Galactooligosaccharide Composition

Abstract

A prebiotic oligosaccharide composition, comprising: (a) at least 8% by weight of Gal-(β1-3)-Gal-(β1-4)-X, based on the total weight of the oligosaccharide compounds present in the composition; a (b) at least 3% by weight of Gal-(β1-3)-Gal-(β1-3)-X b and (c) at least 5% by weight of Gal-(β1-3)-Gal-(β1-2)-X. c wherein X a , X b and X c and (b) are each independently selected from monosaccharides. These compositions contain relatively high amounts of oligosaccharide compounds (a), (b) and (c) and relatively high amounts of β1-3 Gal-Gal linkages compared to known oligosaccharide compositions. These particular characteristics of the compositions are believed to provide benefits to the gut health of consumers, for example, because these compositions provide increased production of butyric acid in the gut of consumers compared to known compositions. Methods for preparing the compositions and uses of the compositions as dietary supplements or medicaments are also disclosed.

Description

[Technical field]

[0001] Field The present invention relates to compositions comprising oligosaccharide compounds, methods for preparing said compositions and the use of said compositions as dietary supplements. In particular, the present invention relates to oligosaccharide compositions having relatively high amounts of certain beneficial oligosaccharides. [Background technology]

[0002] background The human gut microbiota (or microflora) comprises genera of pathogenic, benign and beneficial microorganisms. The predominance of the former can lead to intestinal disorders that can be both acute (e.g. gastroenteritis) and chronic (e.g. inflammatory bowel disease, irritable bowel syndrome and some intestinal cancers). In humans, the large intestinal microbiota is acquired at birth. Breast-fed infants have a predominance of bifidobacteria, which easily outcompete other genera of bacteria. This is because components of human milk stimulate the growth of bifidobacteria. However, many factors, such as environment, drug treatment and diet, especially the consumption of highly processed foods, can affect the composition and activity of the intestinal microbiota over an individual's lifetime (see “Role of the gut microbiota in nutrition and health”, Valdes, AM et al., BMJ, 2018, 361).

[0003] Certain components of gut microbiota are involved in the pathogenesis of intestinal diseases.For example, mycobacteria are associated with Crohn's disease, ulcerative colitis can be caused by sulfate-reducing bacteria, and bacteria may be involved in the development of intestinal cancer.It would be beneficial if the selective growth of indigenous beneficial gut bacteria could be promoted by the intake of prebiotics.Prebiotics are substrates that are selectively utilized by host microorganisms to provide health benefits.The use of such prebiotics can have the ongoing beneficial effect of suppressing the growth of pathogenic microbiota.

[0004] Attempts have been made to influence the balance of gut microbiota in favor of beneficial microorganisms, such as bifidobacteria, by adding one or more such microbial strains to a suitable food medium with the intention of providing a health benefit to the host. Such live microbial feed supplements are known as probiotics. However, it is difficult to guarantee the survival of live bacteria in food after digestion, and as a result, the actual effect of probiotics may be limited. Therefore, prebiotics may be a more promising option to positively influence gut microbiota in patients (see Walter J, Maldonado-Gomez MX, Martinez I. “To engraft or not to engraft: an ecological framework for gut microbiome modulation with live microbes”, Curr Opin Biotechnol. 2018, 49, 129-139).

[0005] As noted above, an alternative approach to dietary manipulation of the gut microbiota is the use of prebiotics, defined as non-digestible food components that beneficially affect the subject by selectively stimulating the growth and / or activity of one or a limited number of bacteria in the gut, thereby resulting in improvements in host health.

[0006] One group of compounds classified as prebiotics is galactooligosaccharides, which are galactose-containing oligosaccharides produced by the transgalactosylase activity of β-galactosidase enzyme from lactose.Known galactooligosaccharide prebiotic products contain a mixture of many oligosaccharide compounds in various ratios, and not all of them have the desired beneficial effect on the gut microbiota of consumers.Although such products have shown some beneficial results in improving the gastrointestinal health of patients, there is still a need for further improved oligosaccharide compositions to fully realize the potential benefits of such prebiotics to consumers. Summary of the Invention

[0007] In particular, it is an object of the present invention to provide a composition comprising an oligosaccharide compound that addresses at least one disadvantage of the prior art, whether specified herein or elsewhere, or to provide an alternative to existing compositions. For example, it may be an object of the present invention to provide a composition comprising an oligosaccharide compound that contains a higher proportion of certain beneficial oligosaccharides than known oligosaccharide compositions.

[0008] According to aspects of the present invention there are provided compositions methods and uses as set out in the accompanying claims. Other features of the invention will be apparent from the dependent claims and the description which follows. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows the results of butyric acid analysis when the compositions of inventive Example 2, Comparative Example 1 and a control blank sample were subjected to dialysis using a 0.5 kDa membrane to obtain 5 g / l samples which were then mixed with feces obtained from three healthy human adult subjects (Donors A, B and C) and the resulting mixtures were shaken under anaerobic conditions and monitored for colonic fermentation products, including butyric acid, over a 48 hour period (at collection points of 6, 24 and 48 hours). [Diagram 2]Figure 2 shows the effect of different novel oligosaccharides on the bacterial counts (log colony forming units (CFU / mL)) of Lactobacillus species (LAB, A), Bacteroidae and Prevotellaceae families (BAC, B), Clostridium coccoides-Eubacterium rectal group (EREC, C), Roseburia subcluster (RREC, D), Atopobium-Coriobacterium species (ATO, E), Clostridium cluster IX (PROP, F), Faecalis prausnitzii (FPRAU, G), Desulfovibrio species (DSV, H) and Clostridium histolyticum (CHIS, I) detected by flow FISH in pH-controlled batch culture experiments. [Diagram 3] FIG. 3 shows the effect of Lactobacillus species (LAB, A), Bacteroidae and Prevotellaceae families (BAC, B), Clostridium coccoides-Eubacterium rectal group (EREC, C), Roseburia subcluster (RREC, D), Atopobium-Coriobacterium species (ATO, E), Clostridium cluster IX (PROP, F), Faecalis prausnitzii (FPRAU, G), Desulfovibrio spp. (DSV, H), and Clostridium histolyticum (CHIS, I) detected by flow FISH in pH-controlled batch culture experiments of Examples 4-9 on bacterial counts (log colony forming units (CFU / mL)). [Figure 4] Figure 4 shows the concentrations of bacterial metabolites: acetate (A), propionate (B), butyrate (C) and lactate (D) detected by GC-MS at 0, 8 and 24 h in samples taken from pH-controlled batch culture studies testing newly synthesized oligosaccharides and controls. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] According to a first aspect of the present invention, there is provided a composition comprising an oligosaccharide compound, the oligosaccharide compound comprising: Based on the total weight of the oligosaccharide compounds present in the composition, (a) at least 8% by weight of Gal-(β1-3)-Gal-(β1-4)-X a ; (b) at least 3% by weight of Gal-(β1-3)-Gal-(β1-3)-X b ; and (c) at least 5% by weight of Gal-(β1-3)-Gal-(β1-2)-X c wherein X a , X b and X c are each independently selected from monosaccharides.

[0011] These types of oligosaccharides, which have a specific bond between galactose unit (designated Gal) and terminal monosaccharide, are considered to be particularly beneficial for maintaining healthy human intestinal microflora, and therefore beneficial to the health of subjects.Known oligosaccharide compositions either do not contain each of these types of oligosaccharides or contain a lower content of these oligosaccharides.Therefore, the composition of this first aspect is considered to provide consumers with advantages in establishing and maintaining human intestinal microflora when used as prebiotics.

[0012] The weight percentage of a particular oligosaccharide compound discussed herein is based on the total weight of the oligosaccharide compounds present in the total composition. Thus, when determining the specific weight percentage, only the fraction of the composition provided by oligosaccharides, either component (a), (b), (c) or any other oligosaccharide present, is considered. For this determination of oligosaccharide content, apart from lactose, disaccharides are included. The composition of this first embodiment may contain other non-oligosaccharide components, including monosaccharides and lactose. These components are not considered when determining the specific weight percentage of the oligosaccharide compound discussed herein. The specific amount of oligosaccharide compounds in a composition can be referred to as the weight percentage of the oligosaccharide fraction of the composition of this first embodiment.

[0013] The oligosaccharide compounds (a), (b) and (c) are each independently selected from sugars; a , X b and X c Each contains X a , X b and X c The group X may be considered to be a sugar unit. a , X b and X c can be considered to be the terminal sugar of an oligosaccharide compound. a , X b and X c is preferably independently selected from monosaccharides. Any suitable monosaccharide unit capable of forming an oligosaccharide with the galactose unit of compounds (a), (b) and (c) is selected from X a , X b and X c Preferably, X a , X b and X c are each independently selected from the following monosaccharides: Suitable monosaccharide units are selected from glucose (Glc), fucose (Fuc), arabinose (Ara), xylose (Xyl), rhamnose (Rha), mannose (Man), galactose (Gal), ribose (Rib), lyxose (Lyx), allose (All), altrose (Alt), gulose (Gul), idose (Ido), talose (Tal), psicose (Psi), fructose (Fru), sorbose (Sor), tagatose (Tag), galactosamine (GalN), glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) or mixtures thereof. Thus, each of compounds (a), (b) and (c) may comprise a mixture of oligosaccharide compounds having different respective X groups, e.g. either Glc or Fuc X groups.

[0014] In some embodiments, the group X a , X b and X c are independently selected from the monosaccharides listed above, preferably independently selected from Glc, Fuc, Ara, Xyl, Rha and Man or mixtures thereof.

[0015] The sugar units of the oligosaccharides in the composition of this first aspect may have either the D or L enantiomeric form. Suitably, the Gal sugar units in components (a), (b) and (c) all have the D enantiomeric form. Components (a), (b) and (c) may thus be as follows: (a)D-Gal-(β1-3)-D-Gal-(β1-4)-X a ; (b)D-Gal-(β1-3)-D-Gal-(β1-3)-X b ; and (c)D-Gal-(β1-3)-D-Gal-(β1-2)-X c .

[0016] Preferably, Gal and X a , X b and X c The sugar units all have the D enantiomeric form. Components (a), (b) and (c) may therefore be as follows: (a)D-Gal-(β1-3)-D-Gal-(β1-4)-DX a ; (b)D-Gal-(β1-3)-D-Gal-(β1-3)-DX b ; and (c)D-Gal-(β1-3)-D-Gal-(β1-2)-DX c .

[0017] In some embodiments, X a , X b and X c Each of is Glc. Thus, compounds (a), (b) and (c) may be galactooligosaccharide compounds (GOS), and the compositions of this first aspect may be referred to as galactooligosaccharide compositions. Such galactooligosaccharides may be formed by converting lactose to the mentioned oligosaccharides using a suitable galactosidase enzyme. In such an embodiment, components (a), (b) and (c) are preferably as follows: (a)Gal-(β1-3)-Gal-(β1-4)-Glc; (b) Gal-(β1-3)-Gal-(β1-3)-Glc; and (c) Gal-(β1-3)-Gal-(β1-2)-Glc.

[0018] Suitably, the Gal and Glc sugar units all have the D enantiomeric form. Components (a), (b) and (c) may therefore be as follows: (a)D-Gal-(β1-3)-D-Gal-(β1-4)-D-Glc; (b) D-Gal-(β1-3)-D-Gal-(β1-3)-D-Glc; and (c) D-Gal-(β1-3)-D-Gal-(β1-2)-D-Glc.

[0019] In some embodiments, X a , X b and X c Each of (a), (b) and (c) may comprise a mixture of Glc and one or more of the other sugar units listed above, such as Fuc, Ara, Xyl, Rha and Man. Thus, each of (a), (b) and (c) may comprise a mixture of oligosaccharide compounds having either Glc or one of the other sugar units listed above as the X group. In such an embodiment, components (a), (b) and (c) may be formed by converting a mixture of monosaccharides selected from lactose and suitable further sugars, such as fucose, arabinose, xylose, rhamnose and mannose, into oligosaccharides. Suitably, X is a mixture of monosaccharides selected from the group consisting of glycerol, ... a , X b and X c Each of these is a mixture of Glc and Fuc.

[0020] Other oligosaccharides in a composition comprising the oligosaccharide compound of this first aspect may, in addition to components (a), (b) and (c) discussed above, also contain the saccharide unit "X" groups referred to above.

[0021] The composition of this first aspect comprises: (a) at least 8% by weight of Gal-(β1-3)-Gal-(β1-4)-X a Suitably, the composition comprises at least 9% by weight of component (a), or at least 10% by weight of component (a).

[0022] Suitably, the composition comprises up to 35% by weight of component (a), up to 30% by weight of component (a) or up to 25% by weight of component (a).

[0023] Suitably, the composition comprises from 8 to 35% by weight of component (a), from 8 to 25% by weight of component (a) or from 10 to 20% by weight of component (a).

[0024] The composition of this first aspect comprises: (b) at least 3% by weight of Gal-(β1-3)-Gal-(β1-3)-X b Preferably, the composition comprises at least 4% by weight of component (b) or at least 5% by weight of component (b). Preferably, the composition comprises at least 6% by weight of component (b) or at least 8% by weight of component (b).

[0025] Suitably, the composition comprises up to 25% by weight of component (b), up to 20% by weight of component (b) or up to 15% by weight of component (b).

[0026] Suitably, the composition comprises from 3 to 25% by weight of component (b), from 4 to 20% by weight of component (b) or from 4 to 10% by weight of component (b).

[0027] The composition of this first aspect comprises: (c) at least 5% by weight of Gal-(β1-3)-Gal-(β1-2)-X c Preferably, the composition comprises at least 6% by weight of component (c). Preferably, the composition comprises at least 8% by weight of component (c).

[0028] Suitably, the composition comprises up to 25% by weight of component (c), up to 20% by weight of component (c) or up to 15% by weight of component (c).

[0029] Suitably the composition comprises from 5 to 25% by weight of component (c), from 5 to 20% by weight of component (c) or from 6 to 10% by weight of component (c).

[0030] The above amounts are based on the total weight of the oligosaccharide compound present in the composition.

[0031] Suitably, in the composition of this first aspect: Based on the total weight of the oligosaccharide compounds present in the composition, component (a) is present in an amount of at least 10% by weight; component (b) is present in an amount of at least 6% by weight; and Component (c) is present in an amount of at least 6% by weight.

[0032] Suitably, in the composition of this first aspect: Based on the total weight of the oligosaccharide compounds present in the composition, component (a) is present in an amount of up to 35% by weight; component (b) is present in an amount of up to 25% by weight; and Component (c) is present in an amount up to 25% by weight.

[0033] Suitably, in the composition of this first aspect: Based on the total weight of the oligosaccharide compounds present in the composition, Component (a) is present in an amount of 8 to 25% by weight; component (b) is present in an amount of 3 to 25% by weight; and Component (c) is present in an amount of from 5 to 20% by weight.

[0034] Suitably, in the composition of this first aspect: Based on the total weight of the oligosaccharide compounds present in the composition, Component (a) is present in an amount of 8 to 25% by weight; component (b) is present in an amount of 6 to 25% by weight; and Component (c) is present in an amount of from 6 to 20% by weight.

[0035] In some embodiments, the composition of this first aspect comprises: Based on the total weight of the oligosaccharide compounds present in the composition, (a) 8 to 25 wt % Gal-(β1-3)-Gal-(β1-4)-Glc; (b) 3 to 25% by weight of Gal-(β1-3)-Gal-(β1-3)-Glc; and (c) 5 to 20% by weight of Gal-(β1-3)-Gal-(β1-2)-Glc Includes.

[0036] In some embodiments, the composition of this first aspect comprises: Based on the total weight of the oligosaccharide compounds present in the composition, (a) 8 to 25 wt % Gal-(β1-3)-Gal-(β1-4)-Glc; (b) 6 to 25% by weight of Gal-(β1-3)-Gal-(β1-3)-Glc; and (c) 6 to 20% by weight of Gal-(β1-3)-Gal-(β1-2)-Glc Includes.

[0037] Preferably the weight percent ratio of compound (a) to compound (b) is from 1:1 to 3:1, preferably from 1.5:1 to 2.5:1.

[0038] Preferably the weight percent ratio of compound (a) to compound (c) is from 1:1 to 3:1, preferably from 1.5:1 to 2.5:1.

[0039] Preferably the weight percent ratio of compound (b) to compound (c) is from 2:1 to 1:2, preferably from 1.5:1 to 1:1.5.

[0040] Suitably, in the composition of this first aspect, the oligosaccharide compound is: Based on the total weight of the oligosaccharide compounds present in the composition, (d) at least 3% by weight of Gal-(β1-3)-Gal-(β1-3)-Gal-(β1-4)-X d wherein X d is selected from monosaccharides.

[0041] X d X a , X b and X c The monosaccharide units may be selected from the same monosaccharide units as those described above for

[0042] In some embodiments, X d is Glc.

[0043] Preferably, the Gal sugar unit of component (d) has the D enantiomeric form. Preferably, Gal and X d All of the sugar units have the D enantiomeric form. Component (d) can therefore be: D-Gal-(β1-3)-D-Gal-(β1-3)-D-Gal-(β1-4)-DX d .

[0044] In some embodiments, X d is a mixture of Glc and one or more other monosaccharide units, such as Fuc, Ara, Xyl, Rha and Man, preferably Fuc.

[0045] Suitably, the composition comprises at least 4% by weight of component (d) or at least 5% by weight of component (d).

[0046] Suitably, the composition comprises up to 25% by weight of component (d), up to 20% by weight of component (d), or up to 15% by weight of component (d).

[0047] Suitably, the composition comprises from 3 to 25% by weight of component (d), from 4 to 20% by weight of component (d), or from 4 to 10% by weight of component (d).

[0048] Suitably, the amount of component (d) present in the composition is lower than the amount of component (c) present in the composition.

[0049] Suitably, in the composition of this first aspect: Based on the total weight of the oligosaccharide compounds present in the composition, Component (a) is present in an amount of 8 to 25% by weight; component (b) is present in an amount of 3 to 25 wt. %; Component (c) is present in an amount of 5 to 20% by weight; and Component (d) is present in an amount of from 3 to 25% by weight.

[0050] In some embodiments, the composition of this first aspect comprises: Based on the total weight of the oligosaccharide compounds present in the composition, (a) 8 to 25 wt % Gal-(β1-3)-Gal-(β1-4)-Glc; (b) 3 to 25 wt % Gal-(β1-3)-Gal-(β1-3)-Glc; (c) 5 to 20% by weight of Gal-(β1-3)-Gal-(β1-2)-Glc; and (d) 3 to 25% by weight of Gal-(β1-3)-Gal-(β1-3)-Gal-(β1-4)-Glc Includes.

[0051] Suitably, in the composition of this first aspect, the oligosaccharide compound is: Based on the total weight of the oligosaccharide compounds present in the composition, (e) at least 5% by weight of Gal-(β1-4)-Gal-(β1-4)-X e wherein X e is selected from monosaccharides.

[0052] X e X a , X b and X c The monosaccharide units may be selected from the same monosaccharide units as those described above for

[0053] Preferably, the Gal sugar unit of component (e) has the D enantiomeric form. Preferably, Gal and X eAll of the sugar units have the D enantiomeric form. Component (e) can therefore be: D-Gal-(β1-4)-D-Gal-(β1-4)-DX e .

[0054] In some embodiments, X e is Glc.

[0055] In some embodiments, X e is a mixture of Glc and one or more other monosaccharide units, such as Fuc, Ara, Xyl, Rha and Man, preferably Fuc.

[0056] Suitably the composition comprises at least 6% by weight of component (e) or at least 7% by weight of component (e).

[0057] Suitably, the composition comprises up to 25% by weight of component (e), up to 20% by weight of component (e) or up to 15% by weight of component (e).

[0058] Suitably the composition comprises from 5 to 25% by weight of component (e), from 5 to 20% by weight of component (e) or from 6 or 10% by weight of component (e).

[0059] Suitably, the amount of component (e) present in the composition is lower than the amount of component (a) present in the composition.

[0060] Suitably, in the composition of this first aspect: Based on the total weight of the oligosaccharide compounds present in the composition, Component (a) is present in an amount of 8 to 25% by weight; component (b) is present in an amount of 3 to 25 wt. %; Component (c) is present in an amount of 5 to 20 wt. %; Component (d) is present in an amount of 3 to 25% by weight; and Component (e) is present in an amount of from 5 to 25% by weight.

[0061] In some embodiments, the composition of this first aspect comprises: Based on the total weight of the oligosaccharide compounds present in the composition, (a) 8 to 25 wt % Gal-(β1-3)-Gal-(β1-4)-Glc; (b) 3 to 25 wt % Gal-(β1-3)-Gal-(β1-3)-Glc; (c) 5 to 20% by weight of Gal-(β1-3)-Gal-(β1-2)-Glc; and (d) 3 to 25% by weight of Gal-(β1-3)-Gal-(β1-3)-Gal-(β1-4)-Glc; and (e) 5 to 25% by weight of Gal-(β1-4)-Gal-(β1-4)-Glc Includes.

[0062] In the composition of this first aspect, X a , X b , X c , X d and X e are each independently selected from the monosaccharides listed above.

[0063] In the composition of this first aspect, X a , X b , X c , X d and X e may each independently be selected from Glc, Fuc, Ara, Xyl, Rha and Man or mixtures thereof.

[0064] In some embodiments, X a , X b , X c , X d and X e are Glc, respectively.

[0065] In some embodiments, X a , X b , X c , X d and X e Each of X contains Fuc. a, X b , X c , X d and X e are mixtures of Glc and Fuc, respectively.

[0066] The oligosaccharide compounds of the composition of this first aspect preferably have a relatively high proportion of β1-3 Gal-Gal linkages - primarily due to the presence of components (a), (b), (c) and optionally (d) in the proportions discussed herein. The inventors have found that such a relatively high proportion of β1-3 Gal-Gal linkages may be particularly advantageous for the uses of the compositions discussed herein.

[0067] Preferably, 35 to 55% of the Gal-Gal bonds in the oligosaccharide compound are 1-3 bonds, preferably 40 to 55%, preferably 40 to 50%.

[0068] Preferably, between 40 and 60% of the Gal-Gal bonds in the oligosaccharide compound are 1-4 bonds, preferably between 45 and 55%, preferably between 45 and 52%.

[0069] Preferably, between 8 and 20% of the Gal-X bonds in the oligosaccharide compound are 1-3 bonds, preferably between 10 and 18%, preferably between 12 and 16%, preferably where X is Glc.

[0070] Preferably, 15 to 25% of the Gal-X bonds in the oligosaccharide compound are 1-4 bonds, preferably 16 to 24%, preferably 17 to 22%, preferably where X is Glc.

[0071] Preferably, 30 to 45% of the Gal-X bonds in the oligosaccharide compound are 1-2 bonds, preferably 32 to 43%, preferably 34 to 41%, preferably where X is Glc.

[0072] Preferably, between 20 and 36% of the Gal-X bonds in the oligosaccharide compound are 1-6 bonds, preferably between 22 and 34%, preferably between 25 and 32%, preferably where X is Glc.

[0073] Suitably, the Gal-X, bond referred to above, such as for example the Gal-Glu bond, is a β linkage, ie a β-glycosidic bond.

[0074] Preferably, the composition comprises at least 25% by weight of trisaccharides based on the total weight of the composition. Preferably, the composition comprises at least 28% by weight of trisaccharides or at least 30% by weight of trisaccharides.

[0075] Preferably the composition comprises up to 70% by weight of trisaccharides, preferably up to 60% by weight of trisaccharides or up to 50% by weight of trisaccharides.

[0076] Preferably the composition comprises from 25 to 70% by weight of trisaccharides, preferably from 30 to 60% by weight of trisaccharides or from 30 to 50% by weight of trisaccharides.

[0077] Preferably, the composition comprises at least 10% by weight of tetrasaccharides, based on the total weight of the composition. Preferably, the composition comprises at least 12% by weight of tetrasaccharides or at least 15% by weight of tetrasaccharides. Preferably, the composition comprises at least 20% by weight of tetrasaccharides or at least 25% by weight of tetrasaccharides.

[0078] Preferably, the composition comprises up to 40% by weight of tetrasaccharides, preferably up to 25% by weight of tetrasaccharides or up to 20% by weight of tetrasaccharides.

[0079] Preferably the composition comprises from 10 to 40% by weight of tetrasaccharide, preferably from 10 to 30% by weight of tetrasaccharide or from 12 to 25% by weight of tetrasaccharide.

[0080] Preferably, the composition comprises from 30 to 50% by weight of trisaccharides and from 10 to 25% by weight of tetrasaccharides, based on the total weight of the composition.

[0081] As mentioned above, the determination of the disaccharides present in the composition excludes lactose.Thus, in some embodiments, the composition preferably comprises up to 40% by weight of disaccharides, preferably up to 30% by weight of disaccharides or up to 20% by weight of disaccharides.Preferably, the content of lactose in the composition of this first aspect is minimized.Preferably, the composition is substantially free of lactose.Preferably, the composition does not contain lactose.

[0082] Preferably the composition comprises from 10 to 40% by weight of disaccharides, preferably from 10 to 30% by weight of disaccharides or from 10 to 20% by weight of disaccharides.

[0083] Suitably, the composition comprises from 10 to 40% by weight of disaccharides, from 30 to 60% by weight of trisaccharides and from 10 to 25% by weight of tetrasaccharides, based on the total weight of the composition.

[0084] The following description relates to the content of disaccharides in the oligosaccharide compound: Preferably, the oligosaccharide compound contains up to 40% by weight of disaccharides, preferably up to 30% by weight of disaccharides or up to 20% by weight of disaccharides.

[0085] Preferably, the oligosaccharide compound comprises 0 to 40% by weight of disaccharides, preferably 10 to 30% by weight of disaccharides or 10 to 20% by weight of disaccharides.

[0086] Preferably, the oligosaccharide compound comprises at least 25% by weight of trisaccharides, based on the total weight of the oligosaccharide compounds present in the composition. Preferably, the oligosaccharide compound comprises at least 30% by weight of trisaccharides, or at least 33% by weight of trisaccharides.

[0087] Preferably, the oligosaccharide compound comprises up to 75% by weight of trisaccharides, preferably up to 65% by weight of trisaccharides or up to 55% by weight of trisaccharides.

[0088] Preferably, the oligosaccharide compound comprises from 25 to 75% by weight of trisaccharides, preferably from 30 to 65% by weight of trisaccharides or from 34 to 55% by weight of trisaccharides.

[0089] Preferably, the oligosaccharide compound comprises at least 10% by weight of tetrasaccharides, based on the total weight of the oligosaccharide compounds present in the composition. Preferably, the oligosaccharide compound comprises at least 12% by weight of tetrasaccharides or at least 15% by weight of tetrasaccharides.

[0090] Preferably, the oligosaccharide compound comprises up to 45% by weight of tetrasaccharides, preferably up to 35% by weight of tetrasaccharides or up to 30% by weight of tetrasaccharides.

[0091] Preferably, the oligosaccharide compound comprises from 10 to 45% by weight of tetrasaccharides, preferably from 10 to 35% by weight of tetrasaccharides or from 15 to 30% by weight of tetrasaccharides.

[0092] Preferably, the oligosaccharide compounds comprise from 0 to 40% by weight of disaccharides and from 30 to 75% by weight of trisaccharides, based on the total weight of the oligosaccharide compounds present in the composition.

[0093] Preferably, the oligosaccharide compounds comprise from 0 to 40% by weight of disaccharides, from 30 to 75% by weight of trisaccharides and from 10 to 45% by weight of tetrasaccharides, based on the total weight of the oligosaccharide compounds present in the composition.

[0094] The composition of this first aspect preferably comprises at least 50% by weight of oligosaccharide compounds comprising components (a), (b), (c) and optionally (d) and (e).Preferably, the composition comprises at least 55% by weight of oligosaccharide compounds, preferably at least 60% by weight, based on the total weight of the composition.

[0095] Preferably the composition comprises up to 100% by weight of oligosaccharide compounds, preferably up to 95%, up to 90% or up to 85% by weight of oligosaccharide compounds.

[0096] Preferably the composition comprises from 50 to 100% by weight of oligosaccharide compounds, preferably from 55 to 95% or from 60 to 85% by weight of oligosaccharide compounds.

[0097] In some embodiments, the composition is in the form of a syrup. The syrup preferably comprises at least 50% by weight of oligosaccharide compounds, at least 55% by weight of oligosaccharide compounds, or at least 60% by weight. Preferably, the syrup comprises 50 to 75% by weight of oligosaccharide compounds, preferably 55 to 70% by weight or 60 to 70% by weight of oligosaccharide compounds.

[0098] The syrup may contain significant amounts of monosaccharides, such as glucose and / or galactose: the syrup may contain 15 to 30% by weight of monosaccharides, preferably 20 to 28% by weight or 21 to 25% by weight of monosaccharides, such as glucose and / or galactose.

[0099] The syrup preferably contains from 20 to 30% by weight water, preferably from 22 to 28% by weight water.

[0100] The syrup may also contain lactose, for example from 4 to 14% by weight of lactose.

[0101] In some embodiments, the composition is in the form of a powder. The powder preferably comprises at least 60% by weight of the oligosaccharide compound, preferably at least 70% by weight or at least 75% by weight of the oligosaccharide compound. Preferably, the powder comprises 60 to 100% by weight of the oligosaccharide compound, preferably 70 to 95% by weight or 75 to 90% by weight of the oligosaccharide compound.

[0102] The powder preferably contains reduced amounts of monosaccharides, such as glucose and / or galactose, compared to the syrups discussed above. The powder may contain 1 to 10% by weight of monosaccharides, preferably 2 to 8% by weight of monosaccharides or 3 to 7% by weight of monosaccharides, preferably about 5% by weight of e.g. glucose and / or galactose.

[0103] The powder preferably contains from 1 to 10% by weight water, preferably from 3 to 6% by weight water.

[0104] The composition of this first aspect may be purified to remove monosaccharides and optionally disaccharides from the composition.

[0105] The composition of this first aspect may be fractionated to separate the oligosaccharide components of the composition according to their molecular weight, for example to remove disaccharides from the composition or to isolate trisaccharides from other oligosaccharide components. This may be done by any suitable method known in the art, for example high performance liquid chromatography. The composition produced by such fractionation may be referred to as an oligosaccharide fraction or a GOS fraction.

[0106] In such embodiments, the composition (i.e. the oligosaccharide fraction) preferably comprises at least 70% by weight of trisaccharides, tetrasaccharides and higher oligosaccharides, preferably at least 80% or at least 90% by weight. Such higher oligosaccharides have a degree of polymerization of 5 and above. Preferably, the composition comprises at least 95% by weight of trisaccharides, tetrasaccharides and higher oligosaccharides. Preferably, the composition consists or consists essentially of trisaccharides, tetrasaccharides and higher oligosaccharides.

[0107] In such an embodiment, the composition suitably comprises from 40 to 70% by weight trisaccharides, preferably from 45 to 70% by weight trisaccharides or from 50 to 70% by weight trisaccharides.

[0108] Preferably, the oligosaccharide compound comprises from 15 to 50% by weight of tetrasaccharides, preferably from 15 to 40% by weight of tetrasaccharides or from 20 to 40% by weight of tetrasaccharides.

[0109] Preferably the oligosaccharide compound comprises from 5 to 25% by weight of higher oligosaccharides, preferably from 5 to 20% by weight of higher oligosaccharides or from 10 to 20% by weight of higher oligosaccharides.

[0110] Preferably, the composition comprises 40 to 70% by weight of trisaccharides, 15 to 40% by weight of tetrasaccharides and 5 to 25% by weight of higher oligosaccharides, based on the total weight of the composition. Preferably, the composition comprises 50 to 70% by weight of trisaccharides, 20 to 40% by weight of tetrasaccharides and 10 to 20% by weight of higher oligosaccharides, based on the total weight of the composition.

[0111] In such an embodiment, the composition (i.e. the oligosaccharide fraction) suitably comprises the above components (a), (b) and (c) in the following amounts: Based on the total weight of the oligosaccharide compounds present in the composition, (a) at least 8% by weight of Gal-(β1-3)-Gal-(β1-4)-X a ; (b) at least 3% by weight of Gal-(β1-3)-Gal-(β1-3)-X b ; and (c) at least 5% by weight of Gal-(β1-3)-Gal-(β1-2)-X c (In the formula, X a , X b and X c are each independently selected from monosaccharides.

[0112] Suitably, the composition (i.e. the oligosaccharide fraction) comprises the above components (a), (b) and (c) in the following amounts: (a) at least 10% by weight of Gal-(β1-3)-Gal-(β1-4)-X a ; (b) at least 5% by weight of Gal-(β1-3)-Gal-(β1-3)-X b ; and (c) at least 7% by weight of Gal-(β1-3)-Gal-(β1-2)-X c .

[0113] The composition may include component (d) and / or component (e) above.

[0114] The composition preferably contains the components in the ratios discussed above.

[0115] In some embodiments, the composition of this first aspect is a trisaccharide and tetrasaccharide fractionation product (which may be referred to as the DP3 / DP4 fraction).

[0116] In such embodiments, the composition (i.e. the DP3 / DP4 fraction) suitably comprises at least 70% by weight of tri- and tetrasaccharides, suitably at least 80% or at least 90% by weight. Suitably the composition consists or consists essentially of tri- and tetrasaccharides.

[0117] In such an embodiment, the composition preferably comprises from 50 to 80% by weight trisaccharides, preferably from 55 to 75% by weight trisaccharides or from 60 to 75% by weight trisaccharides.

[0118] Preferably, the oligosaccharide compound comprises from 20 to 50% by weight of tetrasaccharides, preferably from 25 to 45% by weight of tetrasaccharides or from 25 to 40% by weight of tetrasaccharides.

[0119] Preferably, the composition comprises 50 to 80% by weight of trisaccharides and 20 to 50% by weight of tetrasaccharides, based on the total weight of the composition. Preferably, the composition comprises 60 to 75% by weight of trisaccharides and 25 to 40% by weight of tetrasaccharides, based on the total weight of the composition.

[0120] In such an embodiment, the composition (i.e. the oligosaccharide fraction) suitably comprises the above components (a), (b) and (c) in the following amounts: Based on the total weight of the oligosaccharide compounds present in the composition, (a) at least 8% by weight of Gal-(β1-3)-Gal-(β1-4)-X a ; (b) at least 3% by weight of Gal-(β1-3)-Gal-(β1-3)-X b ; and (c) at least 5% by weight of Gal-(β1-3)-Gal-(β1-2)-X c (In the formula, X a , X b and X care each independently selected from monosaccharides.

[0121] Suitably, the composition (i.e. the oligosaccharide fraction) comprises the above components (a), (b) and (c) in the following amounts: (a) at least 10% by weight of Gal-(β1-3)-Gal-(β1-4)-X a ; (b) at least 5% by weight of Gal-(β1-3)-Gal-(β1-3)-X b ; and (c) at least 7% by weight of Gal-(β1-3)-Gal-(β1-2)-X c .

[0122] The composition may include component (d) and / or component (e) above.

[0123] The composition preferably contains the components in the ratios discussed above.

[0124] In some embodiments, the composition of this first aspect is a trisaccharide fractionation product (which may be referred to as a DP3 fraction). Such trisaccharide fractionation products may be obtained by known fractionation methods as mentioned above. Such compositions preferably comprise at least 70% by weight of trisaccharides, preferably at least 80% by weight or at least 90% by weight. Preferably, the composition comprises at least 95% by weight of trisaccharides.

[0125] In such an embodiment, the composition (i.e. the oligosaccharide fraction) suitably comprises the above components (a), (b) and (c) in the following amounts: Based on the total weight of the oligosaccharide compounds present in the composition, (a) at least 8% by weight of Gal-(β1-3)-Gal-(β1-4)-X a ; (b) at least 3% by weight of Gal-(β1-3)-Gal-(β1-3)-X b ; and (c) at least 5% by weight of Gal-(β1-3)-Gal-(β1-2)-X c (In the formula, Xa , X b and X c are each independently selected from monosaccharides.

[0126] Suitably, the composition (i.e. the oligosaccharide fraction) comprises the above components (a), (b) and (c) in the following amounts: (a) at least 10% by weight of Gal-(β1-3)-Gal-(β1-4)-X a ; (b) at least 5% by weight of Gal-(β1-3)-Gal-(β1-3)-X b ; and (c) at least 7% by weight of Gal-(β1-3)-Gal-(β1-2)-X c .

[0127] The composition may include component (e) above.

[0128] The composition preferably contains the components in the ratios discussed above.

[0129] The composition of this first aspect may be incorporated into a product for consumption by a consumer to improve gut health by promoting the growth of beneficial bacteria, such as bifidobacteria, in the consumer's gut. Such a product may be selected from the group consisting of dairy products (e.g., liquid milk, dry milk powder such as whole milk powder, skimmed milk powder, fat-filled milk powder, whey powder, infant formula, ice cream, yoghurt, cheese, fermented milk products), beverages, infant foods, cereals, breads, biscuits, confectionery, cakes, food supplements, dietary supplements, animal feed, poultry feed or indeed any other food or beverage.

[0130] The composition of this first aspect may be incorporated into a synbiotic composition, which is suitably a mixture comprising a live microorganism and a substrate or substrates selectively utilized by the host microorganism to confer a health benefit on the host, i.e. probiotics and prebiotics.

[0131] According to a second aspect of the invention there is provided a pharmaceutical or nutraceutical composition comprising a composition according to the first aspect and at least one carrier, excipient or diluent.

[0132] The composition of the first aspect of the present invention, which is included in the pharmaceutical composition or nutraceutical composition, is a composition comprising the above-mentioned oligosaccharide compound. The composition comprising the oligosaccharide compound may have any of the preferred features and advantages described above with respect to the first aspect.

[0133] Suitable further ingredients of pharmaceutical or nutraceutical compositions, as well as methods for preparing such pharmaceutical or nutraceutical compositions, are known in the art.

[0134] According to a third aspect of the present invention there is provided the use of a composition according to the first aspect as a dietary supplement.

[0135] The composition of the first aspect is a composition comprising an oligosaccharide compound as described above. The composition comprising an oligosaccharide compound may have any of the preferred features and advantages described above with respect to the first aspect.

[0136] The use of this third aspect preferably increases butyric acid production in the intestine of dietary supplement consumers.The term butyric acid is used to refer to the butyric acid and its derivatives, such as butyric acid salts and esters, produced by the intestinal microflora in mammals.Specifically, members of the Bifidobacterium and Lactobacillus genera of intestinal microflora ferment oligosaccharides for use as energy source.As the by-product of this fermentation, intestinal microflora produces lactic acid and short-chain fatty acids (SCFAs), such as acetic acid, butyric acid, and propionic acid.

[0137] Preferably, the use of this third aspect increases butyric acid production in the intestine of a dietary supplement consumer compared to the normal level of butyric acid production in said consumer, preferably compared to a placebo dietary supplement that does not contain a composition comprising an oligosaccharide compound.Preferably, the use increases butyric acid production in the intestine of a dietary supplement consumer compared to an alternative composition comprising an oligosaccharide compound having a lower amount of components (a), (b) and / or (c) as specified herein.

[0138] Suitably, use of this third aspect promotes the growth of beneficial bacteria such as Bifidobacterium and Lactobacillus in the consumer's intestine, which in turn may lead to an increase in butyric acid production as discussed above.

[0139] According to a fourth aspect of the invention there is provided a composition according to the first aspect or a pharmaceutical or nutraceutical composition according to the second aspect for use as a medicament.

[0140] The composition for use of this fourth aspect may provide the increase in butyrate production discussed above in relation to the third aspect.

[0141] Although the effects of butyrate in the intestine are not fully understood, butyrate is believed to have several beneficial effects on human health, including maintaining immune homeostasis, regulating systemic inflammation, regulating local and / or organ-specific inflammation, including intestinal inflammation, as well as providing a primary energy source for intestinal cell regeneration. Furthermore, butyrate can induce immune tolerance and thus increased production of such butyrate may be of value in the treatment of allergic or other immune hypersensitivity diseases, autoimmune diseases, stem cell transplantation (see “Short chain fatty acids as potential therapeutic agents in human gastrointestinal and inflammatory disorders”, Gill PA, van Zelm MC, Muir JG, Gibson PR., Aliment Pharmacol Ther. 2018, 48(1), pages 15-34), graft-versus-host disease (see “The microbe-derived short-chain fatty acids butyrate and propionate are associated with protection from chronic GVHD”, Markey KA, et al., Blood 2020, 136(1), pages 130-136) and cancer (see “The Clinical Link between Human Intestinal Microbiota and Systemic Cancer Therapy”, Aarnoutse R, Ziemons J, Penders J, Rensen SS, de Vos-Geelen J, Smidt ML. Int J Mol Sci. 2019, 17, page 4145) may be useful for managing and / or treating and / or alleviating symptoms and / or reducing side effects of current treatment regimens.

[0142] Thus, a fourth aspect of the invention may provide a composition of the first aspect for use in the therapeutic management and / or treatment and / or alleviation of symptoms of an allergic disease.

[0143] A fourth aspect of the invention may provide a composition of the first aspect for use in the therapeutic management and / or treatment and / or alleviation of symptoms of immune hypersensitivity.

[0144] A fourth aspect of the invention may provide a composition of the first aspect for use in the therapeutic management and / or treatment and / or alleviation of symptoms of an autoimmune disease.

[0145] A fourth aspect of the invention may provide a composition of the first aspect for use in the therapeutic management and / or treatment and / or alleviation of symptoms of stem cell transplantation.

[0146] A fourth aspect of the invention may provide a composition of the first aspect for use in the therapeutic management and / or treatment and / or alleviation of symptoms of graft versus host disease.

[0147] A fourth aspect of the invention may provide a composition of the first aspect for use in the therapeutic management and / or treatment and / or alleviation of symptoms of cancer.

[0148] Butyric acid is also believed to play a role in maintaining colonic epithelium; in regulating glucose homeostasis, lipid metabolism, and appetite regulation; and in regulating the immune system and inflammatory response (as described in Morrison, DJ; Preston, T. "Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism", Gut Microbes 2016, 7, 189-200). Therefore, increasing butyric acid production in the gut using the composition of the present invention is believed to provide several such health benefits to consumers.

[0149] Suitably, the composition for use according to this fourth aspect promotes the growth of Bifidobacteria in the intestine of a subject.

[0150] The composition of the first aspect is a composition comprising an oligosaccharide compound as described above. The composition comprising an oligosaccharide compound may have any of the preferred features and advantages described above with respect to the first aspect.

[0151] The use of this fourth aspect as a medicine may include preventing the attachment of pathogens or toxins produced by pathogens to the intestinal wall of a subject.The composition may be administered to a patient following a course of antibiotic treatment and / or chemotherapy and / or radiation therapy, which often alters or even destroys normal healthy intestinal flora, or following surgery on the intestine to re-establish the normal microflora of healthy intestines in the intestine.The composition may be used in combination with one or more species of beneficial intestinal microflora, preferably selected from the group of Akkermansia, Bacteroides, Bifidobacterium, Lactobacillus and Parabacteroides, such as Bifidobacterium animalis, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum (including B. infantis) and / or Megamonas.

[0152] In the uses of the third and fourth aspects, the composition may modulate the abundance of said beneficial gut microflora.

[0153] In the use of the third and fourth aspects, the composition can regulate the abundance of bacterial genera present in the gastrointestinal tract. In some embodiments, the composition regulates the abundance of bacterial genera present in one or both of the small intestine and the large intestine. In some embodiments, the composition regulates the abundance of the predominant bacterial genera in the small intestine selected from the group consisting of Achromobacter, Agrobacterium, Blautia, Burkholderia, Coprococcus, Cryocola, Enterococcus, Eubacterium, Hordemannia, Lactococcus, Mycobacterium, Pseudoramibacter, Ralstonia, Sphingomonas, Streptococcus, and Turicibacter. In some embodiments, the composition modulates the abundance of a bacterial genera predominant in the large intestine selected from the group consisting of Anaerotruncus, Akkermansia, Bacteroides, Bilophila, Butyricimonas, Odoribacter, Parabacteroides, Phascolarctobacterium, Prevotella, and Ruminococcus.

[0154] According to a fifth aspect of the present invention there is provided a method for preparing a composition comprising an oligosaccharide compound according to the first aspect, the method comprising: (i) providing a source of sugar compounds; (ii) treating the source of sugar compounds with one or more galactosidase enzymes to at least partially convert the source of sugar compounds to oligosaccharide compounds. The process includes the steps of:

[0155] Suitably, the steps of the method of this fifth aspect are carried out in the order of step (i) followed by step (ii).

[0156] Preferably, the source of sugar compounds comprises lactose, lactulose or epilactose. Preferably, the source of sugar compounds comprises lactose. The source of sugar may be lactose, for example lactose syrup, which may be derived from cow's milk. The lactose may be heat treated.

[0157] In some embodiments, no additional sugars, such as monosaccharides or disaccharides, are added to the sugar source, hi such embodiments, the method produces galactooligosaccharide compounds.

[0158] In some embodiments, the source of sugar compounds comprises at least one additional sugar.Preferably, the at least one additional sugar provides an oligosaccharide compound with alternative terminal monosaccharide units as discussed above.Preferably, the at least one additional sugar is a source of such monosaccharide units.The at least one additional sugar that is a source of such monosaccharide units can be a monosaccharide or a higher sugar, such as a disaccharide. The at least one additional sugar may be a source of monosaccharide selected from glucose (Glc), fucose (Fuc), arabinose (Ara), xylose (Xyl), rhamnose (Rha), mannose (Man), galactose (Gal), ribose (Rib), lyxose (Lyx), allose (All), altrose (Alt), gulose (Gul), idose (Ido), talose (Tal), psicose (Psi), fructose (Fru), sorbose (Sor), tagatose (Tag), galactosamine (GalN), glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) or mixtures thereof. The at least one additional sugar may be one or more of the monosaccharides listed above.

[0159] In such embodiments, the method produces oligosaccharides having one or more of the above monosaccharides as terminal sugar units.

[0160] In some embodiments, the at least one additional sugar may be selected from fucose, arabinose, xylose, rhamnose, mannose, or mixtures thereof. The sugar source may include lactose and a source of one or more of the above monosaccharides. In such embodiments, the method produces oligosaccharides having as a terminal sugar unit selected from Glc, Fuc, Ara, Xyl, Rha, and Man, or mixtures thereof.

[0161] Step (ii) of the method comprises treating the source of sugar compounds with at least one galactosidase enzyme. The galactosidase enzyme can be an alpha-galactosidase enzyme or a beta-galactosidase enzyme, depending on whether alpha or beta bonds are required between the sugar units of the oligosaccharide compounds. Preferably, the enzyme exhibits galactosyltransferase (transgalactoside) activity and forms alpha or beta bonds between the sugar units in the source of sugar compounds. This results in the synthesis of oligosaccharide compounds with two or more galactose units derived from lactose. Preferably, step (ii) is carried out until the conversion of the source of sugars to oligosaccharide compounds is complete.

[0162] Step (ii) may involve treating the source of sugar compounds with one or more further enzymes which are not galactosidase enzymes.

[0163] Suitably the method comprises the step (iii) of separating the galactosidase enzyme from the composition comprising the oligosaccharide compound. Step (iii) may comprise removing the enzyme by filtration, for example by nanofiltration.

[0164] The composition comprising the oligosaccharide compound produced in step (iii) may be heat treated.

[0165] In some embodiments, the composition is evaporated to reduce the water content and provide the final composition comprising the oligosaccharide compound as a syrup, as discussed above with respect to the first embodiment.

[0166] In some embodiments, glucose is removed from the composition produced by step (iii) prior to evaporation. This preferably reduces the glucose content of the composition from 20-30% by weight to less than 10% by weight, preferably about 5% by weight or less. As discussed above with respect to the first embodiment, the water content of the composition is then reduced by evaporation and the product is dried to provide the final composition comprising the oligosaccharide compounds as a powder.

[0167] It will also be appreciated by those skilled in the art that a composition comprising an oligosaccharide compound according to the first aspect, i.e. containing a particular amount of a particular oligosaccharide compound, can be prepared by combining the oligosaccharides obtained and isolated from different sources in the required amounts. EXAMPLES

[0168] Example 1 - Oligosaccharide syrup A composition comprising an oligosaccharide compound according to the present invention in the form of a syrup was obtained by the following procedure: Lactose was rehydrated with potable water to obtain a working solution of between 35-65% solids by weight. The lactose solution was heat treated and then cooled to 40-65°C. The pH of the solution was adjusted to pH 5.5-7.5. Beta-galactosidase enzyme was then added to the solution in a closed vessel to catalyze the transfer of galactose molecules and subsequently react with lactose to produce the oligosaccharide compound. The progress of the reaction was monitored by measuring the production of glucose. The reaction was allowed to proceed for a period between 8 and 26 hours. The reaction was then stopped by high heat treatment. The reaction mixture was cooled and filtered by carbon filtration to remove the enzyme. The mixture was then dried by evaporation to reduce the water content to about 22-28% by weight to obtain the product as a syrup.

[0169] Example 2 - Oligosaccharide Powder A composition containing an oligosaccharide compound according to the invention in the form of a powder was obtained by a modification of the above procedure. After removal of the enzyme, the mixture was further filtered to remove a significant portion of glucose and other monosaccharides, reducing the monosaccharide content from about 23% by weight to about 5% by weight. The water content of the composition was then reduced by evaporation and the product was dried to obtain a powder having a water content of about 3-6% by weight.

[0170] Example 3 - Fractionation Isolation of the different fractions (DP2, DP3, DP4 or DP5) from the samples of Example 2 was performed using a 5×70 cm BioGel P2 column using water as the eluent. The column was operated at room temperature (21° C.) with a flow rate of 40 to 100 mL / h. Depending on the run, 0.5, 0.75, 1.0, 1.5 or 2.0 mL of a 0.5 g / mL solution of Example 2 in ultrapure water was loaded. The column was loaded with this solution a total of 15 times to obtain sufficient material for the low amount fraction, i.e., DP5. Fractions of 5 mL were collected after an excluded volume of approximately 725 mL. Analysis using thin layer chromatography (TLC) and HPAEC-PAD was performed to ensure that molecules with the same DP were pooled.

[0171] Pooled fractions were frozen and lyophilized. The dried material from all runs was combined and reconstituted. After a second cycle of freezing and lyophilization, the dried material was stored at 4° C. for further analysis or experimentation.

[0172] Comparative Example 1 A commercially available composition containing an oligosaccharide compound was obtained in powder form.

[0173] Oligosaccharide Analysis Samples of Inventive Example 2 and Comparative Example 1 were analyzed to determine their oligosaccharide content by the following procedure.

[0174] material and method A sample of each of the dry powders of Example 2 and Comparative Example 1 was dissolved in water to obtain a solution having a concentration of 40 g / l for analysis.

[0175] Gel Permeation Chromatography An HPLC system equipped with a Rezex RSO and RI detector and in-line desalting (for removal of charged substances such as salts and proteins) was used for aqueous GPC separation of the components of the samples. Separation was performed at high temperature (80°C). The separation range of the Rezex RSO column is from DP1 (monosaccharides) to approximately DP10. All samples were analyzed undiluted (at 40 g / L). Prior to analysis, all sample solutions were treated at 100°C for 10 min to remove any microbial or enzymatic activity.

[0176] GOS Fingerprinting HPAEC-PAD (High Performance Anion Exchange Chromatography) equipped with a PA-1 column was used for the separation of mono- and oligosaccharides of the various samples. Efforts were made to achieve the separation quality described by van Leeuwen et al., Carbohydrate Research 2016, 425, 48-58. A commercial maltooligosaccharide mixture and Comparative Example 1 were also injected for comparison of the chromatograms with those reported by Van Leeuwen et al. On this basis, peak annotation was performed for many peaks. Samples used for GPC were diluted 100 times with DMSO before injection.

[0177] result Gel Permeation Chromatography (GPC-RI) Table 1 shows the DP (degree of polymerization) composition results for samples using the Rezex-RSO system based on RI calibration with glucose (values ​​are expressed as g / L in sample). All material eluting in the >DP5 window was combined.

[0178] [Table 1]

[0179] The concentration information can be used to calculate the relative weight percentages of different DP fractions of oligosaccharides contained in a sample, where DP=2 refers to disaccharides, DP=3 refers to trisaccharides, etc., as shown in Table 1.

[0180] GOS Fingerprinting To identify individual galactooligosaccharides in the samples (GOS fingerprinting), a gradient was developed that gave a separation comparable to that reported in van Leeuwen et al., Carbohydrate Research 2016, 425, 48-58. Peak annotation was performed in the chromatograms of all GOS samples based on the peak annotations made in van Leeuwen et al. for the oligosaccharide compounds. A retention window of approximately 15 seconds was applied for peak annotation.

[0181] Table 2 shows information on the HPAEC-PAD peak areas of all annotated peaks, together with information on the indicated incubation conditions, sample concentrations, dilutions and injection volumes.

[0182] [Table 2]

[0183] The peak area was assumed to correspond approximately to the amount of each oligosaccharide compound present in the composition. If a particular oligosaccharide compound was not identified, then "unknown" and a number were entered in the table. Identified compounds are identified either by name or by a number corresponding to the number assigned to a particular galactooligosaccharide in van Leeuwen et al., Carbohydrate Research 2016, 425, 48-58. A list of these galactooligosaccharides and their corresponding numbers is provided below.

[0184] The oligosaccharide components (a)-(e) discussed above, in which each X group is Glu, correspond to the following numbered entries in Table 2 above: (a)D-Gal-(β1-3)-D-Gal-(β1-4)-D-Glu - 12 (b)D-Gal-(β1-3)-D-Gal-(β1-3)-D-Glu - 29 (c)D-Gal-(β1-3)-D-Gal-(β1-2)-D-Glu - 30 (d)D-Gal-(β1-3)-D-Gal-(β1-3)-D-Gal-(β1-4)-D-Glu - 31 (e) D-Gal-(β1-4)-D-Gal-(β1-4)-D-Glu - 11.

[0185] Prebiotic effect – butyric acid production The prebiotic effect of the compositions of the present invention was investigated and compared to a comparative GOS composition using the following procedure.

[0186] The compositions of inventive Example 2, Comparative Example 1 and control blank sample were subjected to dialysis using a 0.5 kDa membrane to obtain 5 g / l samples, which were then mixed with feces obtained from three healthy human adult subjects (Donors A, B and C). The mixtures were shaken under anaerobic conditions and monitored over a 48-hour period for colonic fermentation products, including butyric acid (at collection points of 6, 24 and 48 hours). The distribution of oligosaccharides in the mixtures was also monitored over this period using the method described above with respect to Table 2. The results show that Example 2 was well fermented by all donors, mainly during the 0-24 hour period, and that butyric acid production was increased at the 6 and 48 hour time points compared to Comparative Example 1 and the control. The results of the butyric acid analysis are shown in FIG. 1. The results of the oligosaccharide analysis for the samples at different time points are shown in Table 3. These results indicate that the oligosaccharides in the samples were actively consumed by the bacterial flora present in the fecal samples during the experiment.

[0187] To assess whether the treatment effect on gut microbiota activity was statistically significant, three two-tailed T-tests were performed between Example 2 and the control, Comparative Example 1 and the control, and Example 2 and Comparative Example 1 to obtain p-values. Benjamini-Hochberg false discovery rate (FDR) was also used in this analysis. The difference between the treatment effects was considered significant when the obtained p-value was smaller than the reference value. Table 4 below shows the difference in the average butyric acid production for the compared samples over 48 hours, and an asterisk indicates whether the difference was considered significant according to the above analysis. These results show that the increase in butyric acid production provided by Example 2 during the 0-48 hour period was statistically significant compared to the control and Comparative Example 1.

[0188] [Table 3-1]

[0189] [Table 3-2]

[0190] [Table 4]

[0191] Examples 4-9 - Oligosaccharides with alternative terminal monosaccharides Example compositions 4-9 comprising oligosaccharide compounds having alternative terminal monosaccharides (i.e., alternatives to glucose) according to the present invention were prepared by the procedure described above for Example 1 using the substrates noted in Table 5 below - namely lactose and the combinations of additional substrates noted for Examples 4-8, as well as lactulose alone for Example 9. A significantly higher molar excess of acceptor (substrate) was intentionally utilized to promote the formation of GOS compounds containing alternative terminal monosaccharides.

[0192] [Table 5]

[0193] Characterization and Quantification of Examples 4-9 Using HPAEC-PAD The GOS compositions of Examples 4-9 were separated and quantified by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using a CarboPac PA210 (2×150 mm) analytical column with a CarboPac-PA1 (4×50 mm) guard column. The operating parameters were set as follows: the temperature of the column was 30° C.; elution was performed using gradient concentrations of sodium hydroxide and sodium acetate at a flow rate of 0.2 ml / min. The eluents were as follows: A-Milli-Q water, B-500 mM NaOH, C-500 mM NaOH combined with 600 mM NaOAc, D-Milli-Q water. The DP1, DP2 and higher order GOS peaks were quantified based on calculating the peak areas and using calibration curves constructed using available standards. Glucose, galactose and lactose were calibrated in the range of 5-300 μg / ml. The peak of the novel GOS, DP2, was quantified based on lactose calibration, and the peaks of the novel GOS, DP3 and DP4, were quantified with reference to the external maltooligosaccharide ladder (DP1–8).

[0194] Purification of GOS on activated carbon To remove monosaccharides, the GOS composition was purified with activated charcoal according to the methodology described by Julio-Gonzaelez et al (Food Research International, Vol. 129, March 2020, 108811) with some modifications. The GOS reaction mixture was combined with activated charcoal using the ratio: 6 g charcoal for 0.5 g total sugars. The ratio was adjusted to the final concentration with ethanol (either 1%, 3% or 5% v / v) for monosaccharide desorption. The resulting mixture was stirred at 25° C. for 30 min, 2 h, 4 h and overnight to determine the optimal desorption time, and then filtered under vacuum through Whatman No.1 paper (Whatman International Ltd., Maidstone, UK) (step 1). This process was repeated twice to ensure the removal of total monosaccharides (step 2). Desorption of oligosaccharides from the washed activated carbon was carried out by adding an appropriate amount of ethanol (50%, v / v) and stirring the mixture for 120 min followed by filtration (step 3). The resulting suspension of GOS in ethanol was freeze-dried to obtain a powder that was subsequently tested as a microbial growth substrate in fermentation evaluations.

[0195] HPAEC-PAD chromatography showed that Examples 4-9 contained different GOS compounds than Example 1 due to the incorporation of the respective alternative terminal monosaccharides in the compounds. The formation of these different GOS compounds in the respective product mixtures demonstrates that the beta-galactosidase enzyme can utilize alternative acceptor molecules in addition to lactose to provide alternative terminal monosaccharides to the GOS compounds.

[0196] Table 6 shows the percentage amounts of different types of GOS compounds prepared in Examples 4 to 9. Quantification of HPAEC-PAD peaks was based on calibration curves using available standards. a Total GOS: all quantified transgalactosylated carbohydrate peaks identified in HPAEC-PAD excluding remaining glucose, galactose and lactose. bTotal GOS(-acceptor): Same as before, except excluding the remaining amount of acceptor molecules in the calculation. c All quantified new peaks appearing in the chromatograms of the nGOS:lactose / new acceptor reaction were also absent in the lactose only Example 1 reaction - ignoring peaks purely derived from lactose as evident in the Example 1 chromatogram. d nGOS(-acceptor): Same as before, except that the remaining amount of acceptor molecules is excluded in the calculation. e nGOS in total GOS: Percentage of quantified new GOS peak from the total GOS produced in each reaction - to distinguish from the amount of GOS purely derived from lactose. f All reactions were terminated at the optimal time point of 24 h, except for LactulOS, where the highest yield of novel fructosylated GOS was observed at 12 h. Standard deviations were derived from two independent experiments (provided in brackets).

[0197] [Table 6]

[0198] The best yields from were obtained in Example 8 (58.2%) and Example 9 (42.2%). The other examples showed significantly lower yields (20-30%) compared to Example 1 when calculated relative to the total sugars in the reaction. However, adjusting the same yields relative to the actual transgalactosylated sugars present in the final matrix by removing the quantified weight of excess acceptor molecules provides a clearer result (Table 6, second column). The total GOS(-acceptor) reaction yields in Examples 4-8 were comparable to the overall GOS yields observed in Examples 1 and 9 (55-70%). The most abundant formation of novel hybrid transgalactosylated acceptor species was observed in Example 5 (36.7%) and Example 7 (35.8%) (Table 6, fourth column), suggesting that L-arabinose and L-rhamnose are the best alternative acceptor substrates for the enzyme.

[0199] Following purification using activated carbon, as described above, the relative abundance of simple sugars was significantly reduced to approximately equivalent levels in all carbohydrate mixtures. The remaining amount of simple sugars from the total carbohydrate in all preparations barely exceeded the 5% level following this purification step (Table 7). Thus, the relative abundance of transgalactosylated oligosaccharides in the resulting mixtures was significantly elevated, enriching the more important polymeric GOS carbohydrate molecules. In view of the subsequent in vitro fermentation studies, the removal of simple sugars was essential to minimize the impact on simple sugar fermentation in the early phase (<8 h) after fecal inoculation. Instead, the explicit aim of such fermentation simulations is to establish the true differences in the fermentation profiles between the newly synthesized GOS species, especially in the higher DP range.

[0200] [Table 7]

[0201] Examples 4-9 - Fecal fermentation studies Fecal sample preparation Fresh fecal samples were collected 1 hour before inoculation from three adult male and female healthy donors who had not taken antibiotics for the previous 3 months and had no history of gastrointestinal disorders. Anaerobic conditions were maintained using a sachet (Thermo Scientific TM The feces were maintained in a collection vessel using a 2.5 L Oxoid AnaeroGen (Oxoid AnaeroGen, Basingstoke, UK). Fecal slurry was prepared by diluting the faeces in a 1:10 ratio in phosphate saline buffer (PBS, pH 7.0) and mixing in a stomacher for 2 min. Batch culture experiments were performed in duplicate for each donor.

[0202] Batch fermentation The method used was as described by Olano-Martin et al. 2000 ('In vitro fermentability of dextran, oligodextran and maltodextrin by human gut bacteria.' British Journal of Nutrition, 83(3), 2000, p.247-255). 9 mL of autoclaved nutrient medium was added to a fermentation vessel with a working volume of 10 mL. The medium was brought up to 1 L with ddH20, (g / L -1 ): peptone water, 2; yeast extract, 2; NaCl, 0.1; K2HPO4, 0.04; KH2PO4, 0.04; MgSO4.7H2O, 0.01; CaCl2.6H2O, 0.01; NaHCO3, 2; hemin (a few drops of 1 mol 1 -1 NaOH), 0.05; cysteine ​​HCl, 0.5; bile salts, 0.5; Tween 80, 2 and 10 μL vitamin K. The medium was flushed with N2 overnight to create anaerobic conditions. The vessels were maintained at 37°C via a circulating water bath, and the pH was maintained between 6.7 and 6.9 to mimic conditions in the distal colon using a pH controller (Electrolab, Tewkesbury, UK) connected to a 0.25 M solution of HCl and NaOH. Immediately prior to fecal sample inoculation, 0.1 g of test substrate (1%, w / v) was added to the vessel before adding 1 mL (v / v) of fecal slurry (Table 1). Samples were removed from the fermenter immediately after inoculation of the slurry (0 h) and after 8 and 24 h for bacterial enumeration and metabolite analysis. Fermentation experiments were performed in duplicate for each substrate using fecal inocula from three adult volunteers.

[0203] 3. Preparation of Samples for Bacterial Enumeration by Flow Cytometry-Fluorescence In Situ Hybridization (FISH) Samples (750 μL) were removed from the in vitro fermentation vessels at 0, 8 and 24 h and immediately placed on ice before centrifugation at 13,000×g for 5 min and the supernatant discarded. Pelleted bacteria were fixed for 4 h at 4° C. in a 1:3 (v / v) ratio of PBS and 4% (w / v) filtered paraformaldehyde (PFA, pH 7.2). Samples were washed twice with filtered PBS, resuspended in 300 μL of PBS and ethanol (1:1, v / v) and stored at −20° C. for up to 3 months.

[0204] Flow-FISH analysis Hybridization was performed using the method described by Rigottier-Gois et al. 2003 ('Fluorescent hybridisation combined with flow cytometry and hybridisation of total RNA to analyse the composition of microbial communities in human faeces using 16S rRNA probes.' FEMS Microbiology Ecology, 43(2), 2003, p.237-245) using genus- and group-specific 16S rRNA-targeted oligonucleotide probes (Eurofins Genomics, Wolverhampton, UK). The primers used were commercially available Ato291, Bac303, Bif164, Chis150, DSV687, Erec482, Lab158, Rrec584, Fprau655, Prop853 and Eub338. Samples were screened using a flow cytometer (Accuri C6, BD Biosciences, USA) with Accuri CFlow software.

[0205] Once thawed, the samples were then vortexed for 10 seconds and 75 μL of the sample was added to 500 μL of PBS in a 1.5 mL tube, vortexed again, and centrifuged at 13,000×g for 3 minutes. The supernatant was removed and discarded. 100 μL of Tris-EDTA buffer containing lysozyme (1 mg / mL) was added to the tube, mixed using a pipette, and then incubated in the dark for 10 minutes. The samples were vortexed and centrifuged at 13,000×g for 3 minutes, and the supernatant was removed. 500 μL of PBS was added to the tube and the pellet was resuspended using a pipette, then vortexed and centrifuged at 13,000×g for 3 minutes. The supernatant was removed and the pellet was resuspended in 150 μL of hybridization buffer (0.9 M NaCl, 0.2 M Tris-HCl (pH 8.0), 0.01% sodium dodecyl sulfate, 30% formamide), vortexed, and centrifuged at 13,000 × g for 3 min. The supernatant was again removed and the pellet was resuspended in 1 mL of hybridization buffer. 4 μL of oligonucleotide probe solution (50 ng / μL) (see Supplementary Table 1) was added to the 1.5 mL centrifuge tube with 50 μL of sample, vortexed, and incubated overnight at 36 °C. Once hybridization was complete, 125 μL of hybridization buffer was added to each tube, vortexed, centrifuged at 13,000 × g for 3 min, and the supernatant was carefully removed. 175 μL of wash buffer (0.064 M NaCl, 0.02 M Tris / HCl (pH 8.0), 0.5 M EDTA (pH 8.0), 0.01% sodium dodecyl sulfate) kept at 40° C. was added to each tube to resuspend the pellet. The tubes were then vortexed and incubated in the dark at 38° C. for 20 min to remove non-specific binding of primers. Samples were then centrifuged at 13,000×g for 3 min, the supernatant removed, and 300 μL of PBS added and then vortexed. Samples were stored refrigerated at 4° C. in the dark prior to flow cytometry analysis.

[0206] Gas chromatography-mass spectrometry (GC-MS) for SCFA and lactate analysis A 1.5 mL sample from each vessel was taken at 0, 8 and 24 hours, centrifuged at 13,000×g for 10 minutes, and the supernatants were stored at −20° C. until required for metabolite analysis. Samples were prepared for GC-MS using the SCFA derivatization method as described by Richardson et al., 1989 ('Simultaneous determination of volatile and non-volatile acidic fermentation products of anaerobes by capillary gas chromatography.' Letters in Applied Microbiology, 9(1), 1989, p.5-8). Once the samples were removed from −20° C. storage and thawed, they were vortexed and centrifuged again at 13,000×g for 10 minutes. A 1 mL aliquot of sample supernatant was then transferred into a flat-bottom glass tube with 50 μL of internal standard solution (0.1 M 2-ethylbutyric acid). 500 μL of concentrated hydrochloric acid (HCl) and 3 mL of diethyl ether were added to the samples in each glass tube, which were then vortexed for 1 min and centrifuged at 2000×g for 10 min. 400 μL of the pooled ether extract was then added to 50 μL N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) in a GC screw-cap vial. External samples of 1 M acetic acid, butyric acid, propionic acid and lactic acid were also prepared using the Richardson SCFA derivatization method. Samples were left at room temperature for 72 h to allow complete derivatization of metabolites. An Agilent / HP 6890 gas chromatograph (Hewlett Packard, UK) equipped with an HP-5MS 30 m×0.25 mm column with 0.25 μm coating (crosslinked (5%-phenyl)-methylpolysiloxane, Hewlett Packard, UK) was used for metabolite analysis. Quantification of samples was obtained through calibration curves of lactate, acetate, propionate, and butyrate at concentrations between 6.25 mM and 100 mM from external standards.

[0207] Results – Bacterial populations In the figures and description that follow, Examples 4-9 are referred to below with reference to the additional substrates used in the preparation of these compositions. Example 4 - FucOS Example 5 - AraOS Example 6 - XylOS Example 7 - RhaOS Example 8 - SucOS Example 9 - LactulOS

[0208] The sample designated B-GOS (galactose-containing oligosaccharides) was prepared using 500 mg / ml lactose as the substrate.

[0209] The differences in bacterial populations observed for Examples 4-9 are shown in Figure 2. Figure 2 shows the effect of different novel oligosaccharides on the bacterial counts (log colony forming units (CFU / mL)) of Lactobacillus species (LAB, A), Bacteroidae and Prevotellaceae families (BAC, B), Clostridium coccoides-Eubacterium rectal group (EREC, C), Roseburia subcluster (RREC, D), Atopobium-Coriobacterium species (ATO, E), Clostridium cluster IX (PROP, F), Faecalis prausnitzii (FPRAU, G), Desulfovibrio spp. (DSV, H) and Clostridium histolyticum (CHIS, I) detected by flow FISH in pH-controlled batch culture experiments. Samples were collected at 0, 8 and 24 hours. Error bars are standard error of the mean. Significant differences (p ≤ 0.05) are * Displayed by.

[0210] The batch culture experiments were repeated using fresh stool samples from the same donors used in the first set of experiments. There were no significant differences between the bacterial populations in the donors between the two technical replicates at all three sampling time points, 0, 8 or 24 hours.

[0211] FIG. 2 shows the effect of Examples 4-9 on total bacterial counts (A) and Bifidobacterium species (B) (log colony forming units (CFU / mL) detected by flow FISH cytometry in pH-controlled batch cultures. Samples were taken at 0, 8 and 24 hours. One-way ANOVA was applied to test for main interactions between groups. Error bars are standard error of the mean. Significant differences (p≦0.05 (F test)) were * The negative controls did not have any additional carbon source.

[0212] An increase in total bacteria count (FIG. 2A) was observed after 8 hours for all test oligosaccharides and the positive control, with statistically significant increases detected after 24 hours of fermentation in Examples 4-9 (p≦0.05). The greatest increase in total bacteria was seen at 24 hours in Example 6, Log 10 The result was 8.64±0.99 CFU / mL.

[0213] A significant increase in the number of Bifidobacterium species (FIG. 2B) was observed after 24 hours of fermentation for Example 1 (p=0.013), Example 4 (FucOS) (p=0.006), Example 9 (LactulOS) (p=0.006), Example 8 (SucOS) (p=0.042) and Example 6 (XylOS) (p=0.040). The highest percentage increase was 63% from 0 to 24 hours, which was observed for AraOS (Log 10 6.08(±1.14) to Log 10 7.12(±1.44).

[0214] FIG. 3 shows the effect of flow FISH on bacterial counts (log colony forming units (CFU / mL)) of Lactobacillus spp. (LAB, A), Bacteroidae and Prevotellaceae families (BAC, B), Clostridium coccoides-Eubacterium rectal group (EREC, C), Roseburia subcluster (RREC, D), Atopobium-Coriobacterium spp. (ATO, E), Clostridium cluster IX (PROP, F), Faecalis prausnitzii (FPRAU, G), Desulfovibrio spp. (DSV, H) and Clostridium histolyticum (CHIS, I) detected by flow FISH in pH-controlled batch culture experiments of Examples 4-9. Samples were collected at 0, 8 and 24 hours. Error bars are standard error of the mean. Significant differences (p≦0.05) were * The negative controls again did not have any additional carbon source.

[0215] A significant increase in Lactobacillus numbers (Figure 3A) was observed following 24 hours of fermentation in the treatments Ex. 8 (SucOS) (p=0.049) and Ex. 6 (XylOS) (p=0.036). In vessels treated with B-GOS, Ex. 7 (RhaOS) and Ex. 6 (XylOS), there was a significant increase in the Clostridium coccoides-Eubacterium lectal group, whereas the only treatment in which a significant increase in the Roseburia subcluster population was observed was Ex. 7 (RhaOS) (p=0.05) at 24 hours of fermentation (Figure 3D). There were no significant differences in Clostridium cluster IX (PROP), Faecalibacterium prausnitzii (FPRAU) or Desulfovibrio sp. (DSV), regardless of treatment (Figures 3F, 3G and 3H, respectively).

[0216] There was a significant increase in Atopobium-coriobacterium (ATO) in vessels containing B-GOS, Example 9 (LactulOS), Example 7 (RhaOS) and Example 6 (XylOS) (Figure 3E). Clostridium histolyticum (CHIS) populations were also significantly increased at 24 hours in fermentation vessels treated with Bimuno® (Figure 3I) (p=0.042). However, the number of Clostridium histolyticum populations (Log 10 5.67 (±0.14) is the Log 10 This was significantly lower than the total bacterial count (Figure 3A) at 8.56 ± (0.17), and the level was close to the limit of detection.

[0217] Results - Bacterial Metabolites Figure 4 shows the concentrations of bacterial metabolites: acetate (A), propionate (B), butyrate (C) and lactate (D) detected by GC-MS at 0, 8 and 24 h in samples taken from pH-controlled batch culture studies testing newly synthesized oligosaccharides and controls. Error bars are standard error of the mean.

[0218] Concentrations of acetate, propionate, butyrate and lactate increased following 8 and 24 hours of fermentation compared to 0 hours for all examples and positive controls tested in batch culture fermentation (Figure 4). Acetate was the most prevalent SCFA produced in all batch culture fermentation, reaching the highest concentration compared to other metabolites measured (Figure 4A). The greatest increase in acetate was observed at 24 hours in Example 6 (XylOS), increasing from 0.4 mM at 0 hours to a concentration of 36.8 mM. An increase in acetate was also seen in the samples of Example 1, Example 5 (AraOS) and Example 4 (FucOS) over the 24 hours of fermentation.

[0219] The SCFAs propionic acid and butyric acid were also detected in all examples, but at lower concentrations, and increased from 0 to 24 hours. The largest increase in propionic acid was seen in Example 4 (FucOS), reaching 10.9 mM. A significant increase in this metabolite was observed after 24 hours of fermentation in Example 7 (RhaOS) and Example 6 (XylOS). Fermentation of Example 9 (LactulOS), Example 4 (FucOS) and Example 9 (LactulOS) led to an increase in butyric acid levels at 24 hours, while enhanced lactate levels were identified after 24 hours in the vessels fermenting Example 5 (AraOS) and Example 6 (XylOS) and after 8 hours in Example 8 (SucOS).

[0220] These results indicate that the GOS compositions of Examples 4-9, which contain GOS compounds with alternative terminal monosaccharides, increased Bifidobacterium populations. Furthermore, the bacterial counts from the FISH data, in combination with the SCFA results from these fermentations, suggest that some of these compositions of Examples 4-9 may stimulate bacteria other than the classical probiotic genera Bifidobacterium and Lactobacillus, and thus provide promising prebiotic activity.

[0221] In summary, the present invention provides a method for the preparation of oligosaccharide compounds, e.g., oligosaccharide compounds, comprising: (a) at least 8% by weight of Gal-(β1-3)-Gal-(β1-4)-X, based on the total weight of the oligosaccharide compounds present in the composition; a (b) at least 3% by weight of Gal-(β1-3)-Gal-(β1-3)-X b and (c) at least 5% by weight of Gal-(β1-3)-Gal-(β1-2)-X. c wherein X a , X b and X cThe present invention provides compositions comprising galactooligosaccharide compounds, each of which is independently selected from monosaccharides. These compositions contain relatively high amounts of oligosaccharide compounds (a), (b) and (c) and relatively high amounts of β1-3 Gal-Gal linkages compared to known oligosaccharide compositions. These particular characteristics of the compositions are believed to provide benefits to the gut health of consumers, for example, because these compositions provide increased production of butyric acid in the gut of consumers compared to known compositions.

[0222] While several preferred embodiments have been shown and described, it will be recognized by those skilled in the art that various changes and modifications may be made without departing from the scope of the invention, as defined in the appended claims.

[0223] Throughout this specification, the term "comprising" or "comprises" means including the specified component(s), but not to the exclusion of the presence of other components. The term "consisting essentially of" or "consists essentially of" means including the specified component, but excluding other components, except for materials present as impurities, unavoidable materials present as a result of the process used to provide the component, and components added for purposes other than achieving the technical effect of the invention. Typically, when referring to a composition, a composition consisting essentially of a set of components will contain less than 5% by weight, typically less than 3% by weight, and more typically less than 1% by weight of the unspecified component.

[0224] The terms "consisting of" or "consists of" mean the inclusion of the specified components, but excluding the addition of other components.

[0225] Whenever appropriate, depending on the context, use of the terms "comprises" or "comprising" can also be interpreted as including or including the meaning "consists essentially of" or "consisting essentially of," and can also be interpreted as including the meaning "consists of" or "consisting of."

[0226] For the avoidance of doubt, when the amount of a component in a composition is stated in weight percent, this means the weight percent of the particular component in relation to the total composition to which it refers. For example, "wherein the oligosaccharide compound comprises up to 35% by weight of disaccharides" means that 35% by weight of the oligosaccharide compounds in the composition is provided by disaccharides.

[0227] Any features described herein may be used individually or in combination with each other as appropriate, and in the combinations specifically described in the appended claims. Any features for each aspect or exemplary embodiment of the present invention described herein should also be read as applicable to any other aspect or exemplary embodiment of the present invention as appropriate. In other words, a person skilled in the art reading this specification should consider any features for each exemplary embodiment of the present invention as interchangeable and combinable between different exemplary embodiments.

[0228] Attention is directed to all articles and documents related to this application, filed contemporaneously with or prior to this specification, and which are open to public inspection together with this specification, and the contents of all such articles and documents are incorporated herein by reference.

[0229] All of the features disclosed in this specification (including any accompanying claims, and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive.

[0230] Each feature disclosed in this specification (including any accompanying claims and drawings), unless expressly stated otherwise, may be replaced by alternative features serving the same, equivalent or similar purpose. That is, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0231] The invention is not limited to the details of the embodiment or embodiments described above. The invention extends to any novel, or any novel combination of features disclosed in this specification (including any accompanying claims, and drawings), or to any novel, or any novel combination of method or process steps so disclosed.

Claims

1. A composition comprising an oligosaccharide compound, wherein the oligosaccharide compound is: Based on the total weight of the oligosaccharide compounds present in the composition, (a) at least 8% by weight of Gal-(β1-3)-Gal-(β1-4)-X ａ ; (b) at least 3% by weight of Gal-(β1-3)-Gal-(β1-3)-X ｂ ; and (c) at least 5% by weight of Gal-(β1-3)-Gal-(β1-2)-X ｃ Includes, in the formula, X ａ , X ｂ and X ｃ Each of these is independently selected from monosaccharides, forming a composition.

2. Oligosaccharide compounds: Based on the total weight of oligosaccharide compounds present in the composition, (d) at least 3% by weight of Gal-(β1-3)-Gal-(β1-3)-Gal-(β1-4)-X ｄ Includes, in the formula, X ｄ The composition according to claim 1, wherein , is selected from monosaccharides.

3. Based on the total weight of oligosaccharide compounds present in the composition, Component (a) is present in an amount of up to 35% by weight; Component (b) is present in an amount of up to 25% by weight; and Component (c) is present in an amount of up to 25% by weight. The composition according to claim 1 or claim 2.

4. The composition according to claim 1 or claim 2, wherein the weight percentage ratio of compound (a) to compound (b) is 1:1 to 3:

1.

5. The composition according to claim 1 or claim 2, wherein the weight percentage ratio of compound (a) to compound (c) is 1:1 to 3:

1.

6. The composition according to claim 1 or claim 2, wherein the weight percentage ratio of compound (b) to compound (c) is 2:1 to 1:

2.

7. Oligosaccharide compounds: Based on the total weight of oligosaccharide compounds present in the composition, (e) At least 5% by weight of Gal-(β1-4)-Gal-(β1-4)-X ｅ Includes, in the formula, X ｅ The composition according to claim 2, wherein is selected from monosaccharides.

8. X ａ , X ｂ , X ｃ , X ｄ and X ｅ The composition according to claim 7, wherein each of these is independently selected from glucose, fucose, arabinose, xylose, rhamnose, mannose, galactose, ribose, lyxose, allose, altrose, gross, idose, talose, psicose, fructose, sorbose, tagatose, galactosamine, glucosamine, and N-acetylglucosamine or a mixture thereof.

9. X ａ , X ｂ , X ｃ , X ｄ and X ｅ The composition according to claim 7, wherein each of them is Glc.

10. X ａ , X ｂ , X ｃ , X ｄ and X ｅ The composition according to claim 7, wherein each of the elements comprises Fuc.

11. The composition according to claim 1 or claim 2, wherein 40 to 55% of the Gal-Gal bonds in the oligosaccharide compound are 1-3 bonds.

12. The composition according to claim 1 or claim 2, comprising at least 50% by weight of an oligosaccharide compound, preferably in the form of a syrup.

13. The composition according to claim 1 or 2, comprising at least 75% by weight of an oligosaccharide compound, preferably in the form of a powder.

14. The composition according to claim 1 or claim 2, wherein the oligosaccharide compound comprises at least 25% by weight of a trisaccharide.

15. The composition according to claim 1 or claim 2, wherein the oligosaccharide compound comprises at least 10% by weight of a tetrasaccharide.

16. The composition according to claim 1 or claim 2, comprising 80% by weight of trisaccharides, tetrasaccharides and higher oligosaccharides.

17. The composition according to claim 1 or claim 2, comprising 80% by weight of trisaccharides.

18. A pharmaceutical composition or nutritional supplement composition comprising the composition according to claim 1 or claim 2 and at least one carrier, excipient, or diluent.

19. Use of the composition according to claim 1 or claim 2 as a nutritional supplement.

20. The composition according to claim 1 or claim 2 for use as a nutritional supplement.

21. The use according to claim 19, wherein the use increases butyrate production in the intestines of consumers of the dietary supplement.

22. The composition according to claim 20 for use in increasing butyrate production in the intestines of consumers of nutritional supplements.

23. A composition according to claim 1 or claim 2 for use as a pharmaceutical agent.

24. A method for preparing a composition comprising the oligosaccharide compound described in claim 1 or claim 2, wherein the method is: (i) To provide a source of sugar compounds; (ii) Treating the source of the sugar compound with one or more galactosidase enzymes to at least partially convert the source of the sugar compound into an oligosaccharide compound. A method that includes the following steps.

25. The method according to claim 24, wherein the source of the sugar compound comprises lactose, lactulose, or epilactose.

26. The method according to claim 24, wherein the source of the sugar compound comprises further sugars selected from glucose, fucose, arabinose, xylose, rhamnose, mannose, galactose, ribose, lyxose, allose, altrose, growth, idose, talose, psicose, fructose, sorbose, tagatose, galactosamine, glucosamine and N-acetylglucosamine or mixtures thereof.

27. ​​The method according to claim 25, wherein the source of the sugar compound comprises further sugars selected from glucose, fucose, arabinose, xylose, rhamnose, mannose, galactose, ribose, lyxose, allose, altrose, growth, idose, talose, psicose, fructose, sorbose, tagatose, galactosamine, glucosamine and N-acetylglucosamine or mixtures thereof.

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