically from about 675-680 kcal/liter to about 800-810 kcal/liter. Generally, the 720-810 kcal/liter formulas are more commonly used in preterm or low birth weight infants, and the 675-680 to 700 kcal/liter formulas are more often used in term infants. Non-infant and adult nutritional formulas may have any caloric density suitable for the targeted or intended population.

The nutritional composition of the present disclosure may be packaged and sealed in single or multi-use containers, and then stored under ambient conditions for up to about 36 months or longer, more typically from about 12 to about 24 months. For multi-use containers, these packages can be opened and then covered for repeated use by the ultimate user, provided that the covered package is then stored under ambient conditions (e.g., avoid extreme temperatures) and the contents used within about one month or so.

A nutritional composition of the invention finds various uses in human nutrition and (preventive) therapy. In one embodiment, it is used for providing nutrition to an infant, preferably a preterm infant. In humans, preterm birth typically refers to the birth of a baby of less than 37 weeks gestational age. The cause for preterm birth is in many situations elusive and unknown; many factors appear to be associated with the development of preterm birth, making the reduction of preterm birth a challenging proposition. Preterm infants usually show physical signs of prematurity in reverse proportion to the gestational age. As a result, they are at risk for numerous medical problems affecting different organ systems. Respiratory problems are common, specifically the respiratory distress syndrome (RDS or IRDS). Gastrointestinal and metabolic issues can arise from hypoglycemia, feeding difficulties, rickets of prematurity, hypocalcemia, inguinal hernia, and necrotizing enterocolitis (NEC). Typical infections in preterms include sepsis, pneumonia, and urinary tract infection.

The presence of anti-pathogenic ocl,2-fucosylated HMO in a composition of the invention makes it particularly effective for use in a method for the prophylaxis and/or treatment of symptoms which are connected with the association and/or adhesion of a pathogenic substance to the epithelia. The method may comprise administering to a subject in need thereof the

composition in an amount of at least 100 mg/kg body weight per day, preferably at least 500 mg/kg body weight per day.

Also provided is the use of a synthetic infant formula comprising at least one HMO-protective compound selected from the group consisting of prebiotic oligosaccharides, maltodextrin and lactoferrin, as a dietary supplement for an infant receiving breast milk. For example, the synthetic formula is used in amount to make up between about 5 and 40% of the total diet of the infant based on the total volume of breast milk and infant formula. This approach is advantageously used for the nutrition of a preterm infant, e.g. in a hospital setting. A further aspect related to the use of a synthetic infant formula comprising at least one compound selected from the group consisting of prebiotic oligosaccharides, maltodextrin and lactoferrin, to enhance a health- promoting effect of human milk oligosaccharide (HMO), preferably ocl,2- fucosylated HMO.

Preferred are ocl,2-fucosylated HMO comprisings at least four monosaccharide units, for example, wherein said ocl,2-fucosylated HMO is selected from the group consisting of difucosyllactose (DF-L), lacto-N- fucopentaose I (LNFP I), lacto-N-difucohexaose I (LNDFH I), fucosyllacto-N- hexaose I (F-LNH I), difucosyllacto-N-hexaose I (DF-LNH I), trifucosyllacto-N- hexaose I (TF-LNH I), fucosyllacto-N-neohexaose I or II (F-LNnH I / II) and trifucosyl-para-lacto-N-heaose I (TF-para-LNH I). In one embodiment, the ocl,2-fucosylated HMO has four monosaccharide units, like DF-L. In another embodiment, the structure has five or more monosaccharide units, like LNFP I. In another embodiment, the ocl,2-fucosylated HMO has six or more monosaccharide units like LNDFH I. In yet another embodiment, the HMO has seven monosaccharide units, such as F-LNH I or F-LNnH I or II. In still a further embodiment, the HMO has eight or more monosaccharide units, for example DF-LNH I. An exemplary structure consisting of nine monosaccharide units is TF-para-LNH I.

As said, the protective effect of prebiotics, maltodextrin or lactoferrin prolongs the presence of HMOs in the gut, thereby promoting e.g. their beneficial effects. Beneficial or health-promoting effects include body weight gain (of special importance for preterm infants), increased resistance against pathogenic bacteria and stimulation or establishment of beneficial gut flora.

Typically, establishing beneficial gut flora comprises populating the gut with bifidobacteria or lactobacilli or both. Enhanced growth of bifidobacteria and lactobacilli typically occurs at the expense of other groups of potentially harmful bacteria such as Clostridia, Enterobacteriaceae, and others. Therefore, a composition comprising of the invention can selectively feed the saccharolytic bacteria (bifidobacteria and lactobacilli), allowing them to dominate the gut and compete with potentially harmful bacteria by creating an acidic

environment that is less favorable to pathogens. In one specific embodiment, an infant formula comprising GOS (and/or FOS) and ocl,2-fucosylated HMO is used to enhance populating the gut with Bifidobacteria. For example, the synthetic infant formula comprises GOS and/or FOS, preferably in an amount of 0.4 to 0.8 g/dL. In another embodiment, an infant formula comprising maltodextrin, preferably in an amount of 0.4 to 0.8 g/dL, and ocl,2-fucosylated HMO is used to enhance populating the gut with Lactobacilli.

In a preferred aspect, the invention provides the use of a synthetic infant formula comprising at least one compound selected from the group consisting of prebiotic oligosaccharides, maltodextrin and lactoferrin, to enhance the effect of human ocl,2-fucosylated HMO comprising at least four monosaccharide units on body weight gain in an infant. The invention also provides the use of specific fucosylated HMOs, preferably LNT -based HMOs, more preferably LNDFH I and/or LNFP II, to promote body weight gain in an infant, preferably a preterm infant.

Further aspects of the invention relate to methods for enhancing a health-promoting effect of human milk oligosaccharide (HMO) in an infant, comprising administering to an infant a synthetic infant formula comprising at least one compound selected from the group consisting of prebiotic

oligosaccharides, maltodextrin and lactoferrin. Preferably, the HMO is an ocl,2-fucosylated HMO, more preferably an ocl,2-fucosylated HMO comprising at least four monosaccharide units. For example, said ocl,2-fucosylated HMO is selected from the group consisting of difucosyllactose (DF-L), lacto-N- fucopentaose I (LNFP I), lacto-N-difucohexaose I (LNDFH I), fucosyllacto-N- hexaose I (F-LNH I), difucosyllacto-N-hexaose I (DF-LNH I), trifucosyllacto-N- hexaose I (TF-LNH I), fucosyllacto-N-neohexaose I or II (F-LNnH I / II) and trifucosyl-para-lacto-N-heaose I (TF-para-LNH I). The method for instance comprises promoting body weight gain, increased resistance against

pathogenic bacteria, stimulation of beneficial gut flora, preferably of bifidobacteria and/or lactobacilli. Further therapeutic applications include enhancing or inducing tolerance e.g. tolerance to a given dietary antigen to prevent or treat a food allergy or food hypersensitivity. In one embodiment, the prebiotics, maltodextrin or lactoferrin are used to enhance prevention of allergy or food intolerance in infants.

In a preferred aspect, the method enhances body weight gain in an infant, in particular a premature infant. In one embodiment, the method comprises administering a synthetic infant formula comprising GOS and/or FOS, preferably in an amount of 0.4 to 0.8 g/dL. In another embodiment, the synthetic infant formula comprises maltodextrin, preferably in an amount of 0.4 to 0.8 g/dL. In yet another embodiment, the synthetic infant formula comprises lactoferrin, preferably in an amount of 0.05 to 0.2 g/dL. The method preferably also comprises feeding the infant with breast milk. The synthetic formula may be mixed with breast milk prior to administration.

LEGENDS TO THE FIGURES

Figure 1. CE-LIF electropherograms of oligosaccharides from feces of preterm babies who got (A) >60% breast feeding and infant formula supplemented with GOS (A6) or lactoferrin (A7, A10). (B) >60% breast feeding and maltodextrin- containing infant formula. (C/D) 100% breast feeding.

Figure 2. Relative quantification of LNT and fucosylated LNT -based HMOs (LNFP II; LNFP I[co-migrates with LNFP III]; LNDFH I) from feces of breast- and mixed-fed preterm babies. Quantification is based on CE-LIF data.

Figure 3. Comparison of fecal microbiota analysed by I-Chip in the feces of (A) preterm babies who got 100% infant formula supplemented with GOS and preterm babies who got >60% breast feeding and infant formula supplemented with GOS. (B) preterm babies who got 100% infant formula supplemented with lactoferrin and preterm babies who got >60% breast feeding and infant formula supplemented with lactoferrin. (C) preterm babies who got 100% infant formula containing maltodextrin and preterm babies who got >60% breast feeding and infant formula containing maltodextrin.

Figure 4. Correlation between fecal excretion of LNFP II and body weight gain of preterm babies.

Figure 5. Correlation between fecal excretion of LNDFH I and body weight gain of preterm babies. EXPERIMENTAL SECTION

The gastrointestinal fate of the complex HMOs remains vague. In the 1980s and early 1990s, several studies have been performed in order to investigate fecal oligosaccharides from breast-fed babies. Blood group A active

oligosaccharides were found in the feces of a single blood group A breast-fed baby and a gastrointestinal metabolization of the feeding-related HMOs was supposed. On the other hand, no HMO metabolization products were found for a single blood group B breast-fed baby. In a third study, no indication for gastrointestinal HMO-metabolization was found as HMO-profiles similar to the respective breast milks were observed. In other studies HMOs and blood group characteristic oligosaccharides were detected in urine of breast-fed babies and lactating women, pointing out their gastrointestinal absorbance and importance on a systemic level. Clearly, further research is needed in order to understand the gastrointestinal fate of complex HMOs and their possible conjugation with blood group antigenic structures. The application of novel analytical methods, which provide low detection limits and require limited preparative sample work in combination with a short analysis time, may open new possibilities in this context. It may help to establish an advanced scientific underpinning of the feeding-guidelines set up for neonates. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF), combined with mass spectrometry, has been shown to be a suitable tool for the analysis of HMOs in breast milk and baby feces. Materials and Methods

Set-up study and sample collection

Twenty-seven fecal samples of approximately two months old preterm infants (born after 27-35 weeks of gestation and a birth weight between 770 g and 2285 g) were selected from a clinical study on the investigation of the effect of the supplementation of preterm infant formula with prebiotic GOS or lactoferrin on the performance of preterm babies. The study was performed at the level III neonatal intensive care unit of the Isala clinics, Zwolle, The Netherlands. Enteral feeding of the babies was started after birth as soon as possible and full enteral feeding was established 5-19 days after birth. Fecal samples were taken 6 weeks after full enteral feeding was established and samples were frozen at -20°C until analysis.

The babies received either exclusively expressed human milk from their own mother (n = 5) or preterm infant formula (n = 6) [supplemented with 0.4 g/dL GOS (Frisolac prematuur, FrieslandCampina DOMO, Zwolle, The Netherlands), or without GOS (Frisolac prematuur, GOS replaced by maltodextrin (0.4 g/dL), or supplemented with 0.1 g/dL lactoferrin] . Sixteen babies got mixed feeding (breast milk and formula). Breast milk meals contributed to > 80%, > 60% or < 55% of the total number of feedings, which the babies received during the study period (Table 2). The babies were randomly assigned to one of the three formula groups. The study was performed double-blind and was approved by the medical ethical review board of the hospital. Written informed consent was obtained from all the parents. Except for baby A2, the blood group of the babies was known. Amongst the twenty-seven babies studied were two twin pairs and one triplet pair, but they were not further specified in this study. Table 2: Overview baby codes

Analysis of fecal oligosaccharide profiles (CE-LIF/CE-LIF-MS For CE-LIF analysis, the carbohydrates present in the fecal extracts were derivatized with the fluorescent 9-aminopyrene-l,4,6-trisulfonate (APTS) overnight at room temperature as reported elsewhere. ^{6 7} One nanomole xylose was added as internal standard and mobility marker. CE-LIF was performed on a ProteomeLab PA 800 characterization system (Beckman Coulter,

Fullerton, CA), equipped with a laser induced fluorescence detector (LIF) (excitation: 488 nm, emission: 520 nm) (Beckman Coulter) and a polyvinyl alcohol-coated capillary (50 μηι x 50.2 cm (Beckman Coulter), detector after 40 cm), kept at 25 °C. Samples were loaded hydrodynamically (4 s at 0.5 psi, representing approx. 14 nL sample solution) on the capillary. Separation was performed in the reversed polarity mode (30 kV, 20 min) in a 25 mM acetate buffer containing 0.4% polyethylene oxide provided in the ProteomeLab Carbohydrate Labeling and Analysis Kit (Beckman Coulter). Due to the low pK _a of the sialic acid residues (pK _a 2.6) and in order to provide the same buffer-pH as for CE-MS analysis, the separation buffer was adjusted to pH 2.4 by adding 1.2% (v/v) formic acid. ⁸ Peaks were integrated manually using Chromeleon software 6.8 (Dionex, Sunnyvale, CA).

CE-LIF-ESI-MS ⁿ experiments were performed on a P/ACE™ System MDQ (Beckman Coulter) according to Albrecht et al. ⁸ For the fluorescent detection (excitation: 488 nm, emission: 520 nm), a Picometrics ZetaLIF discovery system was used (Picometrics, Toulouse, France). Separation in 0.3% (v/v) formic acid (pH 2.4) was performed on a fused silica capillary (50 μιη x 85 cm (Beckman Coulter), capillary window fitted with an ellipsoid for LIF detection after 60 cm) in reversed polarity mode (20 kV, 15 °C, 40 min). Samples were injected at 10 psi for 2 s. LIF signals were sent to Beckmann 32Karat software via a SSXL4002 converter (Agilent Technologies, Santa Clara, CA). The ESI- MS (LTQ ion trap, Thermo Fisher Scientific Inc., Waltham, MA) was operated in the negative mode using a spray voltage of 1.9 kV and an MS-capillary temperature of 190 °C. The end of the CE capillary was installed in front of the ESI source by leading it through a T-part designed in our laboratory ⁷ and provided the coaxial addition of a sheath liquid (50/50 isopropanol/water) at 2 pL/min. Mass spectra were acquired from m/z 300 to 2000. MS ⁿ was performed in the data dependent mode using a window of 1 m/z and collision energy of 35%. For increasing the S/N ratio, ions of m/z 311, 314 and 329 were excluded from detection in MS ⁿ experiments (mass exclusion list). MS ⁿ data were interpreted using Xcalibur software 2.0.7 (Thermo).

Analysis of fecal microbiota (I-chip):

The composition of the microbiota was analyzed using the Intestinal-Chip (I- chip) as described before (Maathuis et al. (10) and Rose et al.(2010)) for which probe sequences of intestinal micro-organisms were selected via a literature survey. The I-Chip contains roughly 350 probes, some for phylogenetic groups (e.g. Actinomycetes), some for group-level detection (e.g. all Bifidobacterium species) and some for detection of individual species (e.g. Bifidobacterium longum). Some species and groups were covered by more than one probe and the hybridization to these multiple probes correlated very well. Taxonomic selection was confirmed and expanded based on massive parallel sequencing of 16S rDNA. For each species represented on the microarray a unique short oligonucleotide sequence from within the 16S rDNA was selected. Some species are represented by multiple (unique) sequences. Criteria for sequence selection, apart from being unique, included length and melting temperature. Short oligonucleotide sequences (approx. 20 nt) were used for which a one nucleotide mismatch already resulted in an absence (or very strong decrease) of signal after hybridization. For I-Chip analysis, DNA was isolated from luminal samples at the start and at the end of the long-term TIM-2

experiments. In short, DNA was isolated from 200 mg of faecal material using a commercial DNA isolation kit (Agowa(r), Germany) following the

manufacturer's instructions. This method is described in detail elsewhere (10; Rose et al., 2010). Subsequently the DNA was labeled and hybridized to arrays. After washing, the arrays were scanned and analyzed. Imagene 5.6 software (BioDiscovery, Marina del Rey, CA) was used for data analysis.

Signals were quantified by calculating the mean of all pixel values of each spot and calculating the local background around each spot. For each spot a signal to background ratio was calculated. For further analyses spots which had a minimal number of observations more than two times above its local background were selected.

Abbreviations HMOs

BGA: blood group A; DF-L: di-fucosyllactose; DF-LNH: difucosyl-lacto-N- hexaose; DS-LNT: disialyl-lacto-N-tetraose; FL: fucosyllactose; F-LNH: fucosyl- lacto-N-hexaose; GOS: galactooligosaccharides; HMO: human milk

oligosaccharides; Lf: lactoferrin; LNFP: lacto-N-fucopentaose (I, II, III, Y:

different isomers); LNDFH: lacto-N-difucosahexaose; LNT: lacto-N-tetraose; SL: sialyllactose; S-LNT: sialyl-lacto-N-tetraose (different isomers present); TF- LNH: trifucosyl-lacto-N-hexaose.

Results EXAMPLE 1: Prebiotics delay the gastrointestinal metabolization of HMOs

Figures 1A and IB show the profiles of feces of seven mixed-fed babies (2 months old). For five babies (=71%) it was observed that the HMO-profile is still present. For two babies (= 29%) both receiving control-formula: A12:

besides SL, no HMOs present; A3: exceptional HMO-profile present (because of the abundant presence of LNT).

As a comparison, feces of five 100% HMO-fed babies (2 months old) were analyzed. For three babies, an advanced gastrointestinal metabolization was observed. See Figure 1C. Baby Al: only acidic HMOs are present; Baby Al l: only blood-group A characteristic oligosaccharides (m/z 377 and m/z 498) are present; Baby A13: no HMOs present. Figure ID shows that for the other two 100% HMO-fed babies, the HMO-profiles are still present.

These data show that the admixture of prebiotics (lactoferrin, GOS or maltodextrin) has a protective effect on the metabolism of complex HMOs.

EXAMPLE 2: Presence of fucosylated, lacto-N-biose containing HMOs in different sample groups

The simplest HMO-structure which contains lacto-N-biose is LNT. Lacto-N- biose is considered as bifidogenic component of breast milk ¹ . Additionally, fucosylated (especially al,2-fucosylated) HMOs are recognized for their health- beneficial effects via pathogen-inhibition:

- receptor-analogs for Campylobacter (can cause diarrhea) ²

- receptor-analogs for caliciviruses (=norovirus; diarrhea) ³

- breast-fed babies who were infected with stable toxin from E.coli showed less diarrheal symptoms ⁴

- Fucosylated oligosaccharides of human milk protect suckling mice from heat- stabile enterotoxin of Escherichia coli ⁵

Thus, delayed metabolism and therefore the prolonged presence of fucosylated LNT -based HMOs in the gastrointestinal lumen is of special interest.

Unexpectedly, fucosylated HMOs were found to be present especially in the feces of mixed-fed babies. See Figure 2, showing the relative abundance of LNT and fucosylated LNT -based HMOs (LNFP II; LNFP I[co-migrates with LNFP III]; LNDFH I) in baby feces of different feeding groups. Panels A and B (LNT); panels C and D (LNFP II); panels E and F (LNFP I and III); panels G and H (LNDFH I). EXAMPLE 3: Effect of mixed feeding on fecal microbiota

A comparison was made between the fecal microbiota in babies receiving 100% prebiotic (GOS, lactoferrin or maltodextrin) feeding versus babies receiving a mixture of prebiotic and >60% HMO. Figure 3 summarizes the results obtained. Panel A: prebiotic is GOS. Panel B: prebiotic is lactoferrin. Panel C: prebiotic is maltodextrin.

From Figure 3A it can be concluded that a mixed feeding which contains GOS has a beneficial effect for the gastrointestinal growth of Bifidobacteria species. An increased presence of all Bifidobacteria-subgroups was found compared to the babies who got exclusively formula containing GOS. Mixed feeding also increased the presence of the groups Eubacterium and Arthrobacter. To a small extent, higher values of Butyrivibrio and Campylobacter were found for the mixed-fed group as compared to the prebiotic only group.

Figure 3B shows that a mixed feeding which contains lactoferrin yields a lower presence of Bifidobacteria than babies who got exclusively lactoferrin- supplemented formula. To a lower extent, the mixed-fed group showed a decreased presence of Streptococci, Clostridium, Lactobacillus, Arthrobacter, Streptococcus

Figure 3C shows that mixed feeding with a control infant formula comprising maltodextrin has a marked stimulatory effect on the presence of Lactobacilli. Babies who got exclusively control formula showed a lower presence of these lactobacilli-groups in their feces. To a lower extent, the mixed-fed group showed an increase for Bacteroides prevotella group and Butyrivibrio group. EXAMPLE 4: Effect of HMOs on body weight gain

The correlation between delayed metabolism of complex 1,2-fucosylated HMOs on body weight gain in preterm infants was investigated. From Figure 4 it can be concluded that there is a positive association between fecal LNFP II and body weight of a preterm child after 6 weeks treatment when the child is 8 weeks old. LNFPII excretion is increased in the mix-fed group.

Figure 5 shows that there is also a positive correlation between body weight gain and the fecal concentration of LNDFH I. This could be explained by an increased excretion of Campylobacter and Ruminococcus in the feces.

EXAMPLE 5: Infant formulas Tables 3 and 4 show the composition of exemplary nutritional formulas according to the invention, e.g. infant formulas for the age group between 0-6 months, for supporting the delayed metabolization of HMO.

Table 3: Composition of the formulas (per 100 ml)

Formula A: Formula B: Formula C: di- di- di- fucosyllactose fucosyllactose fucosyllactose + GOS + GOS/FOS + lactoferrin

67 67 67

1.4 1.4 1.4

7.6 7.6 7.9

3.5 3.5 3.5

0.1 0.1 0.1

0.4 0.36

0.04

0.1 Vitamins and minerals according to legislation.

Table 4: Composition of the formulas (per 100 ml)

Formula D: Formula E:

lacto-N- lacto-N- fucopentaose I fucopentaose

+ GOS I + GOS +

lactoferrin

Energy, kcal 67 67

Protein (g) 1.4 1.4

Carbohydrates

(g) 7.6 7.6

Fat (g) 3.5 3.5

lacto-N- 0.05 0.05

fucopentaose I

(g)

Vivinal GOS 0.6

(g) 0.6

Lactoferrin (g) 0.1

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