Technical field of the invention

The present invention relates to an improved process for isolating oligosaccharides from milk. In particular, the present invention relates to a method for isolating oligosaccharides from milk while maintaining milk fractions that are used in the preparation of other dairy products.

Background of the invention

Human milk oligosaccharides (HMO) constitute the third most abundant class of molecules in breast milk. Since infants lack the enzymes required for milk glycan digestion, this group of carbohydrate polymers passes undigested to the lower part of the intestinal tract, where they can be consumed by specific members of the infant gut microbiota, and is considered to provide the infant with nutritional and pharmacological activities.

Some analytical studies have indicated that there may be as much as 20 g/L of oligosaccharides in breast milk which makes it the third most concentrated component after lactose and fat. However, unlike lactose and fat, these molecules do not appear to provide energy to infants as they resist the action of human digestive enzymes.

Most human milk oligosaccharides (HMO) are elongation products of lactose, and are synthesized from glucose, galactose, glucosamine, fucose and sialic acid. In addition to being concentrated, HMO are also highly diverse and at least 200 individual structures have been detected using mass spectrometry. This diversity may come from a difference in the compositions of HMO from woman to woman as well as the stage of the lactation circle.

Several interesting bioactivities have been attributed to HMO. For example, they antagonize the binding of some strains of bacteria to epithelial cells, and a large epidemiologic study indicated that sugar epitopes resulting from an individual genotype appear to protect against diarrhoea in breast fed infants. Furthermore, those containing sialic acid may serve as immune modulators. It has also been suggested that HMO may serve as growth factors for colonic microbiota, as it has long been known that breast feeding results in increased levels of faecal bifidobacteria with respect to infant formula. Based on their structure and composition, HMO may affect the gut microbiota consortium either by selectively providing growth factors and energy substrates to some members, or alternatively via binding and eliminating others, or both.

Furthermore, in order to further investigate the activity of HMO and the activities modulated by HMO, it is first necessary to isolate them in large enough quantities to serve as fermentation and or growth factor substrates in microbiological media, and to ensure that they are entirely free of milk mono and disaccharides. Moreover, by isolating specific HMO's or some specific groups of HMOs' a more specific modelling of the HMO's activity may be investigated.

Previously attempts has been made to isolate HMO from other constituents in milk, although mostly for quantification as opposed to the production of substrates for biochemical assays.

Another interesting source of milk oligosaccharides are milk obtained from a ruminant, such as a cow or a bovine. Bovine milk contains oligosaccharides that are analogous to HMO, and it is suggested that bovine milk oligosaccharides has a similar protective role as suggested by human milk oligosaccharides. The oligosaccharides found in bovine milk (BMO) are structurally similar to those in human milk, but their concentration in bovine milk is much lower than the concentration in human milk. Furthermore, human and bovine milk contain large amounts of the acidic oligosaccharides known as sialyloligosaccharides, especially at the early lactation stage. Because mature bovine milk contains only small amounts of these valuable components, up to now it has not been considered a viable source of oligosaccharides e.g. for human supplementation.

The general scheme for isolating milk oligosaccharides has been to first to remove the fat from milk by centrifugation, and then the protein by precipitation with organic solvents; membrane filtration or chromatography. Leaving a mixture of milk oligosaccharides with other constituents like lactose, minerals, glycomacropeptide (CMP), and other residues from the run through or permeate fractions. To isolate the milk oligosaccharides from these other constituents, such as lactose, gel filtration has often been used. This is a slow method that requires long separation times and has a low capacity. Alternatively, methods like enzymatically converting lactose to glucose and galactose to facilitate separation and nanofiltration has been proposed. However, these methods have never shown to be very effective.

The presently provided attempts to isolate oligosaccharides from milk has only been for analytical studies and there has been no focus on industrial process for isolating larger amounts of oligosaccharides for commercial use. Hence, there is an interest in providing a method for isolating oligosaccharides from milk, or specific groups of oligo saccharides from milk in order to make milk oligosaccharides available for analytical purposes and investigation as well as making the oligosaccharides available for commercial applications as an ingredient.

However, even it may be theoretically possible to isolate oligosaccharides from milk it has not yet been industrially and financially interesting . Hence, an improved method for isolating oligosaccharides from milk would be

advantageous, and in particular a faster, a more efficient and/or reliable and specific method for isolating oligosaccharides, or specific fractions of oligosaccharides, from milk would be advantageous.

Summary of the invention

Thus, an object of the present invention relates to an improved method for isolating oligosaccharides from milk. In particular, it is an object of the present invention to provide an improved method for isolating oligosaccharides from milk, in particular a method which is faster, more efficient and/or reliable and specific in the isolation of oligosaccharides, or specific fractions of oligosaccharides, from milk and that solves the abovementioned problems of the prior art with selectivity and volume of the oligosaccharide product provided.

Thus, one aspect of the invention relates to a method for separating one or more oligosaccharide compound(s) from a preliminary dairy source, the method comprises the steps of: (i) Providing the preliminary dairy source;

(ii) Contacting the preliminary dairy source with a chromatographic support allowing one or more oligosaccharide compound(s) present in the preliminary dairy source to be retained by the chromatographic support;

(iii) Obtaining a unretained, flow through fraction from the chromatographic support comprising a secondary dairy source; (iv) Optionally washing the chromatographic support; and

(v) Subjecting the chromatographic support to at least one elution buffer obtaining one or more oligosaccharide compound(s) from the chromatographic support; wherein the chromatographic support comprises one or more adsorbent capable of specifically binding the one or more oligosaccharide compound(s) from the preliminary dairy source.

Another aspect of the present invention relates to a secondary product obtained by the present invention.

Yet another aspect of the present invention relates to an oligosaccharide compound comprising one or more moiety selected from the group consisting of a Hexose moiety (a Hex moiety); a HexNAc moiety; a fucose moiety (a Fuc moiety); and a NeuAc moiety.

Still another aspect of the present invention relates to the use of the oligosaccharide according to the present invention as an ingredient, e.g. for infant formulas, a fortified or functional food or beverages, a health ingredient for human and animal or an over the counter food supplements.

In an even further aspect of the present invention relates to the use of the secondary product according to the present invention for the production of a cheese product, a milk product, a whey product, a milk protein product, or whey protein product.

Detailed description of the invention

As mentioned earlier, the human milk oligosaccharides (HMO's) have been shown to play an important role in the early development of infants and young children, such as the maturation of the immune system. As the role is dependent on the structure and composition of the HMO many different kinds of HMOs are found in the human milk, where each individual oligosaccharide is based on a specific combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose and/or N-acetylglucosamine with many and varied linkages between them, thus accounting for the enormous number of different oligosaccharides in human milk. Almost all human milk oligosaccharides have a lactose moiety at their reducing end while sialic acid and/or fucose (when present) occupy terminal positions at the non-reducing ends. The HMOs can be acidic (e.g. charged sialic acid containing oligosaccharide) or neutral (e.g. fucosylated oligosaccharide).

Thus, it is of interest to provide a method for isolating one or more oligosaccharides from milk. Preferably, the one or more oligosaccharide compound obtained by the method according to the present invention has a significantly reduced content of lactose. A preferred embodiment of the present invention, relates to a method for separating one or more oligosaccharide compound(s) from a preliminary dairy source, the method comprises the steps of:

(i) Providing the preliminary dairy source;

(ii) Contacting the preliminary dairy source with a chromatographic support allowing one or more oligosaccharide compound(s) present in the preliminary dairy source to be retained by the chromatographic support;

(iii) Obtaining a unretained, flow through fraction from the chromatographic support comprising a secondary dairy source;

(iv) Optionally washing the chromatographic support; and (v) Subjecting the chromatographic support to at least one elution buffer obtaining one or more oligosaccharide compound(s) from the chromatographic support; wherein the chromatographic support comprises one or more adsorbent capable of specifically binding the one or more oligosaccharide compound(s) from the preliminary dairy source.

In an embodiment of the present invention the oligosaccharide may be a free

oligosaccharide, hence, oligosaccharides that are not bound as glycoproteins or glycolipids where the oligosaccharides may be attached on the surface of the respective molecules.

In another embodiment of the present invention the concentration of free oligosaccharides may be increased by hydrolysing the covalent bond between the oligosaccharide and the protein, in a glycoprotein, or between the oligosaccharide and the lipid, in a glycolipid, liberating the oligosaccharide. The hydrolysis may be performed by the action of an enzyme.

In a further embodiment of the present invention the one or more oligosaccharide compound(s) comprises 3-30 sugar moieties, such as 5-25 sugar moieties, e.g . 8-22 sugar moieties, such as 10-20 sugar moieties, e.g . 14-18 sugar moieties.

In the present context, the term "primary dairy source" relates to a diary source comprising one or more milk oligosaccharides.

The primary diary source may preferably comprise one or more milk oligosaccharides and casein.

In an embodiment of the present invention the preliminary dairy source may be selected from the group consisting of milk, whole milk, skimmed milk, milk concentrates, reconstituted milk powder, non-pasteurised milk, micro-filtrated milk, pH-adjusted milk, pre-treated dairy source, and whey.

A characteristic feature of the preliminary dairy source may be that the preliminary dairy source has not been subjected to casein precipitation, removal of casein micelles, and/or removal of the casein aggregates, prior to the separation of one or more oligosaccharide compound(s) .

The preliminary dairy source may be obtained as the flow through obtained from a process of separating one or more milk proteins, such as the isolation process of isolation of soluble proteins from aggregated casein-containing mixtures, as described in WO

2016/055064, which is hereby incorporated by reference.

As the concentration of oligosaccharides present in the milk obtained from ruminants are so small (compared to the concentration of oligosaccharides present in human milk, where on the contrary the volume of milk is very small), it may be preferred to make the separation process with as many naturally occurring constituents remaining in the milk as possible in order not to increase the loos of oligosaccharide to be isolated .

Hence, in an embodiment of the present invention the preliminary dairy source does not comprise isolated whey or isolated whey fractions. In a further embodiment of the present invention the preliminary dairy source may be obtained from a ruminant, such as a cow, a goat, a sheep, or a buffalo; or from another domesticated non-human mammal.

In order to simplify the process, to keep costs low and/or to introduce as little chance in the preliminary dairy source as possible, making it as suitable as possible for further processing in providing, directly or indirectly, a dairy product.

In an embodiment of the present invention the preliminary dairy source may preferably not be subjected to a change in pH, a change in hydrophobicity, a change in conductivity (e.g. addition of salts) or a combination hereof before being contacted with the chromatographic support in step (ii) .

The preliminary dairy source may preferably be preserved as much as possible in order to provide as many valuable products from the preliminary dairy source as possible.

Preferably, the preliminary dairy source may be used for providing at least one oligosaccharide compound ; one or more milk proteins; a casein product and/or a dairy product. In an embodiment of the present invention, the preliminary dairy source may be used for providing at least one oligosaccharide compound ; one or more milk proteins; and a dairy product.

In a further embodiment of the present invention the preliminary dairy source has not been subjected to pasteurisation.

Oligosaccharides are found in the human and animal milk, and each individual oligosaccharide may be based on a specific combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose and/or N-acetylglucosamine coupled by different linkages.

In an embodiment of the present invention the one or more oligosaccharide compound(s) comprises at least one moiety selected from the group consisting of a Hexose moiety (a Hex moiety) ; a HexNAc moiety; a fucose moiety (a Fuc moiety) ; and a NeuAc moiety. Preferably the one or more oligosaccharide compound(s) comprises at least one moiety selected from the group consisting of a Hexose moiety (a Hex moiety) ; a HexNAc moiety; a fucose moiety (a Fuc moiety) ; and a NeuAc moiety as the terminal moiety of the oligosaccharide. Most preferably, the one or more oligosaccharide compound(s) comprises at least one fucose moiety (a Fuc moiety) as the terminal moiety of the oligosaccharide. In a further embodiment of the present invention, the terminal moiety of the

oligosaccharide may be the preferred target available for interacting with the adsorbent and/or the ligand .

The Hex moiety according to the present invention may be selected from a glucose residue, a galactose residue or a mannose residue.

The HexNAc moiety according to the present invention may be a N-acetylglucosamine residue or a N-acetylgalactosamine residue.

The fucose moiety according to the present invention may be a deoxyhexose residue.

The NeuAc moiety according to the present invention may be a N-acetyl neuraminic acid residue or a sialic acid residue.

In an embodiment of the present invention the one or more oligosaccharide compound(s) may be a HexNAc moiety (such as a N-acetylglucosamine residue or a N- acetylgalactosamine residue) ; a fucose moiety (such as a deoxyhexose residue) ; and/or a NeuAc moiety (such as a N-acetyl neuraminic acid residue or a sialic acid residue) .

In a preferred embodiment of the present invention the one or more oligosaccharide compound(s) may be a fucose moiety (such as a deoxyhexose residue) ; and/or a NeuAc moiety (such as a N-acetyl neuraminic acid residue or a sialic acid residue) .

One challenge when being interested in separating oligosaccharides from milk may be the presence of enzymes capable of inactivating or degrading oligosaccharides.

Hence, in an embodiment according to the present invention the preliminary dairy source provided in step (i) may be subjected to a first pre-treatment. The first pre-treatment may involve a step of enzyme inactivation.

In an embodiment of the present invention the enzyme inactivation, involves inactivation of one or more of the enzymes selected from the group consisting of fucose-, sialic acid-, N-Acetylglucosamine-, lacto-N-biose-, glucose- and/or galactose-degrading enzymes in the dairy source.

The enzyme inactivation may involve addition of an enzyme degrading compound, a pre separation step removing the enzyme from the dairy source or a combination hereof. The pre-separation step may preferably involve removing the enzyme from the preliminary dairy source. Such removal may involve a filtration process, a centrifugation process or a chromatographic process.

The adsorbent according to the present invention capable of specifically binding the one or more oligosaccharide compound(s) from the preliminary dairy source may be essential for the effectivity of the invention.

The non-porous material may preferably be selected from a metal oxide; a silicate; a ceramic metal; alumina; or a magnetic material. However, further examples of non-porous materials, (non-porous core materials) are described in WO 2010/037736, which is hereby incorporated by reference.

The metal oxide according to the present invention may be selected from titanium oxide; silicates; ferric oxide, nickel oxide or cobalt oxide.

The magnetic material according to the present invention may be selected from ferric oxide, nickel oxide or cobalt oxide.

In an embodiment of the present invention, the non-porous material may be a high density non-porous material.

In a further embodiment of the present invention the high density non-porous material may have a density of at least 4.0 g/ml, such as at least 10 g/ml, e.g. at least 16 g/ml, such as at least 25 g/ml. Typically, the non-porous core material has a density in the range of about 4.0-25 g/ml, such as about 4.0-20 g/ml, e.g. about 4.0-16 g/ml, such as 12-19 g/ml, e.g. 14-18 g/ml, such as about 6.0-15.0 g/ml, e.g. about 6.0-16 g/ml.

The high density non-porous material according to the present invention may be selected from tungsten carbide or steel.

In an embodiment of the present invention the adsorbent may be a metal oxide capable of specifically binding the one or more oligosaccharide compound(s) from the preliminary dairy source.

In an embodiment of the present invention the adsorbent comprises a particle comprising a non-porous material surrounded by a porous polymeric material. Preferably, the porous polymeric material may be an organic porous polymeric material. The porous polymeric material may be selected from agarose, alginate, chitosan, carrageenan, and/or pectin. In an embodiment of the present invention the one or more adsorbent may have a density of at least 1.3 g/ml, more preferably at least 1.5 g/ml, still more preferably at least 1.8 g/ml, even more preferably at least 2.0 g/ml, even more preferably at least 2.3 g/ml, even more preferably at least 2.5 g/ml, even more preferably at least 2.75 g/ml, even more preferably at least 3.0 g/ml, even more preferably at least 3.5 g/ml, even more preferably at least 4.0 g/ml, even more preferably at least 4.5 g/ml .

The density of the adsorbent according to the present invention relates to the density of an adsorbent in it's fully solvated (e.g . hydrated) state as opposed to the density of a dried adsorbent particle.

The desired density of the one or more adsorbent may be provided by inclusion of a certain proportion or a certain amount of the non-porous material in the porous polymeric material .

In order to improve the capability of the adsorbent to bind the one or more oligosaccharide compound(s) from the preliminary dairy source the adsorbent may comprise one or more ligands capable of specifically binding the one or more oligosaccharide compound(s) from the preliminary dairy source.

In an embodiment of the present invention the ligand capable of specifically binding the one or more oligosaccharide compound(s) from the preliminary dairy source may be selected from affinity ligands; hydrophilic interaction ligands; ion exchange ligands;

chelating ligands; complex bond formation ligands; and/or mixed mode ligands.

In a preferred embodiment of the present invention the affinity ligand may be a lectin or an antibody.

In an embodiment of the present invention the lectin may be a fucose specific lectin, such as an L-fucose specific lectin. The fucose specific lectin may preferably be selected from Aleuria Aurantia Lection (AAL), Aspergillus Oryzae Lectin (AOL), or Pseudomonas aeruginosa Lee B (Lee B) .

In another embodiment of the present invention the lectin may be a sialic acid specific lectin, such as an Neu5Ac specific lectin. The sialic acid specific lectin may preferably be selected from Maackia Amurensis II Lectin (MAL II), or Sambucus Nigra Lectin (SNL) .

The adsorbent and/or the ligand according to the present invention may have a high specificity for one or more oligosaccharide compound(s) from the preliminary dairy source. In the present context, the term "high specificity for one or more oligosaccharide compound(s)" relates to more than 50% (w/w) of the molecules specifically bound to the adsorbent and/or the ligand may be one or more oligosaccharide compound(s), such as more than 55%, e.g. more than 60%, such as more than 65%, e.g. more than 70%, such as more than 75%, e.g. more than 80%, such as more than 90%, e.g. more than 95%.

Preferably, the adsorbent and/or the ligand according to the present invention may have a low specificity for lactose present in the preliminary dairy source. In the present context, the term "low specificity for lactose" relates to less than 10% (w/w) of the molecules specifically bound to the adsorbent and/or the ligand may be lactose, such as less than 7.5%, e.g. less than 5%, such as less than 2.5%, e.g. less than 1%, such as less than 0.5%, e.g. less than 0.1%, such as less than 0.05%, e.g. less than 0.01%. As the milk oligosaccharides comprises different specific terminal sugars, ligands may be provided on the adsorbent in order to specifically binding specifically to these groups of the oligosaccharides.

In the present context, the term "specifically binding" relates to the binding or separation of one individual structure of oligosaccharide over others, or the binding or separation of one particular group of oligosaccharides over at least one other group of oligosaccharides.

Separating one or more oligosaccharide may preferably relate to the isolation of an individual structure of oligosaccharides or a particular group of oligosaccharides. This, means that the method of the present invention may be specific in the binding of oligosaccharides allowing some oligosaccharides to be retained by the chromatographic support and allow other oligosaccharides to flow through the chromatographic support.

In an embodiment of the present invention the chromatographic support may be a membrane chromatography support, or a column chromatography support. Preferably, the column chromatography support includes a Packed Bed Chromatography, stirred tank adsorption, moving bed chromatography, simulated moving bed chromatography, Fluidized Bed Chromatography and/or Expanded Bed Chromatography. In order to quickly process the primary dairy source increased loading speed may be preferred. Hence, in an embodiment of the present invention the preliminary dairy source may be loaded on to the chromatographic support at a flow-rate in the range of 1-50 cm/min; preferably in the range of 5-30 cm/min; more in the range of 10-25 cm/min; even more preferably, in the range of 15-20 cm/min. In an embodiment of the present invention the primary diary source has a pH in the range of pH 6-9, such as in the range of pH 7-8, e.g. in the range of pH 7.3-7.7. The separation process according to the present invention may either be a batch separation process or a continuous separation process.

In an embodiment of the present invention, large-scale production (industrial scale production) may be conducted at a continuous process. When the oligosaccharides are retained by the chromatographic support an elution step at some point during the separation process may be necessary. By providing at least two chromatographic supports and placing them in parallel, such continuous separation may be provided where the flow of primary dairy source may be shifted from one chromatographic support, when this chromatographic material is loaded and ready for elution, to the other chromatographic support. Alternatively, moving bed chromatography, simulated moving bed

chromatography or the like may be used.

The unretained, flow through fraction from the chromatographic support comprising a secondary dairy source obtained in step (iii) of the process according to the present invention may be collected.

In an embodiment of the present invention the secondary dairy source may be used, directly or the secondary dairy source may be further processed, for providing a dairy product.

Further processing of the secondary dairy source may result in at least one fraction of the secondary dairy source and the further processing may include separation of one or more milk proteins or whey proteins by filtration or chromatography; addition of acid or rennet for providing a curd, suitable for cheese production; addition of microorganisms for fermenting the secondary dairy source providing a fermented dairy product.

The secondary dairy source and/or one or more fractions thereof may be used as an ingredient in a food product, a feed product or a beverage. Preferably, the food product is a dairy product.

In an embodiment of the present invention, the dairy product may be selected from the group consisting of milk; powdered milk; UHT milk; whole milk; buttermilk; skim milk; whey; casein; milk and/or whey proteins; condensed milk; evaporated milk; cheese; curds; cultured milk; yogurt; creme fraiche; fromage frais; ice-cream; infant formulas; and nutritional products.

A preferred embodiment of the present invention relates to a secondary product obtainable be a method according to the present invention. Preferably, the secondary dairy source may be the secondary product.

In an embodiment of the present invention the secondary product may comprise casein; one or more milk proteins; one or more whey proteins; lactose; minerals; fat and water, if originally present in the primary dairy source. Preferably, the secondary product may comprise alpha-casein, beta-casein and kappa-casein. Preferably, the secondary product may comprise alpha-casein and kappa-casein.

In a further embodiment of the present invention the secondary product may have the same pH value (or substantially the same pH value) as the preliminary dairy source, the same hydrophobicity (or substantially the same hydrophobicity) as the preliminary dairy source; and/or the same conductivity (or substantially the same conductivity) as the preliminary dairy source.

In an even further embodiment of the present invention the secondary product may be depleted (or substantial depleted) from fucose-, sialic acid-, N-Acetylglucosamine-, lacto- N-biose-, glucose- and/or galactose-degrading enzymes.

The secondary product according to the present invention may be used for the production of a cheese product, a milk product, a whey product, a milk protein product, or whey protein product.

The oligosaccharide compound specifically bound to the chromatographic support may be released from the chromatographic support by subjecting the chromatographic support to at least one elution buffer obtaining one or more oligosaccharide compound(s) from the chromatographic support, as described in step (v) .

In an embodiment of the present invention the elution in step (v) may be a sequential elution of one or more oligosaccharides providing one of more oligo saccharide fractions.

The elution buffer may comprise a carbohydrate compound, such as sorbitol, lactose, and/or fucose. In an embodiment of the present invention the pH value of the elution buffer is in the range of pH 6-9. Such as in the range of pH 7-8, e.g. about pH 7.4.

In another embodiment of the present invention the pH value of the elution buffer is substantially the same pH value as the pH value of the primary diary source when contacted with the chromatographic support. In the present context the term "substantially the same pH value" relates to a difference in the pH value of less than pH 0.5, such as less than pH 0.25, e.g. less than pH 0.1.

A preferred embodiment of the present invention relates to an oligosaccharide compound comprising one or more moiety selected from the group consisting of a Hexose moiety (a Hex moiety); a HexNAc moiety; a fucose moiety (a Fuc moiety); and a NeuAc moiety.

The Hex moiety of the oligosaccharide compound may preferably be selected from a glucose residue, a galactose residue or a mannose residue.

The HexNAc moiety of the oligosaccharide compound may preferably be a N- acetylglucosamine residue or a N-acetylgalactosamine residue.

The fucose moiety of the oligosaccharide compound may preferably be a deoxyhexose residue.

The NeuAc moiety of the oligosaccharide compound may preferably be a N-acetyl neuraminic acid residue or a sialic acid residue.

In an embodiment of the present invention the one or more oligosaccharide compound(s) comprises a fucose moiety (such as a deoxyhexose residue); and a NeuAc moiety (such as a N-acetyl neuraminic acid residue or a sialic acid residue).

In a further embodiment of the present invention the one or more oligosaccharide compound(s) may comprise 3-30 sugar moieties, such as 5-25 sugar moieties, e.g. 8-22 sugar moieties, such as 10-20 sugar moieties, e.g. 14-18 sugar moieties.

Preferably, the content of free lactose present in the oligosaccharide compound according to the present invention may be less than 5 mg/I, such as less than 4 mg/I, e.g. less than 3 mg/I, such as less than 2 mg/I, e.g. less than 1 mg/I, such as less than 0.5 mg/I, e.g. less than 0.1 mg/I. Preferably, the content of glucose present in the oligosaccharide compound according to the present invention may be less than 5 mg/I, such as less than 4 mg/I, e.g. less than 3 mg/I, such as less than 2 mg/I, e.g. less than 1 mg/I, such as less than 0.5 mg/I, e.g. less than 0.1 mg/I.

Preferably, the content of galactose present in the oligosaccharide compound according to the present invention may be less than 5 mg/I, such as less than 4 mg/I, e.g. less than 3 mg/I, such as less than 2 mg/I, e.g. less than 1 mg/I, such as less than 0.5 mg/I, e.g. less than 0.1 mg/I.

The oligosaccharide compound according to the present invention may be used as an ingredient, e.g. for infant formulas, a fortified or functional food or beverages, a health ingredients for human and animal or an over the counter food supplements. The oligosaccharide compound according to the present invention may be used as a prebiotic ingredient.

In the context of the present invention, the term "prebiotic" means non-digestible carbohydrates that beneficially affect the host by selectively stimulating the growth and/or the activity of healthy bacteria such as bifidobacteria in the colon of humans.

Without being bound by theory, the oligosaccharides of the nutritional composition of the present invention may act at different levels to support the natural defenses of the developing infant or young child : (i) boost commensals to obtain better colonization resistence, (ii) boost innate immunity to counteract pathogens and/or (iii) act directly on pathogens as decoy to deviate the pathogens from their natural target. It is also believed that the fucosylated oligosaccharide(s) and the particular N-acetylated oligosaccharide(s) act synergically for these purposes. They could exert synergistic protection in that they target numerous diverse adhesins and quorum sensing mechanisms of pathogens, thereby reducing their virulence and competiveness, and they favour the establishment and competitiveness and metabolic activity of commensal microbiota leading to colonization resistance.

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety. References

WO 2016/055064 WO 2010/037736