WO2018048305A1 - Prebiotic branched galacto-oligosaccharides (gos) - Google Patents

Abstract

The invention relates to galacto-oligosaccharide (GOS) compositions and the use thereof. Provided is the use of a GOS composition comprising branched and linear GOS species having a degree of polymerization (DP) of 3, wherein the branched DP3 GOS species are present in excess of linear DP3 GOS species, for inducing mucin glycan utilization pathways in beneficial gut bacteria in an animal. Preferred compositions comprise the branched DP3 GOS species β-D-Galp-(1-2)-[β-D- Galp-(1-6)-]D-Glcp, β-D-Galp-(1-3)-[β-D-Galp-(1-6)-]D-Glcp β-D-Galp-(1-4)-[β- D-Galp-(1-2)-]D-Glcp, and/or the DP2 GOS species Galβ-1,2-Glc, Gal β-1,3- Glc, Gal-β-1,6-Glc (allolactose) and Gal-β-1,6-Gal. Also provided is a method for providing a GOS composition comprising branched DP3 species and capable of inducing mucin-glycan pathways in beneficial bacteria.

Description

Title: Prebiotic branched galacto-oligosaccharides (GOS). The invention relates to galacto-oligosaccharide (GOS) compositions and uses thereof. Among others, the invention relates to prebiotic GOS compositions that activate mucin and pectic galactan utilization pathways in e.g. the human gut symbiont Bacteroides

thetaiotao micron.

The human gastrointestinal tract is colonized by millions of bacteria that form a complex ecosystem between the food transiting the gut and epithelial lining of the intestinal tract (Hooper and Macpherson, 2010). Bacterial species are adapted to the dynamic conditions of the

gastrointestinal tract where they must acquire nutrients from a variety of sources that are quickly transiting through the intestinal lumen. Gut bacteria have evolved extensive metabolic systems to harvest nutrients they may encounter in the gut. The food we consume therefore dictates to a large extent what bacterial species are present in our gut flora (David et al.,

2014) . Glycans represent dietary fibres that are consumed by specialized gut bacteria (Rogowski et al., 2015)(Martens et al., 2011) (Milani et al.,

2015) . In the absence of dietary glycans, the mucosal layer represents a carbohydrate-rich alternative nutritional source for gut microbiota (Tailford et al., 2015) (Martens et al., 2008). As such, microbial composition is also dependent on mucins (Li et al., 2015). Mucin is composed of high molecular weight glycoproteins that form a thick gel-like layer on the surface of the gut epithelium which acts as lubrication and a protective barrier between the intestinal lumen (Corfield, 2015). Within mucus, core-glycans are covalently hnked to serine or threonine to form complex O-linked glycan structures (Bergstrom and Xia, 2013). The type of O-glycans produced in the human intestinal tract is primarily dictated by human genetic factors, including expression of MUC genes, secretor type and blood group type (such as Lewis/ABO, etc) (Robbe et al., 2004). Bacteria able to degrade mucin glycans are more easily adaptable to the changing intestinal environment and have an advantage in colonizing the mucosal surface for estabhshing themselves as a core species in the GI tract (Bergstrom and Xia, 2013) (Tailford et al, 2015).

The bacterium Bacteroides thetaiotaomicron is a prominent member of the human microbiota and resides in the distal gut. It is characterized by its complex catabolic systems built up of carbohydrate-active enzymes and transporters that function to degrade a wide range of complex host and dietary polysaccharides into their individual monosaccharide components (Sonnenburg et al., 2005) (Martens et al, 2009a). These catabolic systems are arranged in defined operons called polysaccharide utilization loci (PULs) that make up approximately 20% of the B. thetaiotaomicron genome and target specific polysaccharides for degradation (Martens et al., 2011;

Rogowski et al., 2015). B. thetaiotaomicron is found to contain PULs that target dietary polysaccharides, microbial polysaccharides, and human glycans such as mucins. The expression of PULs targeting mucin glycans was demonstrated to be important in colonization, persistence, and mother- to-infant transmission of B. thetaiotaomicron (Martens et al., 2008). Human milk oligosaccharides (hMOS) found in mothers milk facilitated mother-to- infant transmission by inducing the expression of a distinct subset of mucin O-glycan utihzation PULs (Marcobal et al., 2011), suggesting that hMOS can facilitate bacterial transmission to infants by activating mucin glycan utilization pathways.

In humans, mother's natural milk contains approximately 8% (lOg/L) hMOS of which there are approximately 200 structures that have been shown to have beneficial impacts on infant health (Bode, 2012) (Bode, 2015) such as facilitating bacterial colonization (Marcobal and Sonnenburg, 2012) and protection from pathogens (Newburg et al., 2005)(Manthey et al., 2014). Galacto-oligosaccharides (GOS) are non-digestible carbohydrates that have been shown to elicit similar beneficial health effects as hMOS (Ben et al., 2004). Infants fed formulas supplemented with commercial GOS

preparations develop a more natural gut microbiome that more closely resembles the microbiomes of breast-fed infants, when compared to formulas without GOS added (Davis et al., 2010)(Garrido et al., 2013).

GOS are typically synthesized on a large scale via transglycosylation reactions by incubating specific β-galactosidase enzymes with high concentrations of lactose to form complex GOS mixtures that contain many molecules of differing chain length, hnkage type and degree of branching (Prenosil et al., 1987)(van Leeuwen et al., 2016)(Sancler S. van Leeuwen et al., 2014)(Park and Oh, 2010). When consumed, GOS reach the distal colon similar to hMOS where they are degraded by resident bacteria (Gietl et al., 2012), promoting the growth of bacterial families, including

Bifidobacteriaceae and Bacteroidaceae, in the intestine of infants in a similar ratio to that found in the gut microbiota of breast-fed infants.

As said, GOS are highly complex mixtures and very little is known about what components within GOS offer the same hMOS-like effects. In order to allow for more selective and/or effective prebiotic or dietary preparations, the present inventors sought to identify GOS compounds that are responsible for engaging hMOS-like responses.

To that end, B. thetaiotao micron was used as a model bacterium to identify the potential effects of GOS on infant gut associated bacterial species.

Surprisingly, several GOS compounds were identified that are responsible for engaging hMOS-like responses in B. thetaiotaomicron when presented with either hMOS or GOS preparations as a sole carbon source. More specifically, it was found that branched GOS species with a degree of polymerization of 3 (DP3) were particularly effective in eliciting the expression of a broad range of mucin PUL systems.

In addition, two distinct mechanisms directed at GOS metabolism by B. thetaiotao micron were revealed: firstly, extended linear 6-(l→4)-linked GOS molecules were degraded via the action of an extracellular GH53 endo-6- (l→4)-galactanase that is encoded within a galactan utilization PUL.

Secondly, remaining branched GOS and lactose-disaccharide derivatives behaved as inducer molecules for PULs directed at O-glycan and host glycan metabolism similar to hMOS. Together, we show that specific GOS molecules mimic hMOS by inducing the expression of enzymes and transport components from PULs indicated in mucin-O-glycan degradation in B. thetaiotao micron while a specific endo- 6-galactanase directed at dietary fibre catabolism plays an important role in GOS degradation. This is the first study dissecting the molecular details of GOS utilization in a gut commensal bacterial strain and the first detailed identification of specific GOS compounds that elicit hMOS-like responses. These results provide a firm basis for making more selective prebiotic preparations for infant formulas and for broader prebiotic and dietary fibre applications. Accordingly, the invention relates to a composition comprising galacto- oligosaccharides (GOS), wherein branched GOS species having a degree of polymerization (DP) of 3, are present in excess of linear GOS species having a degree of polymerization (DP) of 3.

Provided is the use of a composition comprising galacto- oligosaccharides (GOS) for inducing mucin glycan utihzation pathways in beneficial gut bacteria in an animal, said composition comprising branched and linear GOS species having a degree of polymerization (DP) of 3, wherein the branched DP3 GOS species are present in excess of linear DP3 GOS species. According to the invention, the use is not for the purpose of carrying out therapy on the human or animal body.

The composition for use according to the present invention preferably comprising galacto-oligosaccharides wherein branched galacto- oligosaccharides having DP3 are enriched (e.g., are at least 5%, 10%, 15%, 20%, 30%, 40% more than) compared to the amount by weight of branched DP3 in a (mixed) GOS solution. "A mixed galacto-oligosaccharide solution" refers to a mixture of galacto-oligosaccharides having different DPs, e.g., as is produced using a 6-galactosidase in a transgalactosylation reaction (e.g., as described in Japanese Patent JP 105109 or US Patent No. US 4,957,860). Exemplary mixed galacto-oligosaccharide solutions include, e.g., Vivinal™ GOS (available from Friesland Foods Domo, The Netherlands) and commercial GOS products I-VI as described in Van Leeuwen et al. (2016). In some embodiments, the enriched compositions for use in the invention have less than 10% or less than 5% of sugar monomers (e.g., galactose) and optionally less than 10% or less than 5% of dimeric galacto-oligosaccharides.

A composition for use as disclosed in the present invention is not known or suggested in the art. Typical commercial GOS preparations contain relatively low amounts of branched GOS species. For example, the

commercial syrup Vivinal GOS™ contains only 59% GOS w/w, with lactose, glucose and galactose accounting for the remaining 41%. Among the GOS species present in Vivinal GOS, disaccharides (DP2) and trisaccharides (DP3) are most abundant, representing approximately 33 w% and 39 w%, respectively, based on the total dry weight of all GOS species. Branched DP3 and branched DP4 represent about 8 w% and 2w%, respectively. Other commercially available GOS mixtures are similarly enriched in lower molecular weight GOS species, in particular in GOS di- and trisaccharides, yet the branched structures are present in only minor amounts. The prior art is silent about the beneficial effects of branched DP3 species on mucin glycan utilization pathways in beneficial and human commensal gut bacteria. Yanahar et al. (Biosc. Biotech. Biochem., 59 (6), 102101026 (1995)) investigated the correlation between the structure of GOS species from lactose by Bacillus circulans beta-galactosidase and their use by

bifidobacteria. Eleven structures were identified, among which branched DP3 GOS, that were used by human intestinal bifidobacteria. However, the mere consumption by bifidobacteria as taught by Yanahar et al. does not imply any effect of individual GOS species on mucin utilization. Moreover, it fails to teach or suggest the surprising ability of branched DP3 GOS species to induce mucin glycan utilization pathways in beneficial gut bacteria, thereby opening up novel applications as hMOS mimic, e.g. to promote the colonization and adaptation of mucin-degrading bacteria or to facilitate bacterial transmission to infants. Mucin is a complex amalgam of sugar molecules (core glycans) consisting of several types of monosaccharides in addition to galactose, such as N-acetyl galactosamine, N-acetylglucosamine, fucose, and sialic acid, with diverse glycosidic linkage types. Notably, the GOS molecules herein identified as inducers of mucin utilization are not represented in any known mucin-type structures. Therefore, based on the structural composition of GOS molecules alone, including the structures of Yanahar et al., one would never deduce that GOS could activate mucin utilization pathways. The present finding was therefore completely unexpected and unpredictable based on the fact that the selected GOS species and mucin having completely different carbohydrate structural properties.

WO2005/067962A2 relates to a composition of growth factors and

oligosaccharides from goat milk, to nutritional products containing these oligosaccharides, to a process to obtain that composition, and also to the use of this composition in the preparation of nutritional products and products to be used in the prevention of infections and intestinal disorders. It teaches the DP3 compound 6-GalLac as one of the components that activate expression of the MUC2 and MUC3 genes.

WO2010/105207 provides compositions for stimulating growth of particular Bifidobacteria. In some embodiments, the compositions comprise galacto-oligosaccharides, wherein at least 45% of the galacto- oligosaccharides by weight are tetra or penta galacto-ohgosaccharides or wherein at least 25% of the galacto-oligosaccharides by weight are tetra galacto-oligosaccharides. In some embodiments, the compositions have less than 10% or less than 5% of trimeric (DP3) galacto-oligosaccharides. Thus, WO2010/105207 teaches a clear preference for DP4 and DP5 GOS species. It is silent about the degree of branching.

Preferably, according to the invention, the branched DP3 GOS species are present in an amount of at least 5, at least 10 or at least 15% by weight (w%>), preferably at least 20w%, based on the total dry weight of all GOS species present in the composition. For example, a composition for use according to the invention comprises at least 20w%> of branched DP3 GOS based on the total dry weight of all GOS species present in the composition. Accordingly, in some embodiments, the compositions comprise GOS, wherein at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the galacto-oligosaccharides by weight are branched DP3 galacto- oligosaccharides. All composition percentages as provided herein, unless indicated otherwise, are determined by mass spectrometry (e.g., MALDI- FTICR as described in the Examples). In some embodiments, the

compositions of the present invention comprise GOS, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the GOS by weight are branched DP3 galacto-oligosaccharides. As used herein, a percentage of a particular DP refers to the amount by weight of the particular DP relative to the weight of total GOS in the composition.

In one embodiment, a composition for use according to the invention comprises GOS species comprising β-linked galactosyl residues, in particular β(1-6) hnkages, β(1-3) linkages and/or β(1-2) linkages. In a preferred aspect, it comprises one or more branched DP3 GOS species selected from the group consisting of 6-D-Galp-(l-2)-[6-D-Galp-(l-6)-]D-Glcp, 6-D- Galp-(l-3)-[6-D-Galp-(l-6)-]D-Glcp and 6-D-Galp-(l-4)-[6-D-Galp-(l-2)-]D- Glcp.

As will be understood, a composition provided herein may, in addition to the branched DP3 GOS species, contain one or more further oligosaccharides having a beneficial (e.g. prebiotic, nutritional and/or therapeutic) effect. For example, it may comprise additional GOS molecules such as linear DP3 and/or one or more DP2 GOS species, preferably selected from the group consisting of 6-galactosyl-lactose, 3-galactosyl-lactose, Gal6-

1.2- Glc, Gal 6-1,3-Glc, 6-GalLac, Gal-6-l,6-Glc (allolactose) and Gal-6-1,6- Gal. Hence, in one embodiment, the invention provides a composition comprising branched DP3 GOS species in excess of linear DP3 GOS species, preferably a branched DP3 GOS species selected from the group consisting of 6-D-Galp-(l-2)-[6-D-Galp-(l-6)-]D-Glcp, 6-D-Galp-(l-3)-[6-D-Galp-(l-6)-]D- Glcp and 6-D-Galp-(l-4)-[6-D-Galp-(l-2)-]D-Glcp, and furthermore

comprising DP2 GOS species, preferably one or more selected from the group consisting of Gal6-1,2-Glc, Gal 6-1,3-Glc, 6-GalLac, Gal-6-l,6-Glc and Gal-6-l,6-Gal. In a preferred embodiment, the invention provides the use of a composition comprising the branched DP3 GOS species 6-D-Galp-(l-2)-[6- D-Galp-(l-6)-]D-Glcp, 6-D-Galp-(l-3)-[6-D-Galp-(l-6)-]D-Glcp, 6-D-Galp-(l- 4)-[6-D-Galp-(l-2)-]D-Glcp, and the DP2 GOS species Gal6-1,2-Glc, Gal 6-

1.3- Glc, Gal-6-l,6-Glc (allolactose) and Gal-6-l,6-Gal for inducing mucin glycan utilization pathways in beneficial gut bacteria in an animal. In one embodiment, a composition comprises, in addition to the prebiotic GOS species, one or more further prebiotic ingredients. For example, inulin and/or galactan, e.g. potato galactan may be present. In a specific aspect, all GOS species present in the composition make up at least 25w%, 30w%, 40w% or 50 w%, preferably at least 60 w%, more preferably at least 70 w% based on the dry weight of the composition.

In a further embodiment, the composition is essentially free of

monosaccharides, in particular galactose and/or glucose and/or lactose.

According to the invention, the GOS composition enriched for specific GOS species is used for inducing mucin glycan utihzation pathways in beneficial gut bacteria in an animal. Preferably, the animal is a human, for example a human of less than 5 years old or a human over 50 years old. Beneficial results of inducing mucin glycan utilization pathways include one or more of the following: a) promoting colonization and adaptation of mucin-degrading bacteria; b) facilitating bacterial transmission to infants; c) stimulating colonization of the gut of the animal by at least one beneficial bacterial strain. In particular, inducing mucin glycan utilization pathways using a GOS composition as herein disclosed results in stimulating colonization of the gut of the animal by a Bacteroides and/or a Bifidobacterium strain, preferably B. thetaiotaomicron, Bifidobacterium breve, Bifidobacterium longum bv. infantis and/or Bifidobacterium bifidum. Thus, a composition according to the invention finds its use in many important (human) health applications. For example, the composition is a food product or dietary supplement product. Exemplary food products include an infant formula, a follow-on formula, and a toddler beverage.

Other exemplary food products are those intended for the elderly. The GOS-containing compositions can be administered as a prebiotic formulation i.e., without bacteria, or as a probiotic formulation i.e., with desirable (symbiotic) bacteria. Exemplary beneficial bacteria that can be included in the pro-biotic compositions of the invention include, but are not limited to, a Bacteroides and/or a Bifidobacterium strain, preferably B. thetaiotao micron, Bifidobacterium breve, Bifidobacterium longum bu.

infantis and/or Bifidobacterium bifidum.

In general, any food or beverage that can be consumed by human infants or adults or animals may be used to make formulations containing the prebiotic and probiotic compositions of the present invention. Exemplary foods include those with a semi-hquid consistency to allow easy and uniform dispersal of the prebiotic and probiotic compositions of the invention.

However, other consistencies (e.g., powders, liquids, etc.) can also be used without limitation. Accordingly, such food items include, without limitation, dairy -based products such as cheese, cottage cheese, yogurt, and ice cream. Processed fruits and vegetables, including those targeted for

infants/toddlers, such as apple sauce or strained peas and carrots, are also suitable for use in combination with the galacto-oligosaccharides of the present invention. Both infant cereals such as rice- or oat -based cereals and adult cereals such as Musilix are also suitable for use in combination with the branched DP3 GOS oligosaccharides of the present invention. In addition to foods targeted for human consumption, animal feeds may also be supplemented with the prebiotic and probiotic compositions of the invention. Alternatively, the prebiotic and probiotic compositions for use according to the invention may be used in the form of a supplement to beverage.

Examples of such beverages include, without limitation, infant formula, follow-on formula, toddler's beverage, milk, fermented milk, fruit juice, fruit -based drinks, and sports drinks. Many infant and toddler formulas are known in the art and are commercially available. Other beneficial formulations include the supplementation of animal milks, such as cow's milk.

Alternatively, the prebiotic and probiotic compositions for use of the present invention can be formulated into pills or tablets or encapsulated in capsules, such as gelatin capsules. Tablet forms can optionally include, for example, one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge or candy forms can comprise the compositions in a flavor, e.g., sucrose, as well as pastilles comprising the compositions in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the hke containing, in addition to the active ingredient, carriers known in the art. The inventive prebiotic or probiotic formulations may also contain conventional food supplement fillers and extenders such as, for example, rice flour. In some embodiments, the prebiotic or probiotic composition will further comprise a non-human protein, non-human lipid, non-human carbohydrate, or other non-human component. For example, in some embodiments, the compositions of the invention comprise a bovine (or other non-human) milk protein, a soy protein, a rice protein, beta-lactoglobulin, whey, soybean oil or starch.

The dosages of the prebiotic and probiotic compositions for use according to the present invention will be varied depending upon the requirements of the individual and will take into account factors such as age (infant versus adult), weight, and reasons for loss of beneficial gut bacteria (e.g., antibiotic therapy, chemotherapy, disease, or age). The amount administered to an individual, in the context of the present invention should be sufficient to establish colonization of the gut with beneficial bacteria over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that may accompany the

administration of a prebiotic or probiotic composition of the present invention. In some embodiments, the dosage range will be effective as a food supplement and for reestablishing beneficial bacteria in the intestinal tract. In some embodiments, the dosage of a galacto-oligosaccharide composition of the present invention ranges from about 1 micrograms/L to about 25 grams/L of galacto-oligosaccharides. In some embodiments, the dosage of a galacto-oligosaccharide composition of the present invention is about 100 micrograms/L to about 15 grams/L of galacto-oligosaccharides. In some embodiments, the dosage of a galacto-oligosaccharide composition of the present invention is 1 gram/L to 10 grams/L of galacto-oligosaccharides. Exemplary bacterial dosages include, but are not limited to, 10exp4 to 10exp l2 colony forming units (CFU) per dose. A further advantageous range is 10exp6 to lOexp lO CFU.

The prebiotic or probiotic formulations can be administered to any

individual in need thereof. In some embodiments, the individual is an infant or toddler. For example, in some embodiments, the individual is less than, e.g., 3 months, 6 months, 9 months, one year, two years or three years old. In some embodiments, the individual is an adult. For example, in some embodiments, the individual is an elderly subject, e.g. over 50, 55, 60, 65, 70, or 75 years old. In some embodiments, the individual is immuno- deficient. The invention also relates to a method for providing a composition of the invention being enriched in GOS structures according to the present invention, in particular compositions comprising branched DP3.

In some embodiments, GOS are produced enzymatically as mixtures having different degrees of polymerization from monomelic or dimeric sugars. GOS can be produced, for example, from lactose syrup using the transgalactosylase activity of the enzyme 6-galactosidase by methods well known in the art. Other general GOS production methods include, e.g., production of galacto-oligosaccharide by treating lactose with beta- galactosidase derived from Bacillus circulans (see, e.g., Japanese Patent JP 105109 and production by the reaction between lactose and beta- galactosidase from Aspergillus oryzae (see, e.g., US 4,957,860). See also, e.g., Ito et al, Microbial Ecology in Health and Disease, 3, 285-292 (1990). In addition, as mentioned herein above, commercial GOS products are also available that generally include a wide spectrum of different-sized galacto- oligosaccharides. For example, the mixed galacto-oligosaccharide solution Vivinal™ GOS (available from Friesland Foods Domo, The Netherlands) or any one of the commercial GOS products I -VI as described in Van Leeuwen et al. (2016) can be used as starting material. Thus, to generate the specific purified GOS of the present invention (e.g., being enriched for, branched DP3 species and one or more other preferred GOS species), in some embodiments, the compositions for use according to the present invention can be generated by obtaining a GOS mixture containing a variety of different-sized GOS and then reducing the proportion of galacto-oligosaccharides having a DP and/or degree of branching that is not desired.

In a specific aspect, the invention provides a method for providing a GOS fraction capable of inducing mucin-glycan pathways in beneficial bacteria, said method comprising the steps of:

- providing a mixture of galacto-oligosaccharides (GOS) having varying degrees of polymerization; - optionally removing free lactose from said GOS mixture; - applying said (lactose-free) GOS mixture to an anion exchange resin; - step-wise eluting GOS with increasing degree of

polymerization using a water mobile phase and collecting separate eluent fractions; - analyzing each eluent fraction for the effect on expression of a bacterial gene associated with mucin utilization; and - selecting one or more fraction(s) capable of promoting expression of a gene associated with mucin utilization. In a specific aspect, each eluent fraction is analyzed for the effect on expression of the SusC gene, the SusD gene or a homolog thereof (see experimental section herein below).

Alternatively, or optionally in addition, enzymatic methods can be used to synthesize the GOS species for use in the present invention. In general, any oligosaccharide transglycosylating enzyme or hydrolytic enzyme (with the reaction running in reverse) that converts a substrate into any of the target DP of the galacto-oligosaccharide (or their intermediates) may be used in the practice of this invention. For example, prebiotic galacto- oligosaccharides have been synthesized from lactose using the 6- galactosidase from Lactobacillus reuteri or B. circulans. The reaction employed is known as transgalactosylation, whereby the enzyme 6- galactosidase hydrolyzes lactose, and, instead of transferring the galactose unit to the hydroxyl group of water, the enzyme transfers galactose to another carbohydrate to result in oligosaccharides with a higher degree of polymerization. The transgalactosylation reaction can proceed

intermolecularly or intramolecularly. Intramolecular or direct galactosyl transfer to D-glucose yields regioisomers of lactose.

In a specific embodiment, the conventional process for

enzymatically obtaining a GOS mixture is supplemented with the use of an enzyme capable of degrading one or more less desired GOS species. For example, a (recombinant) endo-6-galactanase such as the GH53 enzyme described in the experimental section herein below or an enzyme having similar properties is suitably used to provide a preferred composition of the present invention. Therefore, also provided is a method for providing a galacto-oligosaccharides (GOS) composition comprising DP2 and branched DP3 GOS species and capable of inducing mucin-glycan pathways in beneficial bacteria, said method comprising providing a mixture of GOS having varying degrees of polymerization and subjecting the mixture to an enzyme capable of degrading one or more less desired GOS species, the enzyme being an endo-6-galactanase GH53 enzyme. Preferably, the enclo-6- galactanase GH53 enzyme is endo-6-galactanase GH53 from B.

thetaiotaomicron, more preferably recombinantly produced B.

thetaiotaomicron endo-6-galactanase GH53. In a specific aspect, the step of providing a mixture of GOS having varying degrees of polymerization comprises a transgalactosylation reaction using β-galactosidase, preferably β-galactosidase from L. reuteri or B. circulans. The invention also provides a GOS composition obtainable by a method as described herein above. See for example Figure 7B for a representative HPAEC-PAD analysis of such composition.

Still further, the invention provides the use of a recombinant endo-6-galactanase GH53 enzyme in the manufacture of a prebiotic composition comprising galacto-oligosaccharides (GOS). Preferably, the endo-6-galactanase GH53 enzyme is recombinantly produced B.

thetaiotaomicron endo-6-galactanase GH53.

Alternatively, conventional chemical methods may be used for the de novo organic synthesis of or conversion of pre-existing oligosaccharides into the galacto-oligosaccharides of the present invention.

Hence, a method of the invention may comprise 1) fractionation of Vivinal GOS or other commercial GOS samples I -VI; 2) use of endo-6- galactanase GH53 to eliminate by hydrolysis other GOS molecules from e.g. Vivinal GOS or from other commercial GOS samples I -VI; 3) enzymatic synthesis from lactose using β-galactosidase enzymes, for instance wild type enzymes such as the one from B. circulans, incubating it more briefly with lactose (or using modified incubation conditions) than what is currently used for Vivinal synthesis (24 h); and / or 4) chemical synthesis of one or more of the preferred 8 GOS molecules as disclosed herein. A still further aspect of the invention relates to a method for stimulating beneficial microflora in an animal, the method comprising administering a sufficient amount of the prebiotic/probiotic composition of the invention to the animal to stimulate colonization of the gut of the animal by at least one beneficial bacterial strain. For example, said method can stimulate gut colonization by a Bacteroides and/or a Bifidobacterium strain, preferably B. thetaiotaomicron, Bifidobacterium breve and/or Bifidobacterium longum bv. infantis. Typically, said animal is a human but the method also finds its use in non-human applications, e.g. as a veterinary treatment.

In one embodiment, the human is less than 5 years old or over 50 years old. The animal subject may be healthy or diseased. For example, the animal, preferably a human subject, may have a condition selected from the group consisting of inflammatory bowel syndrome, constipation, diarrhea, colitis, Crohn's disease, colon cancer, functional bowel disorder, irritable bowel syndrome, and excess sulfate reducing bacteria.

LEGEND TO THE FIGURES

Figure 1: GOS profile analysis of Vivinal GOS (bottom) and purified GOS (top). Compounds were separated by HPAEC-PAD and identified based on previously published information (Sander S van Leeuwen et al., 2014).

Galactose is depicted as light grey circles, glucose is depicted as dark grey circles.

Figure 2: Growth on GOS mixture. Growth curves of B. thetaiotaomicron in a minimally defined medium with carbon sources added at a final concentration of 5 mg/ml. Hungate tubes flushed with gas containing

80%N2, 10%H2, 10% CO2. Readings were taken by placing Hungate tubes in the spectrophotometer at time intervals over a 4 day period. Glucose

(diamonds), purified GOS (triangles) and carbon excluded control (circles). Figure 3: HPAEC-PAD separation and analysis of GOS mixture before (dotted line) and after growth (solid hne) in B. thetaiotao micron chemically defined minimal media plus GOS. Compounds identified in GOS mixture after growth: 6-D-Galp-(l→4)-[6-D-Galp-(l→6)-]D-Glcp and galactobiose. Verification that the branched DP3 compound was 6-D-Galp-(l→4)-[6-D- Galp-(1→6)-]D-Glcp was done by H¹ NMR analysis.

Figure 4: Graphical representation of the pectic galactan PUL from B. thetaiotaomicron. GH2, family 2 glycoside hydrolase, GH53, family 53 glycoside hydrolase, SusE, SusD and SusC-like substrate binding and transport proteins, and hybrid two-component sensor regulator (HCTS). Protein identification numbers are listed above each PUL component.

Figure 5: Graphical representation of the pectic galactan (panel a) and linear GOS (panel b) substrates showing the structural similarities between these two classes of compounds. Galactose is depicted as light grey circles, glucose is depicted as dark grey circles.

Figure 6: Enzymatic activity of BtGH53 on pectic galactan. (a) TLC analysis of products formed in time by BtGH53 incubated with potato galactan. (b) HPAEC-PAD profile of galactan derived products formed in time. Figure 7: Enzymatic activity of BtGH53 on GOS. (a) TLC analysis of the products formed by enzymatic digestion of BtGH53 on GOS mixture. Lane 1: Pectic galactan products standard. Lane 2: GOS, Lane 3: GOS + BtGH53. 10 μΐ of BtGH53 was incubated with 10 μΐ of 1% GOS in reaction buffer for 16 h at 37°C. Afterwards, 2 x 2 μΐ of each reaction was spotted on a TLC plate and run for 4 h in solvent system containing 3: 1: 1 isopropanol, ethyl acetate, water. Spots were visualized using stain containing 20% sulfuric acid, 80% methanol and 0.5% orcinol after heating at 110° C for 20 min. (b) Corresponding HPAEC-PAD analysis of BtGH53 activity on GOS (dotted line: before incubation; solid line: after incubation) showing depletion of higher DP GOS molecules and accumulation of galactobiose.

Figure 8: A schematic comparison of GOS compounds potentially inducing O-glycan utihzation pathways (a) (this study) with core hMOS glycaii structures (b)(Marcobal et ah, 2011) and mucin core glycan structures (c)(Bergstrom and Xia, 2013).

EXPERIMENTAL SECTION Materials and Methods

Bacterial Strains, media and reagents

Bacteroides thetaiotaomicron VPI-5482 was purchased from DSMZ (DSM 2079, ATCC 29148) (Braunschweig, Germany). The Vivinal ® GOS and TS0903 GOS mixtures were provided by FrieslandCampina (NL). 6- GalactosylLactose was purchased from Carbosynth (UK). Pectic galactan (potato) and azo-galactan were purchased from Megazyme (UK). All other media and reagents were purchased from Sigma (Zwijndrecht, Netherlands) unless otherwise stated.

Isolation of hMOS hMOS were isolated from a human milk sample that was collected from a volunteer, collecting a full feeding of at least 100 mL around 1 month postpartum. 10 mL human milk samples were centrifuged at 5000 rpm for 30 min at 4 °C. The clear liquid was applied on a graphitized carbon column (10 g, graphitized carbon black, 20-60 mesh, SigniaAldrich) and washed with 30 mL Milli-Q water. The majority of lactose was removed by washing with 30 mL 2% ACN and hMOS were eluted with 40% ACN, containing 0.05% TFA.

B. thetaiotaomicron growth experiments B. thetaiotao micron growth was carried out as described previously

(Martens et al., 2008). Briefly, overnight cultures of B. thetaiotao micron were grown at 37°C in rich medium (Brain Heart infusion broth + 5 mM L- cysteine) under anaerobic conditions in an anaerobic jar (Oxoid) with a GasPak (BD). The following day, 1 ml of a 50 fold dilution containing B. thetaiotao micron overnight culture was prepared in a carbon-limited minimally defined medium of 100 mM KH2PO4 (pH 7.2), 15 mM NaCl, 8.5 mM (NH₄)2S04, 4 mM L-cysteine, 1.9 mM hematin, 200 mM L-histidine, 100 nM MgCl₂, 1.4 11M FeS0 ^■ 7 H₂0, 50 mM CaCl₂, 1 mg/ml vitamin K3, 5 ng/ml vitamin B12 and individual carbon sources (0.5%, wt/vol). Cultures containing 2 ml total volume were prepared in Hungate tubes under anaerobic conditions using Hungate techniques by flushing sealed culture tubes with 100% CO2 gas (Hungate, 1950). Growth curves were obtained by incubating tubes at 37°C and taking optical density readings at 600 nm (OD600) at ~1 - 2 h time intervals over a 4 day period.

HPAEC-PAD

GOS components and products of enzymatic digestion were analyzed by high-pH anion-exchange chromatography on a Dionex DX500 work station equipped with an ED40 pulsed amperometric detection system (HPAEC- PAD) as described previously (Sander S. van Leeuwen et al., 2014). The oligosaccharides were separated on a CarboPac PA-1 column (250 by 5 mm; Dionex) using a system of buffer A = 0.1 M NaOH, buffer B = 0.6 M NaOAc in 0.1 M NaOH, buffer C = deionized water and buffer D = 50 mM NaOAc. Separation was performed with 10% A, 85% C and 5% D in 25 min to 40%> A, 10% C and 50% D, followed by a 35-min gradient to 75% A, 25% B, directly followed by 5 min washing with 100% B and reconditioning with 10% A, 85% B and 5% D for 7 min.

Proteomics and enzyme activity analysis After growth, cultures were centrifuged to obtain supernatants which subsequently were filtered through a 0.2 μηι-pore-size syringe filter

(Millipore). Identification of proteins in culture supernatants produced by B. thetaiotaomicron were analyzed as described previously (Lammerts van Bueren et al., 2015). For CAZyme activity in culture supernatants, 100 μΐ of culture supernatant was added to 100 μΐ of a 10 mg/ml solution of GOS carbohydi'ate solution in deionized water and incubated at 37°C overnight to allow the enzyme reactions to proceed. Analysis of the activity of the

BtGH53 endo-galactanase on GOS substrates was carried out in Reaction buffer at 37°C and carbohydrate products were analyzed by HPAEC-PAD (as described above) and by TLC.

Thin-layer chromatography (TLC) of GOS carbohydrates and enzymatic products was completed as described in the literature (Koropatkin and Smith, 2010). Briefly, 2 μΐ of each reaction mixture was spotted, dried, and then subsequently spotted again on a sihca gel 60 plate (Millipore). The solvent system used was 3: 1: 1 isopropanol, ethylacetate, and deionized water, and plates were run in a TLC jar for approximately 4 to 5 h. Plates were removed from the jar and dried, and spots were visualized by staining with 20% sulfuric acid plus 0.5% orcinol in methanol and heated at 110°C for half an hour. Plates were scanned and figures prepared using Adobe Photoshop.

Production ofBtGH53

BtGH53 (BT_4668) was amplified from B. thetaiotaomicron VPI-5482 genomic DNA by PCR using the Forward primer

CAGGGACCCGGTGAAGATGGCCCGGTTACAAATCCTCG and Reverse primer CGAGGAGAAGC C CGGTTATTGGATTTTAAAAGC ATCTAGTGC containing a stop codon (underlined). The resulting amplified DNA was cloned into a modified pET15b vector (Novagen) containing a LIC (ligation independent cloning) site via the incorporated LIC-specific primer sequences (indicated in bold). The resulting expression vector pBtGH53 encoded BtGH53 with an additional N-terminal His6 tag. Recombinant BtGH53 was produced in Escherichia coli BL21*DE3 cells (Novagen) harboring the pBtGH53 plasmid. Cells were grown in Luria Bertani (LB) broth containing ampicillin at a concentration of 100 pg/nil at 37°C until an OD600 of 1.1 was reached. Protein overproduction was induced by the addition of 1 mM isopropyl β-D-thiogalactopyranoside and cultures were then further incubated for 16 h at 25°C. Bacterial cells were harvested by centrifugation, resuspended in buffer containing 20 mM Tris pH8.0, 500 mM NaCl, and lysed by sonication. Recombinant His6-tagged BtGH53 was purified from supernatants by immobilized metal affinity chromatography (IMAC) on a nickel-sepharose column (GE Healthcare) preequilibrated with the same buffer and eluted with a gradient of imidazole. Eluted fractions were assessed by SDS-PAGE for the presence of BtGH53 with a size of 39.6 KDa. Pooled fractions of BtGH533 were dialyzed into buffer consisting of 20 mM Tris pH 7.5, 150 mM NaCl (Reaction buffer). The concentration of BtGH53 protein was determined spectrophotometrically at 280 nm using an extinction coefficient of 0.07326 μΜ^{- 1} cm ¹.

Determination ofBtGH53 specific activities BtGH53 endo-galactanase activity was determined using the dyed substrate Azo galactan (Megazyme) according to manufacturer's instructions. A reaction mixture containing 500 μΐ 2% Azo-Galactan in 50 mM phosphate buffer pH 7.0 plus 500 μΐ of 5 μΜ BtGH53 in the same buffer was incubated for 10 min at 37°C. After incubation, the reaction was stopped and the residual high molecular weight polymer was precipitated by the addition of 2.5 ml of cold 100% ethanol. The reactions were centrifuged for 10 min at 2800 g and the absorbance of supernatants was measured at 590 nm.

Activity was calculated using the standard curve supplied by the manufacturer. BtGH53 activity on galactan and GOS substrates was analyzed using TLC and HPAEC-PAD as stated above.

Results

B. thetaiotaomicron consumes distinct components of GOS mixtures For this study, we made use of a purified GOS mixture (TS0903, provided by FrieslandCampina) based upon Vivinal ® GOS. We first analyzed the compounds present in the mixture using HPAEC-PAD and compared this to the composition of Vivinal ® GOS which has been previously reported (Sander S van Leeuwen et al., 2014). We could identify the majority of compounds within this purified GOS mixture, in terms of its carbohydrate content, linkage type and degree of polymerization (Fig 1).

The purified GOS mixture has a considerably higher linear DP3 content than Vivinal GOS (30% versus 15%) (Table 1) with 4-galactosyllactose as major peak (Fig 1). In Vivinal GOS, the DP2 content is significantly greater than in TS0903 GOS (27%. versus 8%.); Vivinal GOS contained much higher amounts of the starting material lactose than purified GOS (19% versus l%i). Also, purified GOS contained greater amounts of DP4 and higher GOS than Vivinal GOS (38%> versus 11%), and a higher proportion of branched compounds (17% versus 7%).

Table 1: Ratios of GOS compounds expressed in a percentage of total GOS mixture as determined by relative peak heights obtained by HPAEC-PAD elution profiles.

Percentage (%) of total mixture

GOS component Vivinal^® GOS TS0903 Purified GOS

Galactose 0.68 2.03

Glucose 15.34 3.92

0-D-Galp-(l- &^ ^ -D-Gai 0 1.32

Lactose 19.23 1.08

|3-D-Galp-(l- 6)-^-D-Galp-(1^4)-D-Glcp 5.60 3.83 (6-galactosyI-lactose)

Linear DP3 ^> 14 83 29.19 fcrat ned J P3 ^c 4J9 $$?

Linear DP4 ^d 6.60 17.45

Branched 0P4 * 1 3 10,14

>DP5 2.80 10.35

³ lactose derivatives -D-Galp-(l- 2)-3-D-Glc, -D-Galp-(l->3)- -D-Glc and -D-Galp-(l- 6)-p- D-Glc.

b includes β-0-63ΐρ-(1->4)-β-0-63ΐρ-(1->4)-0-6Ιΰ , β-0-63ΐρ-(1->4)-β-0-63ΐρ-(1->2)-0-6Ιΰρ and -0-σ3ΐρ-(1->4)-β-0-63ΐρ-(1- 3)-0-6Ιΰρ. c includes β-0-63ΐρ-(1- 3)-[β-0-63ΐρ-(1->6)-]0-6^ρ, β-ϋ-63ΐρ-(1- 2)-[ -0-63ΐρ-(1- 6)-]0-6^ρ and β-0-63ΐρ-(1->4)-[β-0-63ΐρ-(1->2)-]0-6^ρ.

d corresponding DP3 compounds with an additional β-0-63ΐρ-(1- 4)- at the non-reducing end.

We chose to use the purified GOS mixture in our studies because it consisted of the presumable indigestible portion of GOS, i.e. only those compounds which would enter the lower gastrointestinal tract after being exposed to human digestive enzymes in the small intestine.

To investigate metabolic properties of GOS consumption, we grew B.

thetaiotaomicron in a carbon-limited minimal medium containing the purified GOS mixture at 5 mg/ml concentrations. B. thetaiotaomicron exhibited excellent growth on GOS (Fig 2), reaching OD values equivalent to that of an equal amount of glucose suggesting that the majority of the GOS compounds were metabolized. Using HPAEC-PAD analysis of culture supernatants we observed that the majority of GOS compounds within the mixture were consumed by B. thetaiotaomicron. From a comparison of the HPAEC-PAD profiles of the starting mixture with the supernatant after bacterial growth, we identified the 2 compounds remaining in culture supernatants as a branched DP3 compound 6-D-Galp-(l→4)-[6-D-Galp- (1→6)-]D-Glcp and the disaccharide 6-D-Galp-(l→4)-6-D-Gal which appears to be in a shghtly larger quantity than the initial starting material (Fig 3).

Identification of galactan and mucin O-glycan PULS involved in B. thetaiotaomicron GOS consumption

In order to determine which PULs are produced in response to GOS, we took the culture supernatants after growth and analyzed for proteins and enzymes using mass spectrometry as has been previously described

(Lammerts van Bueren et al., 2015), Previously it was found that B.

thetaiotaomicron consumes hMOS using a distinct set of pathways attributed to mucin-O-glycan utilization (Marcobal et al. , 2011) (Table 2).

Since GOS are thought to mimic properties of hMOS, we aimed to identify whether similar features were also activated by GOS in B. thetaiotaomicron

(Table 2). Table 2: SusC/D pairs identified in proteomics analysis of B. thetaiotaomicron culture supernatants after growth on lactose, galacto-oligosaccharides and hMOS. Proteins identified are unique PULs after considering glucose controls.

SusC gene ID Lactose GOS hMOS hMOS (a)

BT_0317 - - + -

BT_0439 - + + -

BT_0867 - - + -

BT_1040 - - + +

BT_1631 - - + -

BT_2032 + + + -

BT_2626 - - - +

BT_2805 - + + +

BT_3958 - + + +

BT_4039 - - + +

BT_4135 - - + +

BT_4247 - + + -

BT_4298 - + + +

BT_4671 - + - -

SusD gene ID Lactose GOS hMOS hMOS (a)

BT_0318 - - + -

BT_0866 - - + -

BT_1039 - + + +

BT_1630 - - + -

BT_2033 + + + -

BT_2365 - + + -

BT_2559 - + + -

BT_2625 - + + +

BT_2806 - - + +

BT_3520 - + + -

BT_3959 - + + +

BT_3984 - - + -

BT_4038 - - + +

BT_4134 - - + +

BT_4246 - + + -

BT_4297 - + + +

BT_4670 - + - - ^a as identified in (Marcobal et al, 2011)

As a confirmation that proteomics is a suitable method for detecting expressed components of PULs, we grew a culture of B. thetaiotaomicron on defined minimal medium containing 5 mg/ml purified hMOS substrates isolated from a donor milk sample as a sole carbon source to compare with previous hMOS qPCR gene expression analysis results (Marcobal et al, 2011).

Within the sets of proteomics data we identified several SusC-like and SusD-like proteins from GOS and hMOS culture filtrates (Table 2).

SusC/SusD homologs (Cho et al , 2001) (Martens et al, 2009b) form the major transport components of PULs and expression of these homologs is an indication of PUL activation by specific carbohydrate inducer substrates. We observed that TS0903 GOS molecules induce expression of several SusC and SusD homologs in B. thetaiotao micron that are associated with hMOS and mucin utihzation PULs (this study and (Marcobal et al , 2011)). In total, GOS induced the expression of five PUL SusC/D homologs that were also upregulated by hMOS and mucin (Marcobal et al, 2011) (Martens et al., 2008). These are BT_2032/BT_2033, BT_3958/BT3959, BT_4246 BT_4247, BT_4297/BT_4298 and BT_2805/BT_2806. In previous studies,

BT_3958/BT_3959 was identified as a mucin O-glycan PUL upregulated by the Core 1 (^saccharide, while BT_4246/BT_4247, BT_4297/BT_4298 and BT_2805/BT_2806 are upregulated in the presence of mucin O-glycans of undefined structure (Martens et al, 2008) (Marcobal et al, 2011). The PUL identified by BT_2032/BT_2033 is a separate PUL that appears to be induced by lactose and had not been previously identified as a PUL upregulated by mucin-O-glycans when compared to the galactose controls (Marcobal et al , 2011). This specific PUL maybe expressed due to catabolite derepression caused by an accumulation of galactose; this PUL remains of undefined function. In addition there were several individual SusC and SusD proteins identified in GOS culture supernatants that are associated with B. thetaiotaomicron hMOS consumption (Table 2). An additional SusC transporter (BT_0439) implicated in degradation of host glycans, and two additional SusD binding protein homologs induced by mucin O-glycans, BT_1039, and N- acetyllactosamine specific BT_2559, were identified.

SusC/D homologs of another prominent single PUL were identified in GOS culture supernatants that were neither found in hMOS nor in mucin culture supernatants. These were identified by the genetic ID loci

BT_4670/BT_4671. These form the transport components of the putative galactan utilization system in B. thetaiotao micron. Galactan utihzation systems are employed by bacterial species for degradation and uptake of pectic galactan (Tabachnikov and Shoham, 2013)(Delangle et al., 2007), a linear polymer of 6-(l→4)-linked galactose, that constitutes a structural component of pectin in plant cell walls. B. thetaiotao micron encodes a PUL system for pectic galactan degradation (BT4667 - BT4673) which includes an encoded extracellular endo-6-(l→4)-galactanase (family GH53) for degrading larger galactan polymers (Fig 4)(Ryttersgaard et al. , 2004).

Previous studies on GOS utilization by the probiotic bacterium

Bifidobacterium breve have also shown that GH53 endo-6-galactanase activity is important for GOS utihzation by Bifidobacteria(O'Connell Motherway et al., 2013). Therefore we went on to investigate what the contribution of this endo-galactanase activity is towards utihzation of GOS substrates.

Influence of pectic galactan associated Endo- -Galactanase

Comparison of GOS with pectic galactan reveals the same structural motifs in pectic galactan and in GOS compounds that have a higher DP and contain extensions of 6-(l→4)-linked galactose residues (Fig 5). Since pectic galactan is a dietary polysaccharide and has not been previously shown to be associated with hMOS effects (Table 2), galactan utihzation PUL induction in B. thetaiotaomicron in the presence of GOS would be separate from mucin utilization - associated function. The proteome analysis from GOS culture supernatants identified the presence of the extracellular family GH53 endo-6-galactanase (BT4668) from the galactan utilization PUL while we did not find this enzyme in hMOS samples (Table 2). To study the potential effects of galactan PUL activation, we investigated the activity of the endo-6-galactanase GH53 enzyme (BT4668) with GOS substrates. We cloned the gene BT4668 encoding endo-galactanase enzyme from B. thetaiotaomicron (herein called BtGH53), expressed it in E. coli and purified the enzyme to test for its activity on GOS. The enzyme was produced in soluble form and in large quantities (approx. 100 mg/L culture) and purified to >95% purity. BtGH53 was found to be active on 6-(l→4)-linked potato galactan (Fig 6) but was not active on larch arabino galactan with 6-(l→3)-linkage type (not shown). At the initial stages (1 min), BtGH53 converted potato galactan into GOS products with a degree of polymerization ranging from DP 1-8 (Fig 6). BtGH53 thus is an endo-acting β-galactanase enzyme. As the reaction progressed, GalDP4 and higher were hydrolyzed further to produce GalDP3, GalDP2 and galactose. At completion (circa 20 h) the main products were galactose (23%) and galactobiose (77%). These results suggest that BtGH53 is an endo-6-galactanase but also has some exo-activity in view of the presence of galactose and activity on GalDP3 substrate. Subsequently we tested for BtGH53 activity on the purified GOS mixture (Fig 7). TLC analysis showed that BtGH53 facilitates hydrolysis of GOS that have a higher degree of polymerization forming galactobiose as the major product (Fig 7a). By further examining the products of

depolymerization using HPAEC-PAD we observed that BtGH53 hydrolyzes linear DP3 and higher GOS compounds (i.e. those compounds that contain at least two 6-(l→4)-linked galactose residues) forming galactobiose as the main product (Fig 7b). As an enclo-6-galactanase BtGH53 thus acts on the linear high DP GOS compounds that share similar motifs to native pectic galactans (Fig 5). Also the branched DP4 GOS in region B were partially hydrolyzed, suggesting that GH53 may be cleaving terminal galactose from these compounds. GH53 enzymes from fungal origin have been observed to have exo-activity (Torpenholt et al., 2011), while the GH53 from Bacillus licheniformis did not (Ryttersgaard et al., 2004). Because we observed that BtGH53 degrades galactan DP3 products to DP2 plus galactose (Fig. 6), the exo-activity on these substrates is plausible, however more experimental evidence would be needed to support this hypothesis.

We observed that the branched DP3 compounds 6-D-Galp-(l→4)-[6-D-Garp- (1→6)-]D-Glcp and 6-D-Galp-(l→2)-[6-D-Galp-(l→6)-]D-Glcp, 6-D-Galp- (l→3)-[6-D-Galp-(l→6)-]D-Glcp and 6-D-Galp-(l→4)-[6-D-Galp-(l→2)-]D- Glcp (region A) and the linear DP3 compound 6-galactosyl-lactose were recalcitrant to BtGH53 activity. The DP2 compounds Gal-6-(l→2)-Glc, Gal- 6-(l→3)-Glc, Gal6-(1→6)-Gal, Gal6-(1→6)-Glc (allolactose) also were not cleaved. Additionally, we observed an accumulation of lactose and Gal-6- (l→2)-Glc/Gal-6-(l→3)-Glc after BtGH53 activity, likely arising from the hydrolysis of higher DP compounds (Fig 7b).

BtGH53 thus facilitates the degradation of linear and elongated branched GOS to produce galactobiose as the major product. The remaining GOS molecules that are not targeted by BtGH53 are DP2 Gal6-(1→2)-Glc and Gal 6-(l→3)-Glc, Gal-6-(l→6)-Glc (allolactose) and Gal-6-(l→6)-Gal, linear DP 3 6-GalLac, and the branched DP3 compounds 6-D-Galp-(l→2)-[6-D-Galp- (1→6)-]D-Glcp, 6-D-Galp-(l→3)-[6-D-Galp-(l→6)-]D-Glcp and β-D-Galp- (l→4)-[6-D-Galp-(l→2)-]D-Glcp (Fig 8a). These eight compounds constitute a class of GOS molecules that are potentially important in promoting the colonization and growth of beneficial gut bacteria in a similar manner as hMOS. Our data provide the first time demonstration that prebiotic GOS mixtures activate the galactan PUL and a subset of mucin glycan utilization PULs in B. thetaiotaomicron.

In this study we performed a detailed analysis of the effects of GOS on the activation of PULs in B. thetaiotaomicron in order to unravel what features of GOS provide the qualities of hMOS mimicry in intestinal microbiota. We found that consumption of GOS by B. thetaiotaomicron, a dominant bacterial species of the distal gastrointestinal tract, results in the activation of a combination of mucin glycan and galactan utilization pathways. Higher DP GOS compounds are metabohzed by an endo-galactanase BtGH53, while small DP2 and branched GOS trisaccharides acted as potential inducers for the expression of mucin glycan degradation PUL-encoded systems in B. thetaiotaomicron. This was indicated by the presence of SusC and SusD homologs in B. thetaiotaomicron culture supernatants which correlated with the bacterium expressing transporters that are involved in the uptake of specific products of glycan degradation (Martens et al., 2009a) (Lammerts van Bueren et al., 2015).

We observed that the galactan utilization pathway plays an important role in metabolism of GOS by B. thetaiotaomicron. Longer DP GOS carrying 6- (l→4)-galactan motifs are degraded by the GH53 endo-6-galactanase, whilst some of the mucin-glycan pathway inducer molecules are only uncovered after endo-6-galactanase activity. Therefore, bacteria that harbor the ability to degrade galactan will uncover some of the underlying mucin inducer molecules buried in the higher DP GOS molecules. Several members of Firmicutes and Bacteroidetes encode GH53 endo-6-galactanase enzymes (Table 3) (www.cazy.org/GH53.html) (Lombard et al., 2014), and

interestingly they are found in many Bifidobacterium and Bacteroides strains that colonize the infant gut. Bifidobacterium strains that encode GH53 enzymes include B. breve, B. longum sub infantis and B. longum sub longum that are also found in the gut of breast-fed infants (Sela, 2011) (Table 3).

Table 3: The presence of GH53 enzymes in microbial strains associated with gut microbiota.

Based on what is known about the composition of hMOS and core mucin glycaiis (Fig 8) (Bergstrom and Xia, 2013) (Marcobal and Sonnenburg, 2012), we hypothesize that the Gal 6-(l→3)-Glc and 6-GalLac molecules structurally mimic the core mucin and hMOS motifs, while the branched DP 3 compound 6-D-Galp-(l→3)-[6-D-Galp-(l→6)-]I)-Glcp structurally mimics the branched core mucin glycan motifs found in core 2 and core 4 glycans. Allolactose (Gal-6-(l→6)-Glc) present in GOS is typically an inducer of the Lac-operon and is degraded by lactose-specific β-galactosidases (Wheatley et al, 2013). The presence of specific polysaccharides will dictate which bacteria dominate in their environment (Rogowski et al., 2015) (Koropatkin et al., 2012). We show herein that specific GOS molecules can induce mucin glycan utilization pathways which may help promote colonization and adaptation of bacterial species in the GI tract. Mucin glycan foraging is essential for persistence of B. thetaiotaomicron in the gut (Martens et al., 2008) while hMOS promote intestinal colonization of B. thetaiotaomicron via mucin-associated pathways (Marcobal et al., 2011). Therefore, the specific GOS molecules identified in this study that trigger mucin glycan utihzation pathways may act as "intestinal adaptation factors" to promote colonization and adaptation of mucin-degrading bacteria. It is likely that the

combination of these effects contributes to the features of GOS as HMO- mimics. The metabolic features of GOS revealed in this study are useful for the future design of fermentable fibres or prebiotic compounds that selectively promote the growth and colonization of beneficial bacteria or for the selection of bacterial strains that may be administered as probiotics in combination with galactooligosaccharides.

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Claims

Claims

1. Use of a galacto-oligosaccharides (GOS) composition comprising branched and linear GOS species having a degree of polymerization (DP) of 3, wherein the branched DP3 GOS species are present in excess of linear DP3 GOS species, for inducing mucin glycan utihzation pathways in beneficial gut bacteria in an animal.

2. The use according to claim 1, wherein said branched DP3 GOS species are present in an amount of at least 15% by weight (w%), preferably at least 20w%>, based on the total dry weight of all GOS species in the composition.

3. The use according to claim 1 or 2, wherein the composition comprises GOS species comprising β-linked galactosyl residues, in particular β(1-6) hnkages, β(1-3) linkages and/or β(1-2) linkages.

4. The use of any one of the preceding claims, wherein the composition comprises one or more branched DP3 GOS species selected from the group consisting of 6-D-Galp-(l-2)-[6-D-Galp-(l-6)-]D-Glcp, 6-D-Galp-(l- 3)-[6-D-Galp-(l-6)-]D-Glcp and 6-D-Galp-(l-4)-[6-D-Galp-(l-2)-]D-Glcp.

5. The use of any one of the preceding claims, wherein the composition comprises the linear DP3 GOS species 6-galactosyl-lactose and/or 3 -galactosyl -lactose.

6. The use of any one of the preceding claims, wherein the composition furthermore comprises DP2 GOS species, preferably one or more selected from the group consisting of Gal6-1,2-Glc, Gal 6- 1,3-Glc, Gal- 6-1,6-Glc (allolactose) and Gal-6- l,6-Gal.

7. The use of any one of the preceding claims, wherein the

composition comprises the branched DP3 GOS species 6-D-Galp-(l-2)-[6-D- Galp-(l-6)-]D-Glcp, 6-D-Galp-(l-3)-[6-D-Galp-(l-6)-]D-Glcp 6-D-Galp-(l-4)-[6- D-Galp-(l-2)-]D-Glcp, and the DP2 GOS species Gal6- 1,2-Glc, Gal 6-l,3-Glc, Gal-6- l,6-Glc (allolactose) and Gal-6- l,6-Gal.

8. The use of any one of the preceding claims, wherein all GOS species present in the composition make up at least 30w%, 40w%, 50 w%, preferably at least 60 w%, more preferably at least 70 w% based on the dry weight of the composition.

9. The use of any one of the preceding claims, wherein the

composition comprises at least one further prebiotic component, preferably galactan.

10. The use of any one of the preceding claims, wherein the

composition is a food product or dietary supplement product, preferably wherein the food product is selected from the group consisting of an infant formula, a follow-on formula, and a toddler beverage.

11. The use of any one of the preceding claims, wherein the

composition further comprises a Bacteroides and/or a Bifidobacterium strain, preferably B. thetaiotaomicron, Bifidobacterium breve,

Bifidobacterium longum bv. infantis and/or Bifidobacterium bifidum.

12. The use according to any one of the preceding claims, wherein the animal is a human, preferably wherein the human is less than 5 years old or wherein the human is an elderly subject, for example over 50 years old.

13. Use of any one of the preceding claims, wherein inducing mucin glycan utilization pathways results in any one or more of the following: a) promoting colonization and adaptation of mucin-degrading bacteria;

b) facilitating bacterial transmission to infants;

c) stimulating colonization of the gut of the animal by at least one beneficial bacterial strain.

14. Use according to claim 13, wherein inducing mucin glycan utilization pathways results in stimulating colonization of the gut of the animal by a Bacteroides and/or a Bifidobacterium strain, preferably B. thetaiotaomicron, Bifidobacterium breve, Bifidobacterium longum bv.

infantis, and/or Bifidobacterium bifidum.

15. A method for providing a galacto-oligosaccharides (GOS) composition comprising DP2 and branched DP3 species and capable of inducing mucin-glycan pathways in beneficial bacteria, said method comprising providing a mixture of GOS having varying degrees of polymerization and subjecting the mixture to an enzyme capable of degrading one or more less desired GOS species, the enzyme being an endo- β-galactanase GH53 enzyme.

16. Method according to claim 15, wherein the endo-6-galactanase GH53 enzyme is endo-6-galactanase GH53 from B. thetaiotaomicron, preferably recombinantly produced B. thetaiotaomicron endo-6-galactanase GH53.

17. Method according to claims 1 or 16, wherein providing said mixture of GOS having varying degrees of polymerization comprises a transgalactosylation reaction using β-galactosidase, preferably 6- galactosidase from L. reuteri or B. circulans.

18. A GOS composition obtainable by a method according to any one of claims 15-17.

19. Use of a recombinant endo-6-galactanase GH53 enzyme in the manufacture of a prebiotic composition comprising galacto-oligosaccharides (GOS).

20. The use according to claim 19, wherein said endo-6-galactanase GH53 enzyme is recombinantly produced B. thetaiotao micron endo-6- galactanase GH53.

21. A method for providing a GOS fraction capable of inducing mucin - glycan pathways in beneficial bacteria, said method comprising the steps of:

- providing a mixture of galacto-oligosaccharides (GOS) having varying degrees of polymerization;

- optionally removing free lactose from said GOS mixture;

- applying said (lactose-free) GOS mixture to an anion exchange resin;

- step-wise eluting GOS with increasing degree of polymerization using a water mobile phase and collecting separate eluent fractions;

- analyzing each eluent fraction for the effect on expression of a gene associated with mucin utihzation; and

- selecting one or more fraction(s) capable of promoting expression of a gene associated with mucin utilization, wherein said gene associated with mucin utilization is SusC, SusD or a homolog thereof.