Oligosaccharide composition

Abstract

The invention relates to a prebiotic oligosaccharide composition. The present invention relates to a composition comprising an oligosaccharide compound, such as a galactooligosaccharide compound, comprising, based on the total weight of oligosaccharide compounds present in the composition: (a) at least 8 wt% of Gal-(beta1-3)-Gal-(beta1-4)-Xa; (b) at least 3 wt% of Gal-([beta] 1-3)-Gal-([beta] 1-3)-Xb; and (c) at least 5 wt% of Gal-(beta1-3)-Gal-(beta1-2)-Xc; wherein Xa, Xb and Xc are independently selected from monosaccharides. These compositions contain relatively high levels of oligosaccharide compounds (a), (b) and (c) and relatively high levels of [beta] 1-3Gal-Gal linkages compared to known oligosaccharide compositions. These special features of the compositions are believed to be beneficial to the gut health of consumers, for example, as these compositions increase the production of butyric acid in the gut of a consumer as compared to known compositions. Also disclosed are methods of making the compositions, as well as the use of the compositions as dietary supplements or medicaments.

Description

Technical Field

[0001] The present invention relates to a composition comprising oligosaccharide compounds, a method for preparing the composition and the use of the composition as a dietary supplement. The present invention particularly relates to an oligosaccharide composition having a relatively high content of specific beneficial oligosaccharides. Background Art

[0002] The human intestinal flora (or microflora) contains pathogenic, benign and beneficial microbial genera. The dominance of the former may lead to intestinal diseases, which can be acute (such as gastroenteritis) or chronic (such as inflammatory bowel disease, irritable bowel syndrome and some intestinal cancers). In humans, the coliform flora is acquired at birth. Bifidobacteria are dominant in breastfed infants and can easily outcompete bacteria of other genera. This is because breast milk components stimulate the growth of bifidobacteria. However, many factors can affect the composition and activity of the intestinal flora throughout an individual's life, such as the environment, drug treatment and diet, especially the consumption of highly processed foods (see "Role of the gut microbiota in nutrition and health", Valdes, AM et al., BMJ, 2018, 361).

[0003] Certain components of the intestinal flora have been implicated in the etiology of intestinal diseases. For example, mycobacteria have been implicated in Crohn's disease; ulcerative colitis may be triggered by sulfate-reducing bacteria, and there may be bacteria involved in the development of intestinal cancer. It would be beneficial if the selective growth of intrinsically beneficial intestinal bacteria could be promoted by the ingestion of prebiotics. Prebiotics are substrates that are selectively utilized by the host microbiome and have a health benefit. The use of such prebiotics may have a sustained beneficial effect of inhibiting the growth of pathogenic flora.

[0004] Attempts have been made to affect the balance of intestinal flora by adding one or more beneficial microorganism strains in appropriate food carriers, so that it is beneficial to beneficial microorganisms, such as bifidobacteria, so as to bring health benefits to the host. This live microbial nutritional supplement is called probiotics. However, it is difficult to ensure that the live bacteria in food survive after digestion, so the actual effect of probiotics may be limited. Therefore, prebiotics may be a more promising option for positively affecting the intestinal flora of patients (see Walter J, Maldonado-Gómez MX, Martínez I. "To engraft or not to engraft: an ecological framework for gut microbiome modulation with livemicrobes", Curr Opin Biotechnol. 2018, 49, 129-139).

[0005] As mentioned above, an alternative approach to dietary manipulation of the gut microbiota is the use of prebiotics, which are 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 intestine, thereby improving the health of the host.

[0006] One group of compounds classified as prebiotics are galacto-oligosaccharides, which are galactose-containing oligosaccharides produced from lactose by the transgalactosidase activity of β-galactosidase. Known galacto-oligosaccharide prebiotic products contain a mixture of various oligosaccharide compounds in varying proportions, not all of which have the desired beneficial effects on the consumer's intestinal flora. Although these products have shown some beneficial results in improving the gastrointestinal health of patients, there is still a need for further improvements in oligosaccharide compositions to fully realize the potential benefits of these prebiotics for consumers. Summary of the invention

[0007] Among other purposes, an object of the present invention is to provide a composition comprising an oligosaccharide compound to address at least one disadvantage of the prior art, whether found herein or elsewhere, or to provide an alternative to existing compositions. For example, an object of the present invention may be to provide a composition comprising an oligosaccharide compound, the composition comprising a higher proportion of specific 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. Further features of the present invention will become apparent from the dependent claims and the following description.

[0009] According to a first aspect of the present invention, there is provided a composition comprising an oligosaccharide compound, wherein the oligosaccharide compound comprises, based on the total weight of the oligosaccharide compound present in the composition:

[0010] (a) at least 8 wt% Gal-(β1-3)-Gal-(β1-4)-X a ;

[0011] (b) at least 3 wt% of Gal-(β1-3)-Gal-(β1-3)-X b ;and

[0012] (c) at least 5 wt% of Gal-(β1-3)-Gal-(β1-2)-X c ;

[0013] Where X a , X b and X c Each is independently selected from monosaccharides.

[0014] These types of oligosaccharides have a specific bond between the galactose unit (denoted as Gal) and the terminal monosaccharide and are considered to be particularly beneficial to maintaining healthy human intestinal flora, and therefore to the health of the subject. Known oligosaccharide compositions either do not contain each of these types of oligosaccharides, or contain lesser amounts of these oligosaccharides. Therefore, the composition of the first aspect is considered to provide an advantage to consumers in establishing and maintaining human intestinal flora when used as a prebiotic.

[0015] The weight percentage of the specific oligosaccharide compound discussed herein is based on the weight of all oligosaccharide compounds present in the whole composition. Therefore, when determining the specific weight percentage, only the composition part provided by the oligosaccharide (component (a), (b), (c) or any other oligosaccharide present) is considered. When determining the oligosaccharide content, in addition to lactose, disaccharides are also included. The composition of the first aspect 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 compound in the composition can refer to the weight percentage of the oligosaccharide part in this first part of the composition.

[0016] The oligosaccharide compounds (a), (b) and (c) respectively contain X a , X b and X c , X a , X b and X c Each is independently selected from sugars. a , X b and X c The groups can be considered as sugar units. Thus, the group Xa , X b and X c It can be considered as the terminal sugar of oligosaccharide compounds. Group X a , X b and X c Any suitable monosaccharide unit capable of forming an oligosaccharide with the galactose units of compounds (a), (b) and (c) can provide X a , X b and X c . Appropriately, X a , X b and X c Each independently selected from the following monosaccharides. Suitable monosaccharide units are selected from glucose (Glc), fucose (Fuc), arabinose (Ara), wood sugar (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. Therefore, each of compounds (a), (b) and (c) can comprise a mixture of oligosaccharide compounds with different corresponding X groups (e.g., Glc or FucX groups).

[0017] In some embodiments, group X a , X b and X c Each is independently selected from the monosaccharides listed above, suitably independently selected from Glc, Fuc, Ara, Xyl, Rha and Man, or a mixture thereof.

[0018] The sugar units of the oligosaccharide in the composition of the first aspect may have the D enantiomeric form or the L enantiomeric form. Suitably, the sugar units in components (a), (b) and (c) all have the D enantiomeric form. Thus components (a), (b) and (c) may be as follows:

[0019] (a)D-Gal-(β1-3)-D-Gal-(β1-4)-X a ;

[0020] (b)D-Gal-(β1-3)-D-Gal-(β1-3)-X b ;and

[0021] (c)D-Gal-(β1-3)-D-Gal-(β1-2)-X c .

[0022] Appropriately, Gal and X a , X b and X c The sugar units are all in the D enantiomeric form. Components (a), (b) and (c) may therefore be as follows:

[0023] (a)D-Gal-(β1-3)-D-Gal-(β1-4)-DX a ;

[0024] (b)D-Gal-(β1-3)-D-Gal-(β1-3)-DX b ;and

[0025] (c)D-Gal-(β1-3)-D-Gal-(β1-2)-DX c .

[0026] In some embodiments, X a , X b and X c Each is Glc.Therefore, compound (a), (b) and (c) can be galacto-oligosaccharide compound (GOS), and the composition of this first aspect can refer to galacto-oligosaccharide composition.This galacto-oligosaccharide can be formed by converting lactose into described oligosaccharide with suitable β-galactosidase.In these embodiments, component (a), (b) and (c) are suitably as follows:

[0027] (a)Gal-(β1-3)-Gal-(β1-4)-Glc;

[0028] (b)Gal-(β1-3)-Gal-(β1-3)-Glc; and

[0029] (c)Gal-(β1-3)-Gal-(β1-2)-Glc.

[0030] Suitably, both the Gal and Glc sugar units are in the D enantiomeric form. Thus components (a), (b) and (c) may be as follows:

[0031] (a) D-Gal-(β1-3)-D-Gal-(β1-4)-D-Glc;

[0032] (b) D-Gal-(β1-3)-D-Gal-(β1-3)-D-Glc; and

[0033] (c) D-Gal-(β1-3)-D-Gal-(β1-2)-D-Glc.

[0034] In some embodiments, Xa , X b and X c Each is a mixture of Glc and one or more other sugar units described above, such as Fuc, Ara, Xyl, Rha and Man. Thus, each of (a), (b) and (c) may comprise a mixture of oligosaccharide compounds, either having Glc or having one of the other sugar units described above as X groups. In these embodiments, components (a), (b) and (c) may be formed by converting a mixture of lactose and a suitable additional sugar (e.g., a monosaccharide selected from fucose, arabinose, xylose, rhamnose and mannose) into oligosaccharides. Suitably, X a , X b and X c Each is a mixture of Glc and Fuc.

[0035] The other oligosaccharides in the composition comprising oligosaccharide compounds of the first aspect, in addition to components (a), (b) and (c) discussed above, may also comprise the sugar unit "X" groups referred to above.

[0036] The composition of the first aspect comprises (a) at least 8 wt% of Gal-(β1-3)-Gal-(β1-4)-X a Suitably, the composition comprises at least 9 wt% component (a) or at least 10 wt% component (a).

[0037] Suitably, the composition comprises at most 35 wt% component (a), at most 30 wt% component (a), or at most 25 wt% component (a).

[0038] Suitably, the composition comprises 8 wt% to 35 wt% component (a), 8 wt% to 25 wt% component (a), or 10 wt% to 20 wt% component (a).

[0039] The composition of the first aspect comprises (b) at least 3 wt% of Gal-(β1-3)-Gal-(β1-3)-X b Suitably, the composition comprises at least 4 wt% of component (b) or at least 5 wt% of component (b). Suitably, the composition comprises at least 6 wt% of component (b) or at least 8 wt% of component (b).

[0040] Suitably, the composition comprises at most 25 wt% component (b), at most 20 wt% component (b), or at most 15 wt% component (b).

[0041] Suitably, the composition comprises from 3 wt% to 25 wt% component (b), from 4 wt% to 20 wt% component (b), or from 4 wt% to 10 wt% component (b).

[0042] The composition of the first aspect comprises (c) at least 5 wt% of Gal-(β1-3)-Gal-(β1-2)-X c Suitably, the composition comprises at least 6 wt% of component (c). Suitably, the composition comprises at least 8 wt% of component (c).

[0043] Suitably, the composition comprises at most 25 wt% component (c), at most 20 wt% component (c), or at most 15 wt% component (c).

[0044] Suitably, the composition comprises 5 wt% to 25 wt% component (c), 5 wt% to 20 wt% component (c), or from 6 wt% to 10 wt% component (c).

[0045] The above amounts are based on the total weight of the oligosaccharide compounds present in the composition.

[0046] Suitably, in the composition of the first aspect, based on the total weight of the oligosaccharide compounds present in the composition:

[0047] Component (a) is present in an amount of at least 10 wt %;

[0048] Component (b) is present in an amount of at least 6 wt%; and

[0049] Component (c) is present in an amount of at least 6 wt%.

[0050] Suitably, in the composition of the first aspect, based on the total weight of the oligosaccharide compounds present in the composition:

[0051] Component (a) is present in an amount of up to 35 wt %;

[0052] Component (b) is present in an amount of up to 25 wt%; and

[0053] Component (c) is present in an amount of up to 25 wt%.

[0054] Suitably, in the composition of the first aspect, based on the total weight of the oligosaccharide compounds present in the composition:

[0055] Component (a) is present in an amount of 8 wt % to 25 wt %;

[0056] Component (b) is present in an amount of 3 wt% to 25 wt%; and

[0057] Component (c) is present in an amount of 5 wt% to 20 wt%.

[0058] Suitably, in the composition of the first aspect, based on the total weight of the oligosaccharide compounds present in the composition:

[0059] Component (a) is present in an amount of 8 wt % to 25 wt %;

[0060] Component (b) is present in an amount of 6 wt% to 25 wt%; and

[0061] Component (c) is present in an amount of 6 wt% to 20 wt%.

[0062] In some embodiments, the composition of the first aspect comprises, based on the total weight of the oligosaccharide compounds present in the composition:

[0063] (a) 8 wt% to 25 wt% of Gal-(β1-3)-Gal-(β1-4)-Glc;

[0064] (b) 3 wt% to 25 wt% of Gal-(β1-3)-Gal-(β1-3)-Glc; and

[0065] (c) 5 to 20 wt% of Gal-(β1-3)-Gal-(β1-2)-Glc.

[0066] In some embodiments, the composition of the first aspect comprises, based on the total weight of the oligosaccharide compounds present in the composition:

[0067] (a) 8 wt% to 25 wt% of Gal-(β1-3)-Gal-(β1-4)-Glc;

[0068] (b) 6 wt% to 25 wt% of Gal-(β1-3)-Gal-(β1-3)-Glc; and

[0069] (c) 6 wt% to 20 wt% of Gal-(β1-3)-Gal-(β1-2)-Glc.

[0070] Suitably, the ratio of wt% of compound (a) to compound (b) is from 1:1 to 3:1; suitably, from 1.5:1 to 2.5:1.

[0071] Suitably, the ratio of wt% of compound (a) to compound (c) is from 1:1 to 3:1; suitably, from 1.5:1 to 2.5:1.

[0072] Suitably, the ratio of wt% of compound (b) to compound (c) is from 2:1 to 1:2; suitably, 1.5:1 to 1:1.5.

[0073] Suitably, in the composition of the first aspect, the oligosaccharide compound comprises, based on the total weight of the oligosaccharide compounds present in the composition:

[0074] (d) at least 3 wt% of Gal-(β1-3)-Gal-(β1-3)-Gal-(β1-4)-X d ;

[0075] Where X d Selected from sugar.

[0076] X d X can be selected from the above description a , X b and X c of the same monosaccharide units.

[0077] In some embodiments, X d It is Glc.

[0078] Suitably, the Gal sugar unit of component (d) has the D enantiomeric form. Suitably, Gal and X d The sugar units are all in the D enantiomeric form. Thus, component (d) may be as follows: D-Gal-(β1-3)-D-Gal-(β1-3)-D-Gal-(β1-4)-DX d .

[0079] In some embodiments, X d is a mixture of Glc and one or more other monosaccharide units (for example Fuc, Ara, Xyl, Rha and Man, suitably Fuc).

[0080] Suitably, the composition comprises at least 4 wt% component (d) or at least 5 wt% component (d).

[0081] Suitably, the composition comprises at most 25 wt% component (d), at most 20 wt% component (d), or at most 15 wt% component (d).

[0082] Suitably, the composition comprises 3 wt% to 25 wt% of component (d), 4 wt% to 20 wt% of component (d), or 4 wt% to 10 wt% of component (d).

[0083] Suitably, the amount of component (d) present in the composition is lower than the amount of component (c) present in the composition.

[0084] Suitably, in the composition of the first aspect, based on the total weight of the oligosaccharide compounds present in the composition:

[0085] Component (a) is present in an amount of 8 wt % to 25 wt %;

[0086] Component (b) is present in an amount of 3 wt % to 25 wt %;

[0087] Component (c) is present in an amount of 5 wt% to 20 wt%; and

[0088] Component (d) is present in an amount of 3 wt% to 25 wt%.

[0089] In some embodiments, the composition of the first aspect comprises, based on the total weight of the oligosaccharide compounds present in the composition:

[0090] (a) 8 wt% to 25 wt% of Gal-(β1-3)-Gal-(β1-4)-Glc;

[0091] (b) 3 wt% to 25 wt% of Gal-(β1-3)-Gal-(β1-3)-Glc;

[0092] (c) 5 wt% to 20 wt% of Gal-(β1-3)-Gal-(β1-2)-Glc, and

[0093] (d) 3 wt% to 25 wt% of Gal-(β1-3)-Gal-(β1-3)-Gal-(β1-4)-Glc.

[0094] Suitably, in the composition of the first aspect, the oligosaccharide compound comprises, based on the total weight of the oligosaccharide compounds present in the composition:

[0095] (e) at least 5 wt% of Gal-(β1-4)-Gal-(β1-4)-X e ;

[0096] Where X e Selected from monosaccharides.

[0097] X e X can be selected from the above description a , X b and X c of the same monosaccharide units.

[0098] Suitably, the Gal sugar unit of component (e) has the D enantiomeric form. Suitably, Gal and X e The sugar units are all in the D enantiomeric form. Thus, component (e) may be as follows: D-Gal-(β1-4)-D-Gal-(β1-4)-DX e .

[0099] In some embodiments, X e It is Glc.

[0100] In some embodiments, X e is a mixture of Glc and one or more other monosaccharide units (for example Fuc, Ara, Xyl, Rha and Man, suitably Fuc).

[0101] Suitably, the composition comprises at least 6 wt% component (e) or at least 7 wt% component (e).

[0102] Suitably, the composition comprises at most 25 wt% component (e), at most 20 wt% component (e), or at most 15 wt% component (e).

[0103] Suitably, the composition comprises from 5 wt% to 25 wt% component (e), from 5 wt% to 20 wt% component (e), or from 6 wt% to 10 wt% component (e).

[0104] Suitably, component (e) is present in the composition in an amount less than the amount of component (a) present in the composition.

[0105] Suitably, in the composition of the first aspect, based on the total weight of the oligosaccharide compounds present in the composition:

[0106] Component (a) is present in an amount of 8 wt % to 25 wt %;

[0107] Component (b) is present in an amount of 3 wt % to 25 wt %;

[0108] Component (c) is present in an amount of 5 wt % to 20 wt %;

[0109] Component (d) is present in an amount of 3 wt% to 25 wt%; and

[0110] Component (e) is present in an amount of 5 wt% to 25 wt%.

[0111] In some embodiments, the composition of the first aspect comprises, based on the total weight of the oligosaccharide compounds present in the composition:

[0112] (a) 8 wt% to 25 wt% of Gal-(β1-3)-Gal-(β1-4)-Glc;

[0113] (b) 3 wt% to 25 wt% of Gal-(β1-3)-Gal-(β1-3)-Glc;

[0114] (c) 5 wt% to 20 wt% of Gal-(β1-3)-Gal-(β1-2)-Glc, and

[0115] (d) 3 wt% to 25 wt% of Gal-(β1-3)-Gal-(β1-3)-Gal-(β1-4)-Glc, and

[0116] (e) 5 to 25 wt% of Gal-(β1-4)-Gal-(β1-4)-Glc.

[0117] In the composition of the first aspect, X a , X b , X c , X d and X e Each is independently selected from the monosaccharides described above.

[0118] In the composition of the first aspect, X a , X b , X c , X d and X e Each is independently selected from Glc, Fuc, Ara, Xyl, Rha and Man or a mixture thereof.

[0119] In some embodiments, X a , X b , X c , X d and X e Each is Glc.

[0120] In some embodiments, X a , X b , X c , X d and X e Each comprises Fuc. Suitably, X a , X b , X c , X d and X e Each is a mixture of Glc and Fuc.

[0121] The oligosaccharide compound of the composition of the first aspect suitably has a relatively high proportion of β1-3Gal-Gal bonds - primarily because components (a), (b), (c) and optionally (d) are present in the proportions discussed herein. The inventors have found that such a high proportion of β1-3Gal-Gal bonds is particularly advantageous for the use of the composition discussed herein.

[0122] Suitably, 35% to 55% (suitably 40% to 55%, suitably 40% to 50%) of the Gal-Gal bonds in the oligosaccharide compound are 1-3 bonds.

[0123] Suitably, 40% to 60% (suitably 45% to 55%, suitably 45% to 52%) of the Gal-Gal bonds in the oligosaccharide compound are 1-4 bonds.

[0124] Suitably, 8% to 20% (suitably 10% to 18%, suitably 12% to 16%) of the Gal-X bonds in the oligosaccharide compound are 1-3 bonds, suitably wherein X is Glc.

[0125] Suitably, 15% to 25% (suitably 16% to 24%, suitably 17% to 22%) of the Gal-X bonds in the oligosaccharide compound are 1-4 bonds, suitably wherein X is Glc.

[0126] Suitably, 30% to 45% (suitably 32% to 43%, suitably 34% to 41%) of the Gal-X bonds in the oligosaccharide compound are 1-2 bonds, suitably wherein X is Glc.

[0127] Suitably, 20% to 36% (suitably 22% to 34%, suitably 25% to 32%) of the Gal-X bonds in the oligosaccharide compound are 1-6 bonds, suitably wherein X is Glc.

[0128] Suitably, the above-mentioned Gal-X' bond (eg Gal-Glu bond) refers to a β bond, ie a β-glycosidic bond.

[0129] Suitably, the composition comprises at least 25 wt% trisaccharide, based on the total weight in the composition. Suitably, the composition comprises at least 28 wt% trisaccharide or at least 30 wt% trisaccharide.

[0130] Suitably the composition comprises at most 70 wt% trisaccharide, suitably at most 60 wt% trisaccharide, or at most 50 wt% trisaccharide.

[0131] Suitably the composition comprises 25 wt% to 70 wt% trisaccharide, suitably 30 wt% to 60 wt% trisaccharide, or 30 wt% to 50 wt% trisaccharide.

[0132] Suitably, the composition comprises at least 10 wt% tetrasaccharide based on the total weight in the composition. Suitably, the composition comprises at least 12 wt% tetrasaccharide or at least 15 wt% tetrasaccharide. Suitably, the composition comprises at least 20 wt% tetrasaccharide or at least 25 wt% tetrasaccharide.

[0133] Suitably the composition comprises at most 40 wt% tetrasaccharide, suitably at most 25 wt% tetrasaccharide, or at most 20 wt% tetrasaccharide.

[0134] Suitably, the composition comprises 10 wt% to 40 wt% tetrasaccharide, suitably 10 wt% to 30 wt% tetrasaccharide, or 12 wt% to 25 wt% tetrasaccharide.

[0135] Suitably, the composition comprises from 30 wt% to 50 wt% trisaccharide and from 10 wt% to 25 wt% tetrasaccharide, based on the total weight of the composition.

[0136] As mentioned above, it is determined that the disaccharides in the composition exclude lactose. Therefore, in some embodiments, the composition suitably comprises at most 40wt% disaccharides, suitably at most 30wt% disaccharides, or at most 20wt% disaccharides. Suitably, the content of lactose in the composition of this first aspect is minimized. Suitably, the composition is substantially free of lactose. Suitably, the composition does not contain lactose.

[0137] Suitably the composition comprises 10 wt% to 40 wt% disaccharide, suitably 10 wt% to 30 wt% disaccharide, or 10 wt% to 20 wt% disaccharide.

[0138] Suitably, the composition comprises from 10 wt% to 40 wt% disaccharide, from 30 wt% to 60 wt% trisaccharide and from 10 wt% to 25 wt% tetrasaccharide, based on the total weight of the composition.

[0139] The following description relates to the content of disaccharides in the oligosaccharide compound. Suitably, the oligosaccharide compound comprises at most 40 wt% disaccharides, suitably at most 30 wt% disaccharides, or at most 20 wt% disaccharides.

[0140] Suitably, the oligosaccharide compound comprises 0wt% to 40wt% disaccharides, suitably 10wt% to 30wt% disaccharides, or 10wt% to 20wt% disaccharides.

[0141] Suitably, the oligosaccharide compound comprises at least 25 wt% trisaccharides, based on the total weight of oligosaccharide compounds present in the composition. Suitably, the oligosaccharide compound comprises at least 30 wt% trisaccharides, or at least 33 wt% trisaccharides.

[0142] Suitably, the oligosaccharide compound comprises at most 75 wt% trisaccharides, suitably at most 65 wt% trisaccharides, or at most 55 wt% trisaccharides.

[0143] Suitably, the oligosaccharide compound comprises 25 wt% to 75 wt% trisaccharides, suitably 30 wt% to 65 wt% trisaccharides, or 34 wt% to 55 wt% trisaccharides.

[0144] Suitably, the oligosaccharide compound comprises at least 10 wt% tetrasaccharide, based on the total weight of oligosaccharide compounds present in the composition. Suitably, the oligosaccharide compound comprises at least 12 wt% tetrasaccharide, or at least 15 wt% tetrasaccharide.

[0145] Suitably, the oligosaccharide compound comprises at most 45 wt% tetrasaccharide, suitably at most 35 wt% tetrasaccharide, or at most 30 wt% tetrasaccharide.

[0146] Suitably, the oligosaccharide compound comprises 10 wt% to 45 wt% tetrasaccharide, suitably 10 wt% to 35 wt% tetrasaccharide, or 15 wt% to 30 wt% tetrasaccharide.

[0147] Suitably, the oligosaccharide compound comprises from 0 wt% to 40 wt% disaccharides and from 30 wt% to 75 wt% trisaccharides, based on the total weight of the oligosaccharide compound present in the composition.

[0148] Suitably, the oligosaccharide compound comprises 0 wt% to 40 wt% disaccharides, 30 wt% to 75 wt% trisaccharides and 10 wt% to 45 wt% tetrasaccharides, based on the total weight of the oligosaccharide compounds present in the composition.

[0149] The composition of the first aspect comprises at least 50 wt% of oligosaccharide compounds, including components (a), (b), (c) and optionally (d) and (e). Based on the total weight of the composition, the composition suitably comprises at least 55 wt% of oligosaccharide compounds, suitably at least 60 wt%.

[0150] Suitably, the composition comprises at most 100 wt% oligosaccharide compounds, suitably at most 95 wt%, at most 90 wt%, or at most 85 wt% oligosaccharide compounds.

[0151] Suitably, the composition comprises 50 wt% to 100 wt% of the oligosaccharide compound, suitably 55 wt% to 95 wt%, or 60 wt% to 85 wt% of the oligosaccharide compound.

[0152] In some embodiments, the composition is in the form of syrup. The syrup suitably comprises at least 50wt% of oligosaccharide compounds, at least 55wt% of oligosaccharide compounds, or at least 60wt%. Suitably, the syrup comprises from 50wt% to 75wt% of oligosaccharide compounds, suitably, 55wt% to 70wt% or 60wt% to 70wt% of oligosaccharide compounds.

[0153] The syrup may comprise a significant amount of monosaccharides, such as glucose and / or galactose. The syrup may comprise 15wt% to 30wt% monosaccharides, suitably 20wt% to 28wt% monosaccharides, or 21wt% to 25wt% monosaccharides, such as glucose and / or galactose.

[0154] The syrup suitably comprises 20 to 30 wt % water, suitably 22 to 28 wt % water.

[0155] The syrup may also contain lactose, for example 4 to 14 wt % lactose.

[0156] In some embodiments, the composition is in the form of a powder. The powder suitably comprises at least 60wt% of an oligosaccharide compound, suitably at least 70wt% or at least 75wt% of an oligosaccharide compound. Suitably, the powder comprises 60wt% to 100wt% of an oligosaccharide compound, suitably 70wt% to 95wt% or 75wt% to 90wt% of an oligosaccharide compound.

[0157] Compared with the syrup discussed above, the powder suitably comprises a reduced content of monosaccharides, such as glucose and / or galactose. The powder may comprise 1 wt% to 10 wt% of monosaccharides, suitably 2 wt% to 8 wt% of monosaccharides, or 3 wt% to 7 wt% of monosaccharides, such as suitably about 5 wt% of glucose and / or galactose.

[0158] The powder suitably comprises from 1 wt% to 10 wt% water, suitably from 3 wt% to 6 wt% water.

[0159] The composition of the first aspect may have been purified to remove monosaccharides and optionally disaccharides from the composition.

[0160] The composition of this first aspect can be graded to separate the oligosaccharide components in the composition according to its molecular weight, for example, to remove disaccharides from the composition or to separate trisaccharides from other oligosaccharide components. This can be carried out according to suitable methods known in the art, for example high performance liquid chromatography. The composition prepared by this classification can refer to the oligosaccharide part or the GOS part.

[0161] In these embodiments, composition (or oligosaccharide part) suitably comprises trisaccharide, tetrasaccharide and higher oligosaccharide of at least 70wt%, suitably, at least 80wt% or at least 90wt%.These higher oligosaccharides have 5 and above polymerization degree.Suitably, composition comprises trisaccharide, tetrasaccharide and higher oligosaccharide of at least 95wt%.Suitably, composition is made up of trisaccharide, tetrasaccharide and higher oligosaccharide, or is made up of trisaccharide, tetrasaccharide and higher oligosaccharide substantially.

[0162] In these embodiments, the composition suitably comprises 40 wt% to 70 wt% trisaccharide, suitably 45 wt% to 70 wt% trisaccharide, or 50 wt% to 70 wt% trisaccharide.

[0163] Suitably, the oligosaccharide compound comprises from 15 wt% to 50 wt% tetrasaccharide, suitably, 15 wt% to 40 wt% tetrasaccharide, or 20 wt% to 40 wt% tetrasaccharide.

[0164] Suitably, the oligosaccharide compound comprises 5wt% to 25wt% higher oligosaccharides, suitably 5wt% to 20wt% higher oligosaccharides, or 10wt% to 20wt% higher oligosaccharides.

[0165] Suitably, based on the gross weight of the composition, the composition comprises 40wt% to 70wt% trisaccharides, 15wt% to 40wt% tetrasaccharides and 5wt% to 25wt% higher oligosaccharides. Suitably, based on the gross weight of the composition, the composition comprises 50wt% to 70wt% trisaccharides, 20wt% to 40wt% tetrasaccharides and 10wt% to 20wt% higher oligosaccharides.

[0166] In these embodiments, the composition (or oligosaccharide portion) suitably comprises components (a), (b) and (c) described above in the following amounts, based on the total weight of the oligosaccharide compounds present in the composition:

[0167] (a) at least 8 wt% Gal-(β1-3)-Gal-(β1-4)-X a ;

[0168] (b) at least 3 wt% of Gal-(β1-3)-Gal-(β1-3)-X b ;and

[0169] (c) at least 5 wt% of Gal-(β1-3)-Gal-(β1-2)-X c ;

[0170] Where X a , X b and X c Each is independently selected from monosaccharides.

[0171] Suitably, the composition (or oligosaccharide fraction) comprises components (a), (b) and (c) described above, suitably in the following amounts:

[0172] (a) at least 10 wt% of Gal-(β1-3)-Gal-(β1-4)-X a ;

[0173] (b) at least 5 wt% of Gal-(β1-3)-Gal-(β1-3)-X b ;and

[0174] (c) at least 7 wt% Gal-(β1-3)-Gal-(β1-2)-X c .

[0175] The composition may comprise component (d) and / or component (e) as described above.

[0176] The composition suitably comprises the components in the ratios described above.

[0177] In some embodiments, the composition of the first aspect is a trisaccharide and tetrasaccharide fractionation product (which may be referred to as a DP3 / DP4 fraction).

[0178] In these embodiments, the composition (or DP3 / DP4 fraction) suitably comprises at least 70 wt% trisaccharides and tetrasaccharides, suitably at least 80 wt%, or at least 90 wt%. Suitably, the composition consists of, or consists essentially of, trisaccharides and tetrasaccharides.

[0179] In these embodiments, the composition suitably comprises 50 wt% to 80 wt% trisaccharide, suitably 55 wt% to 75 wt% trisaccharide, or 60 wt% to 75 wt% trisaccharide.

[0180] Suitably, the oligosaccharide compound comprises 20 wt% to 50 wt% tetrasaccharide, suitably 25 wt% to 45 wt% tetrasaccharide, or 25 wt% to 40 wt% tetrasaccharide.

[0181] Suitably, the composition comprises 50 wt% to 80 wt% trisaccharides and 20 wt% to 50 wt% tetrasaccharides, based on the total weight of the composition. Suitably, the composition comprises 60 wt% to 75 wt% trisaccharides and 25 wt% to 40 wt% tetrasaccharides, based on the total weight of the composition.

[0182] In these embodiments, the composition (or oligosaccharide portion) suitably comprises components (a), (b) and (c) described above in the following amounts, based on the total weight of the oligosaccharide compounds present in the composition:

[0183] (a) at least 8 wt% Gal-(β1-3)-Gal-(β1-4)-X a ;

[0184] (b) at least 3 wt% of Gal-(β1-3)-Gal-(β1-3)-X b ;and

[0185] (c) at least 5 wt% of Gal-(β1-3)-Gal-(β1-2)-X c ;

[0186] Where X a , X b and X c Each is independently selected from monosaccharides.

[0187] Suitably, the composition (or oligosaccharide fraction) comprises components (a), (b) and (c) described above, suitably in the following amounts:

[0188] (a) at least 10 wt% of Gal-(β1-3)-Gal-(β1-4)-X a ;

[0189] (b) at least 5 wt% of Gal-(β1-3)-Gal-(β1-3)-X b ;and

[0190] (c) at least 7 wt% Gal-(β1-3)-Gal-(β1-2)-X c ,

[0191] The composition may comprise component (d) and / or component (e) as described above.

[0192] The composition suitably comprises the components in the ratios described above.

[0193] In some embodiments, the composition of the first aspect is a trisaccharide fractionation product (which may be referred to as a DP3 fraction). This trisaccharide fractionation product may be obtained by the above-mentioned known fractionation method. This composition suitably comprises at least 70wt% trisaccharides, suitably, at least 80wt%, or at least 90wt%. Suitably, the composition comprises at least 95wt% trisaccharides.

[0194] In these embodiments, the composition (or oligosaccharide portion) suitably comprises components (a), (b) and (c) described above in the following amounts, based on the total weight of the oligosaccharide compounds present in the composition:

[0195] (a) at least 8 wt% Gal-(β1-3)-Gal-(β1-4)-X a ;

[0196] (b) at least 3 wt% of Gal-(β1-3)-Gal-(β1-3)-X b ;and

[0197] (c) at least 5 wt% of Gal-(β1-3)-Gal-(β1-2)-X c ;

[0198] Where X a , X b and X c Each is independently selected from sugars.

[0199] Suitably, the composition (or oligosaccharide fraction) comprises components (a), (b) and (c) described above, suitably in the following amounts:

[0200] (a) at least 10 wt% of Gal-(β1-3)-Gal-(β1-4)-Xa ;

[0201] (b) at least 5 wt% of Gal-(β1-3)-Gal-(β1-3)-X b ;and

[0202] (c) at least 7 wt% Gal-(β1-3)-Gal-(β1-2)-X c ,

[0203] The composition may comprise component (e) as described above.

[0204] The composition suitably comprises the components in the ratios described above.

[0205] The composition of the first aspect can be incorporated into a product ingested by a consumer to improve intestinal health by promoting the growth of beneficial bacteria (e.g., bifidobacteria in the intestinal tract of the consumer). Such a product can be selected from the group consisting of dairy products (e.g., liquid milk, milk powder such as whole milk powder, skimmed milk powder, fat-filled milk powder, whey powder, infant formula, ice cream, yogurt, cheese, fermented dairy products), beverages, baby food, cereals, bread, biscuits, candy, cakes, food supplements, dietary supplements, animal feed, poultry feed, or any other food or beverage.

[0206] The composition of the first aspect may be incorporated into a synbiotic composition.Such a synbiotic composition is a mixture suitably comprising live microorganisms and substrates selectively utilised by the host microorganisms which confer a health benefit to the host (ie probiotics and prebiotics).

[0207] According to a second aspect of the present invention, there is provided a pharmaceutical or nutritional composition comprising the composition of the first aspect and at least one carrier, excipient or diluent.

[0208] The composition of the first aspect in the pharmaceutical or nutritional composition is a composition comprising the oligosaccharide compound as described above. The composition comprising the oligosaccharide compound may have any suitable features and advantages described above for the first aspect.

[0209] Suitable other components of pharmaceutical or nutritional compositions and methods of preparing such pharmaceutical or nutritional compositions are known in the art.

[0210] According to a third aspect of the present invention, there is provided use of the composition according to the first aspect as a dietary supplement.

[0211] 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 suitable features and advantages described above for the first aspect.

[0212] The application of this third aspect appropriately increases the production of butyric acid in the intestinal tract of dietary supplement consumers. The term butyric acid is used to refer to butyric acid and its derivatives, such as butyrate and butyrate produced by mammalian intestinal flora. Specifically, members of the genera Bifidobacterium and Lactobacillus of the intestinal flora ferment oligosaccharides for use as an energy source. As a byproduct of this fermentation, the intestinal flora produces lactic acid and short-chain fatty acids (SCFA), such as acetate, butyrate and propionate.

[0213] Suitably, the use of the third aspect increases the production of butyric acid in the intestine of a dietary supplement consumer compared to normal levels of butyric acid production in the body of the dietary supplement consumer, suitably compared to a placebo dietary supplement not containing a composition comprising an oligosaccharide compound. The use suitably increases the production of butyric acid in the intestine of a dietary supplement consumer compared to an alternative composition comprising an oligosaccharide compound, wherein the content of components (a), (b) and / or (c) in the alternative composition is lower than that specified herein.

[0214] Suitably, the use of this third aspect promotes the growth of beneficial bacteria (such as bifidobacteria and lactobacilli) in the consumer's gut, which in turn may result in the increase in butyrate production described above.

[0215] According to a third aspect of the present invention, there is provided a composition according to the first aspect or a pharmaceutical or nutritional composition according to the second aspect for use as a medicament.

[0216] The composition of use of the fourth aspect may provide the increase in butyrate production discussed above in relation to the third aspect.

[0217] Although the role of butyrate in the intestine is not fully understood, butyrate is thought to have multiple beneficial effects on human health, including maintaining immune homeostasis, regulating systemic inflammation, modulating local and / or organ-specific inflammation (including intestinal inflammation), and providing a major energy source for enterocyte regeneration. In addition, butyrate can induce immune tolerance, so this increase in butyrate production can be used to control and / or treat and / or alleviate symptoms and / or alleviate allergic diseases or other immune hypersensitivity reactions, 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”) side effects of current treatment options.

[0218] Thus, a fourth aspect of the present 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.

[0219] A fourth aspect of the present invention may provide the composition of the first aspect for use in therapeutic management and / or treatment and / or alleviation of symptoms of immune hypersensitivity reactions.

[0220] A fourth aspect of the present invention may provide the composition of the first aspect for use in the therapeutic management and / or treatment and / or alleviation of the symptoms of an autoimmune disease.

[0221] A fourth aspect of the present invention may provide the composition of the first aspect for use in therapeutic management and / or treatment and / or alleviation of symptoms of stem cell transplantation.

[0222] A fourth aspect of the present invention may provide the composition of the first aspect for use in the therapeutic management and / or treatment and / or alleviation of the symptoms of graft-versus-host disease.

[0223] A fourth aspect of the present invention may provide the composition of the first aspect for use in the therapeutic management and / or treatment and / or alleviation of symptoms of cancer.

[0224] Butyrate is also believed to play a role in maintaining colonic epithelium, regulating blood glucose homeostasis, regulating lipid metabolism and appetite regulation, as well as the immune system and inflammatory response (as described in Morrison, DJ; Preston, T. "Formation of shortchain fatty acids by the gut microbiota and their impact on humanmetabolism", Gut Microbes 2016, 7, 189-200). Therefore, increasing the production of butyrate in the intestine using the composition of the present invention is believed to provide consumers with several such health benefits.

[0225] Suitably, the composition for use in the fourth aspect promotes the growth of bifidobacteria in the intestinal tract of a subject.

[0226] 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 suitable features and advantages described above for the first aspect.

[0227] This fourth aspect is used as a medicament and can be related to preventing pathogens or toxins produced by pathogens from adhering to the intestinal wall of the subject. The composition can be given to the patient after a course of antibiotic treatment and / or chemotherapy and / or radiotherapy (which often changes or even destroys normal healthy intestinal flora), or after intestinal surgery, so as to reconstruct the normal microbial flora of a healthy intestine in the intestine. The composition can be used in combination with one or more beneficial intestinal flora, appropriately selected from the group consisting of Akkermansia, Bacteroides, Bifidobacterium, Lactobacillus and Parabacteroides, such as Bifidobacterium animalis, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum (including Bifidobacterium infantis) and / or Megamonas.

[0228] In the applications of the third and fourth aspects, the composition can regulate the abundance of the beneficial intestinal flora.

[0229] In the application of the third and fourth aspects, the composition can regulate the abundance of the bacterial genus present in the gastrointestinal tract. In some embodiments, the composition regulates the abundance of the bacterial genus present in one or both of the small intestine or large intestine. In some embodiments, the composition regulates the abundance of the bacterial genus dominant in the small intestine, and the bacterial genus is selected from the group consisting of Achromobacter, Agrobacterium, Blautia, Burkholderia, Coprococcus, Cryocola, Enterococcus, Eubacterium, Holdemania, Lactococcus, Mycobacterium, Pseudomonas, Ralstonia, Sphingomonas, Streptococcus and Turicibacter. In some embodiments, the composition modulates the abundance of a bacterial genus predominant in the large intestine selected from the group consisting of Anaerobic Bacillus, Akkermansia, Bacteroides, Bilophila, Butyricum, Odorobacterium, Parabacteroides, Phascolarctobacterium, Prevotella, and Ruminococcus.

[0230] 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 the steps of:

[0231] (i) providing a source of sugar compounds;

[0232] (ii) treating the sugar compound source with one or more β-galactosidases to at least partially convert the sugar compound source into oligosaccharide compounds.

[0233] Suitably, the method of the fifth aspect is carried out in the order of step (i) followed by step (ii).

[0234] Suitably, the sugar compound source comprises lactose, lactulose or epilactose. Suitably, the sugar compound source comprises lactose. The sugar source may be lactose, for example lactose syrup from cow's milk. The lactose may be heat treated.

[0235] In some embodiments, no other sugars (such as monosaccharides or disaccharides) are added to the sugar source. In these embodiments, the method produces a galacto-oligosaccharide compound.

[0236] In some embodiments, the sugar compound source includes at least one additional sugar. Suitably, at least one additional sugar provides a replacement terminal monosaccharide unit to the oligosaccharide compound, as described above. Suitably, at least one additional sugar is this monosaccharide unit source. At least one additional sugar as this monosaccharide unit source can be a monosaccharide, or can be a higher sugar, such as a disaccharide. At least one additional sugar that can be a monosaccharide source is 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 its mixture. The at least one additional sugar may be one or more of the monosaccharides listed above.

[0237] In these embodiments, the methods produce oligosaccharides having one or more of the above monosaccharides as terminal sugar units.

[0238] In some embodiments, at least one additional sugar can be selected from fucose, arabinose, xylose, rhamnose, mannose or its mixture. Sugar source can comprise lactose and one or more described monosaccharide sources. In these embodiments, method produces the oligosaccharide with the terminal sugar unit from Glc, Fuc, Ara, Xyl, Rha and Man or its mixture.

[0239] Step (ii) of the method involves treating a sugar compound source with at least one galactosidase. The galactosidase may be an alpha-galactosidase or a beta-galactosidase, depending on whether an alpha bond or a beta bond is required between the sugar units of the oligosaccharide compound. Suitably, the enzyme exhibits galactosyltransferase (transgalactosylation) activity and forms an alpha-bond or a beta-bond between the sugar units in the sugar compound source. This results in the synthesis of an oligosaccharide compound having two or more galactose units derived from lactose. Suitably, step (ii) is performed until the conversion of the sugar source to the oligosaccharide compound is complete.

[0240] Step (ii) may involve treating the source of sugar compounds with one or more additional enzymes other than galactosidase.

[0241] Suitably, the method comprises the step (iii) of isolating the galactosidase from the composition comprising the oligosaccharide compound.Step (iii) may involve removing the enzyme by filtration (eg by nanofiltration).

[0242] The composition comprising oligosaccharide compounds produced in step (iii) may be heat-treated.

[0243] In some embodiments, the composition is evaporated to reduce the water content, thereby providing a final composition comprising oligosaccharide compounds in the form of a syrup, as discussed above with respect to the first aspect.

[0244] In some embodiments, glucose is removed from the composition produced in step (iii) prior to evaporation. This suitably reduces the glucose content in the composition from 20wt%-30wt% to less than 10wt%, suitably, about 5wt% or less. The water content of the composition is then reduced by evaporation, and the product is dried to provide a final composition comprising a powdered oligosaccharide compound, as discussed above in the first aspect.

[0245] It should also be understood that those skilled in the relevant art can produce a composition comprising the oligosaccharide compounds according to the first aspect, ie, a composition comprising a specific amount of a specific oligosaccharide compound, by combining the desired amounts of said oligosaccharides obtained and isolated from different sources. Example

[0246] Example 1 - Oligosaccharide Syrup

[0247] The composition comprising oligosaccharide compounds according to the present invention in the form of syrup is obtained by the following steps. Lactose is rehydrated with drinking water to obtain a working solution with a solid content between 35wt% and 65wt%. The lactose solution is heat treated and then cooled to 40-65°C. The pH of the solution is adjusted to pH 5.5-7.5. β-galactosidase is then added to the solution in a closed container and subsequently reacted with lactose to catalyze the transfer of galactose molecules to produce oligosaccharide compounds. The progress of the reaction is monitored by measuring the generation of glucose. The reaction is allowed to proceed for a period of between 8 hours and 26 hours. The reaction is then terminated by high heat treatment. The reaction mixture is cooled and the enzyme is removed by charcoal filtration. The mixture is then dried by evaporation to reduce the water content to about 22wt%-28wt% to provide a product in the form of syrup.

[0248] Example 2 - Oligosaccharide Powder

[0249] The composition comprising oligosaccharide compounds according to the present invention in powder form is obtained by the improvement of the above described method. After removing the enzyme, the mixture is further filtered to remove most of the glucose and other monosaccharides, and the monosaccharide content is reduced from about 23wt% to about 5wt%. The water content of the composition is reduced by evaporation, and the product is dried to provide a powder with a water content of about 3wt%-6wt%.

[0250] Example 3 - Classification

[0251] A 5×70 cm BioGel P2 column was used to separate different fractions (DP2, DP3, DP4 or DP5) from the sample of Example 2 using water as the eluent. The column was operated at a flow rate of 40 to 100 mL / h at room temperature (21° C.). Depending on the flow rate, 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 added. The column was loaded with this solution a total of 15 times to obtain enough low-content fractions, namely DP5. After a void volume of approximately 725 mL, 5 mL fractions were collected. Thin layer chromatography (TLC) and HPAEC-PAD were used for analysis to ensure that molecules with the same DP were pooled together.

[0252] The pooled fractions were frozen and lyophilized. The dry material from all rounds was collected and reconstituted. After a second round of freezing and lyophilization, the dry material was stored at 4°C for further analysis or testing.

[0253] Comparative Example 1

[0254] A commercially available composition comprising an oligosaccharide compound was obtained in the form of a powder.

[0255] Oligosaccharide analysis

[0256] The samples of Example 2 of the present invention and Comparative Example 1 were analyzed by the following steps to determine their oligosaccharide contents.

[0257] Materials and methods

[0258] The dry powder samples of Example 2 and Comparative Example 1 were respectively dissolved in water to provide solutions having a concentration of 40 g / L for analysis.

[0259] Gel Permeation Chromatography

[0260] An HPLC apparatus equipped with a Rezex RSO and RI detector and online desalting (for removal of salts and charged species such as proteins) was used for aqueous GPC separation of sample components. 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 (40 g / L). Prior to analysis, all sample solutions were treated at 100°C for 10 minutes to remove any microbial or enzymatic activity.

[0261] GOS Fingerprint

[0262] HPAEC-PAD (high-performance anion exchange chromatography) equipped with PA-1 columns is used to separate monosaccharides and oligosaccharides of different samples. The method for achieving separation quality is described in van Leeuwen et al., Carbohydrate Research 2016, 425, 48-58. Commercial maltooligosaccharide mixture and comparative example 1 were also injected for comparison with the chromatogram reported by Van Leeuwen et al. Based on this, peak annotations were performed for multiple peaks. The samples used for GPC were diluted 100 times with DMSO before injection.

[0263] result

[0264] Gel Permeation Chromatography (GPC-RI)

[0265] Table 1 shows the DP (degree of polymerization) composition results for samples using the Rezex-RSO system, based on RI calibration with glucose (values ​​in samples are expressed as g / L). All material eluting in the >DP5 window was combined.

[0266] Table 1

[0267]

[0268] The concentration information can be used to calculate the relative weight percentages of the different DP fractions of oligosaccharides contained in the sample, as shown in Table 1, where DP=2 represents a disaccharide, DP=3 represents a trisaccharide, and so on.

[0269] GOS Fingerprint

[0270] To confirm the individual galacto-oligosaccharides (GOS fingerprints) in the samples, 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 annotation of oligosaccharide compounds by van Leeuwen et al. A retention window of approximately 15 seconds was used for peak annotation.

[0271] Table 2 shows the HPAEC-PAD peak area information for all annotated peaks, as well as the incubation conditions, sample concentrations, dilutions, and injection volumes.

[0272] Table 2

[0273]

[0274] Assume that the peak area approximates the amount of each oligosaccharide compound present in the composition. If no specific oligosaccharide compound is identified, then enter "unknown" and a number in the table. The identified compound is identified by the name or number corresponding to the number assigned to a specific galacto-oligosaccharide in vanLeeuwen et al., Carbohydrate Research 2016, 425, 48-58. The list of these galacto-oligosaccharides and their corresponding numbers is provided below.

[0275] The oligosaccharide components (a)-(e) discussed above correspond to the following numbered entries in Table 2 above, wherein each X group is Glu:

[0276] (a)D-Gal-(β1-3)-D-Gal-(β1-4)-D-Glu—12

[0277] (b)D-Gal-(β1-3)-D-Gal-(β1-3)-D-Glu—29

[0278] (c)D-Gal-(β1-3)-D-Gal-(β1-2)-D-Glu—30

[0279] (d)D-Gal-(β1-3)-D-Gal-(β1-3)-D-Gal-(β1-4)-D-Glu—31

[0280] (e)D-Gal-(β1-4)-D-Gal-(β1-4)-D-Glu—11

[0281] Probiotic Effects—Butyrate Production

[0282] The prebiotic effects of the compositions of the present invention were studied and compared to a comparative GOS composition using the following procedure.

[0283] The composition of Example 2 of the present invention, Comparative Example 1 and a control blank sample were dialyzed using a 0.5 kDa membrane to provide a 5 g / L sample, which was then mixed with fecal material obtained from three healthy adult subjects (donors A, B and C). The mixture was shaken under anaerobic conditions and colonic fermentation products including butyric acid were monitored over 48 hours (with 6, 24 and 48 h collection points). During this time, the distribution of oligosaccharides in the mixture was also monitored using the method described in Table 2 above. The results showed that Example 2 was fully fermented by all donors, mainly during the 0-24 hour period, and butyric acid production increased at 6 and 48 hour time points compared to Comparative Example 1 and the control. The results of the butyric acid analysis are shown in Figure 2. Figure 1The results of oligosaccharide analysis of samples at different time points are shown in Table 3. These results indicate that during the experiment, the oligosaccharides in the samples were actively consumed by the microbiota present in the fecal samples.

[0284] In order to evaluate whether the treatment effect on intestinal microbial activity is statistically significant, three two-sided 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. The Benjamini-Hochberg false discovery rate (FDR) was also used for this analysis. When the p value obtained was less than the reference value, the difference between the treatment effects was considered significant. Table 4 below shows the difference in the average butyrate production of the comparison samples within 48 hours, and the asterisk indicates whether the difference is considered significant based on the above analysis. These results show that the increase in butyrate production provided by Example 2 during 0-48 hours is statistically significant compared to the control and Comparative Example 1.

[0285]

[0286]

[0287] Table 4

[0288] D0-48h / output Example 2 - Control Comparative Example 1-Control Example 2-Comparative Example 1 Butyrate (mM) 5.88＊ 4.75＊ 1.13＊

[0289] Example 4-9 - Oligosaccharides with alternative terminal monosaccharides

[0290] Example compositions 4-9 according to the invention comprising oligosaccharide compounds having alternative terminal monosaccharides (i.e., alternatives to glucose) were prepared by the procedure described above for Example 1, using the substrates noted in Table 5 below - i.e., combinations of lactose and additional substrates noted for Examples 4-8, and lactulose alone for Example 9. Significantly higher molar excesses of acceptor (substrate) were intentionally used to promote the formation of GOS compounds comprising alternative terminal monosaccharides.

[0291] Table 5

[0292]

[0293] Characterization and Quantification of Examples 4-9 Using HPAEC-PAD

[0294] The GOS compositions of Examples 4-9 were separated and quantified by high performance anion exchange chromatography (HPAE-PAD) equipped with a pulsed amperometric detector, using a CarboPac PA210 (2×150 mm) analytical column and a CarboPac-PA1 (4×50 mm) guard column. The operating parameters were set as follows: column temperature 30°C; elution was performed using a gradient concentration 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. DP1, DP2 and higher order GOS peaks were quantified based on calculated peak areas and calibration curves constructed using available standards. Glucose, galactose and lactose were calibrated in the range of 5-300 μg / mL. The new GOSDP2 peak was quantified based on the lactose calibration. The new GOS, DP3 and DP4 peaks were quantified with reference to an external maltooligosaccharide gradient (DP1-8).

[0295] Activated carbon purification of GOS

[0296] To remove monosaccharides, the GOS composition was purified with activated carbon according to the method described by Julio-González et al. (Food Research International, Vol. 129, March 2020, 108811) with some modifications. The GOS reaction mixture was mixed with activated carbon, using a ratio of 6 g activated carbon to 0.5 g total sugar. The ratio was adjusted to the final concentration with ethanol (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 through Whatman No. 1 paper (Whatman International Ltd., Maidstone, UK) under vacuum (step 1). The process was repeated a second time to ensure the removal of total monosaccharides (step 2). Oligosaccharides were desorbed from the washed activated carbon by adding an appropriate volume of ethanol (50%, v / v), stirring the mixture for 120 min, and then filtering (step 3). The resulting suspension of GOS in ethanol was freeze-dried to obtain a powder, which was subsequently tested as a microbial growth substrate in a fermentation evaluation.

[0297] HPAEC-PAD chromatograms indicated that Examples 4-9 contained different GOS compounds than Example 1 due to the introduction of respective alternative terminal monosaccharides into the compounds. The formation of these different GOS compounds in the respective product mixtures demonstrated that β-galactosidase can utilize other acceptor molecules besides lactose to provide alternative terminal monosaccharides for GOS compounds.

[0298] 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 a calibration curve constructed using available standards. a Total GOS: all quantitative transgalactosylated carbohydrate peaks identified by HPAEC-PAD in addition to the remaining glucose, galactose and lactose. b Total GOS (- receptor): Same as the previous one except that the remaining amount of receptor molecules is excluded in the calculation. c All of the quantitative new peaks that appear in the chromatograms of the nGOS:lactose / neo-receptor reactions are also absent in the Example 1 reaction containing only lactose - ignore the peaks that are clearly derived from lactose alone in the Example 1 chromatograms. d nGOS(-receptor): Same as the previous one except that the remaining amount of receptor molecules is excluded from the calculation. e nGOS: Percentage of the quantified new GOS peak out of the total GOS produced in each reaction - to distinguish peaks derived solely from lactose. f All reactions were terminated at the optimal 24 h time point, except for lactulose (the highest yield of new fructosylated GOS was observed at 12 h). Standard deviations are from two independent experiments (provided in parentheses).

[0299] Table 6

[0300]

[0301] The best yields were obtained in Example 8 (58.2%) and Example 9 (42.2%). When total sugars in the reaction were calculated, the other examples showed significantly lower yields (20-30%) compared to Example 1. However, adjusting the same yields to the actual transgalactosylated sugars present in the final matrix by removing the excess acceptor molecule quantitative weight provided clearer results (Table 6, column 2). The total GOS (-acceptor) reaction yields in Examples 4-8 were comparable to the total GOS yields observed in Examples 1 and 9 (55-70%). The most abundant formation of new hybrid transgalactosylating acceptor species was observed in Example 5 (36.7%) and Example 7 (35.8%) (Table 6, column 4), indicating that L-arabinose and L-rhamnose are the best alternative acceptor substrates for the enzyme.

[0302] As described above, after purification using activated carbon, the relative abundance of monosaccharides in all carbohydrate mixtures was greatly reduced to almost equivalent levels. After this purification step, the remaining amount of monosaccharides in the total carbohydrates in all compositions hardly exceeded the level of 5% (Table 7). In this way, in the mixtures obtained, the relative abundance of transgalactosylated oligosaccharides was significantly increased, concentrating the more important polymeric GOS carbohydrate molecules. For the subsequent in vitro fermentation experiments, the elimination of monosaccharides was crucial in order to minimize the impact on monosaccharide fermentation in the initial stage after fecal inoculation (<8 hours). On the contrary, the obvious purpose of this fermentation simulation is to establish real differences in the fermentation profiles among the newly synthesized GOS varieties, especially in the higher DP range.

[0303] Table 7

[0304] Example <![CDATA[MONO 前 ]]> <![CDATA[MONO 后 ]]> <![CDATA[Total GOS 前 > <![CDATA[Total GOS 后 > 4 74.5 4.8 20.3 85.0 5 71.5 5.5 22.8 83.8 6 63.3 4.2 32.6 86.7 7 65.8 5.4 28.3 83.7 8 23.3 3.2 58.2 73.3 9 29.4 5.2 42.2 61.4

[0305] Example 4-9 - Feces fermentation study

[0306] Stool sample preparation

[0307] Fresh fecal samples were collected 1 hour before inoculation from three healthy adult male and female donors who had not taken antibiotics for the previous 3 months and had no history of gastrointestinal illness. Anaerobic conditions were maintained in the collection container using a small bag to absorb atmospheric oxygen in a wide-mouth bottle while releasing carbon dioxide, hydrogen, and nitrogen (Thermo Scientific TM , Basingstoke, UK, Oxoid AnaeroGen 2.5 L). Fecal slurries were prepared by diluting feces 1:10 in phosphate buffered saline (PBS, pH 7.0) and mixing in a stomacher for 2 min. Batch culture experiments were performed in duplicate for each donor.

[0308] Batch fermentation

[0309] The method used is 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 contained (g / L): 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; heme (dissolved in a few drops of 1 mol L -1 NaOH), 0.05; cysteine ​​hydrochloride, 0.5; bile salts, 0.5; Tween 80, 2 and 10 μL vitamin K, added to 1 L with ddH2O. The culture medium was flushed with N2 overnight to produce anaerobic conditions. The container was maintained at 37°C by a circulating water bath, and the pH was maintained between 6.7 and 6.9 using a pH controller (Electrolab, Tewkesbury, UK) connected to 0.25M HCl and NaOH solutions to simulate conditions in the distal colon. Just before inoculation of fecal samples, 0.1 g of the test substrate (1%, w / v) was added to the container before adding 1 mL (v / v) of fecal slurry (Table 1). Immediately (0 h) and 8 h and 24 h after inoculation of the slurry, samples were removed from the fermenter for bacterial counts and metabolite analysis. Fermentation experiments were performed in duplicate for each substrate using fecal inocula from three adult volunteers.

[0310] Sample preparation for bacterial enumeration by flow cytometry-fluorescence in situ hybridization (FISH)

[0311] Samples (750 μL) were taken from the in vitro fermentation vessel at 0, 8, and 24 h and immediately placed on ice, then centrifuged at 13,000 × g for 5 min and the supernatant discarded. The precipitated bacteria were fixed for 4 hours at 4 ° C in PBS and 4% (w / v) filtered paraformaldehyde (PFA, pH 7.2) at a ratio of 1:3 (v / v). The samples were washed twice with filtered PBS and resuspended in 300 μL PBS and ethanol (1:1, v / v) and then stored at -20 ° C for up to 3 months.

[0312] Flow-FISH analysis

[0313] 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 (AccuriC6, BD Biosciences, USA) with AccuriC Flow software.

[0314] After thawing, vortex the sample for 10 seconds, add 75 μL of the sample to 500 μL PBS in a 1.5 mL test tube, vortex again, and centrifuge at 13,000 × g for 3 min. Remove the supernatant and discard. Add 100 μL Tris-EDTA buffer containing lysozyme (1 mg / mL) to the test tube, mix with a pipette, and then incubate in the dark for 10 min. Vortex the sample and centrifuge at 13,000 × g for 3 min, and remove the supernatant. Add 500 μL PBS to the tube, resuspend the pellet with a pipette, then vortex and centrifuge at 13,000 × g for 3 min. Remove the supernatant and resuspend the pellet in 150 μL hybridization buffer (0.9 M NaCl, 0.2 M Tris-HCl (pH 8.0), 0.01% sodium dodecyl sulfate, 30% formamide), vortex and centrifuge at 13,000 × g for 3 min. The supernatant was removed again 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 a 1.5 mL centrifuge tube containing 50 μL of sample, vortexed and incubated at 36°C overnight. 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) maintained at 40°C was added to each tube and the pellet was resuspended. The tubes were then vortexed and incubated in the dark at 38°C for 20 min to remove nonspecific binding of the primers. The samples were then centrifuged at 13,000 × g for 3 min, the supernatant removed, 300 μL of PBS added, and then vortexed. Samples were stored refrigerated in the dark at 4 °C prior to flow cytometric analysis.

[0315] Gas chromatography-mass spectrometry (GC-MS) for SCFA and lactate analysis

[0316] 1.5 mL samples were removed from each container at 0, 8, and 24 h, centrifuged at 13,000 × g for 10 min, and the supernatant was stored at 20 ° C until needed for metabolite analysis. Samples were prepared for GC-MS using the SCFA derivatization method 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 min. A 1 mL aliquot of the sample supernatant was then transferred to a flat-bottomed glass tube containing 50 μL of internal standard solution (0.1 M 2-ethylbutyric acid). 500 μL of concentrated hydrochloric acid (HCl) and 3 mL of 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 mixed ether extracts were then added to 50 μL of 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. The samples were placed at room temperature for 72 h to allow complete derivatization of metabolites. Metabolite analysis was performed using an Agilent / HP6890 gas chromatograph (HewlettPackard, UK) equipped with an HP-5MS 30 m × 0.25 mm column with a 0.25 μm coating (cross-linked (5%-phenyl)-methylpolysiloxane, Hewlett Packard, UK). Quantification of samples was obtained by calibration curves of lactate, acetate, propionate, butyrate from external standards with concentrations between 6.25 mM and 100 mM.

[0317] Results—Bacterial Populations

[0318] In the figures and description that follow, reference is made to the additional substrates used in the production of these compositions, Examples 4-9 are referred to as:

[0319] Example 4—FucOS

[0320] Example 5—AraOS

[0321] Example 6—XylOS

[0322] Example 7—RhaOS

[0323] Example 8—SucOS

[0324] Example 9—LactulOS

[0325] Using 500 mg / ml lactose as substrate, a sample labeled B-GOS (galactose containing oligosaccharides) was generated.

[0326] Figure 2 The differences in bacterial populations observed in Examples 4-9 are shown. Figure 2 The effects of different novel oligosaccharides on bacterial counts (log colony forming units (CFU / mL)) of Lactobacillus species (LAB, A), Bacteroidetes and Prevotellaceae (BAC, B), Clostridium coccidioides-Eubacterium straight-shaped group (EREC, C), Roseberyces subgroup (RREC, D), Asterobacterium-Atroviridae (ATO, E), Clostridium cluster IX (PROP, F), Faecalibacterium prausnitzii (FPRAU, G), Desulfovibrio (DSV, H) and Clostridium histolyticum (CHIS, I) detected by flow-FISH in pH-controlled batch culture experiments are shown. Samples were collected at 0, 8 and 24 h. Error bars are mean standard error. Significant differences (p≤0.05) are indicated with *.

[0327] 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 in bacterial populations in the donors between the two technical replicates at all three sampling time points, 0, 8 or 24 h.

[0328] Figure 2 The effects of Examples 4-9 on total bacteria (A) and bifidobacterium species (B) detected by flow-FISH cytometry in pH controlled batch cultures (log colony forming units (CFU / mL) are shown. Samples were collected at 0, 8 and 24 h. One-way ANOVA was used to test for major interactions between groups. Error bars are mean standard error. Significant differences (p≤0.05 (F-test)) are indicated with *. Negative controls had no added carbon source.

[0329] For all tested oligosaccharides and the positive control, an increase in total bacterial counts was observed after 8 h ( Figure 2 A), after 24 h of fermentation, statistically significant increases (p≤0.05) were detected in Examples 4-9. At 24 h, the largest increase in total bacteria count was observed in Example 6, Log 10 It is 8.64±0.99CFU / mL.

[0330] After 24 h of fermentation, a significant increase in the number of Bifidobacterium species was observed for Example 1 (p = 0.013), Example 4 (FucOS) (p = 0.006), Example 9 (LactulOS sugar) (p = 0.006), Example 8 (SucOS) (p = 0.042) and Example 6 (XylOS) (p = 0.040). Figure 2 B). From 0h to 24h, the largest percentage increase was 63%, which was observed in AraOS (Log 10 6.08(±1.14) to Log 10 7.12(±1.44)).

[0331] Figure 3 shows the effect of Examples 4-9 on bacterial counts (log colony forming units (CFU / mL)) detected by flow-FISH in a pH-controlled batch culture experiment on Lactobacillus species (LAB, A), Bacteroidetes and Prevotellaceae (BAC, B), Clostridium coccidioides-straight Eubacterium group (EREC, C), Roseberyces subgroup (RREC, D), Aesthetica-Apotropaeohyde (ATO, E), Clostridium cluster IX (PROP, F), Faecalibacterium prausnitzii (FPRAU, G), Desulfovibrio (DSV, H) and Clostridium histolyticum (CHIS, I). Samples were collected at 0, 8 and 24 h. Error bars are mean standard error. Significant differences (p≤0.05) are indicated by *. Similarly, no carbon source was added to the negative control.

[0332] After 24 h of fermentation, a significant difference in the number of lactobacilli treated with Example 8 (SucOS) (p = 0.049) and Example 6 (XylOS) (p = 0.036) was observed ( Figure 3A In the vessels treated with B-GOS, Example 7 (RhaOS) and Example 6 (XylOS), there was a significant increase in the C. cocci-Eubacterium rectale group, while the only significant increase in the Roseburia subpopulation was observed with Example 7 (RhaOS) (p=0.05) at 24 h fermentation ( Figure 3D Regardless of the treatment, there were no significant differences in the bacterial populations of Clostridium cluster IX (PROP), Faecalibacterium prausnitzii (FPRAU), or Desulfovibrio (DSV) ( Figure 3F , 3G and 3H).

[0333] In a container containing B-GOS, Example 9 (LactulOS), Example 7 (RhaOS) and Example 6 (XylOS), an unfamiliar fungus - Atropurpureus (ATO) ( Figure 3E ) has been significantly improved. (p = 0.042) in the fermentation vessel treated with Clostridium histolyticum (CHIS) at 24 h. Fig. 3I However, the population size of Clostridium histolyticum (log 10 ±5.67(0.14)) was significantly lower than that at the same 24h time point log 10 ±8.56(0.17) total bacteria count ( Figure 3A ), and the level was close to the detection limit.

[0334] Results—Bacterial metabolites

[0335] Figure 4 shows the concentrations of bacterial metabolites in Yangping measured by GC-MS at 0, 8 and 24 hours: acetic acid (A), propionic acid (B), butyric acid (C) and lactic acid (D), samples taken from pH-controlled batch cultures testing newly synthesized oligosaccharides and controls. Error bars are standard error of the mean.

[0336] Compared to 0 h for all examples and positive controls tested in batch culture fermentations, the concentrations of acetate, propionate, butyrate, and lactate increased after 8 h and 24 h of fermentation ( FIG. 4 ). Compared to the other metabolites measured, acetate was the most prevalent SCFA produced in all batch culture fermentations, reaching the highest concentrations ( Figure 4A The maximum increase in acetic acid was observed in Example 6 (XylOS) at 24 h, rising from 0.4 mM at 0 h to a concentration of 36.8 mM. An increase in acetic acid was also observed in the samples of Example 1, Example 5 (AraOS) and Example 4 (FucOS) after 24 h of fermentation.

[0337] The SCFA propionate and butyrate were also detected in all examples, albeit at lower concentrations, with concentrations increasing from 0 to 24 h. The largest increase in propionate was seen in Example 4 (FucOS), reaching 10.9 mM. After 24 h of fermentation, significant increases in this metabolite were observed in Example 7 (RhaOS) and Example 6 (XylOS). Fermentation of Example 9 (LactulOS), Example 4 (FucOS) and Example 9 (LactulOS) resulted in increased butyrate levels at 24 h, while increased lactate levels were identified in the vessels after 24 h of fermentation of Example 5 (AraOS) and Example 6 (XylOS) and in the vessels after 8 h of fermentation of Example 8 (SucOS).

[0338] These results indicate that the GOS compositions of Examples 4-9, comprising GOS compounds with alternative terminal monosaccharides, increased the number of Bifidobacterium. Furthermore, the bacterial numbers in the FISH data; combined with the fermented SCFA results; indicate that some of these compositions of Examples 4-9 stimulated bacteria other than the traditional probiotics, Bifidobacterium and Lactobacillus, and therefore may provide potential prebiotic activity.

[0339] In summary, the present invention provides a composition comprising an oligosaccharide compound, such as a galacto-oligosaccharide compound, which comprises: (a) at least 8 wt % of Gal-(β1-3)-Gal-(β1-4)-X a (b) at least 3 wt% of Gal-(β1-3)-Gal-(β1-3)-X b and (c) at least 5 wt% Gal-(β1-3)-Gal-(β1-2)-X c , based on the total weight of the oligosaccharide compounds present in the composition, wherein X a , X b and X c Each independently selected from monosaccharides. Compared with known oligosaccharide compositions, these compositions contain relatively high content of oligosaccharide compounds (a), (b) and (c) and relatively high content of β1-3Gal-Gal bonds. These special features of the composition are considered to be beneficial to the intestinal health of consumers, for example, because these compositions increase the production of butyric acid in the intestinal tract of consumers compared with known compositions.

[0340] While several preferred embodiments have been shown and described, it will be understood 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.

[0341] Throughout the specification, the term "comprising" or "comprises" means including the specified components, but does not exclude the presence of other components. The term "consisting essentially of" or "consists essentially of" means including the specified components, but excluding other components, except for substances present as impurities, unavoidable substances present as a result of the method for providing the components, and components added for purposes other than achieving the technical effects of the present invention. Typically, when referring to a composition, a composition consisting essentially of a group of components will contain less than 5wt%, typically less than 3wt%, and more typically less than 1wt% of non-specific components.

[0342] The term "consisting of" or "consists of" means including the specified components but excluding the addition of other components.

[0343] Where appropriate, depending on the context, use of the terms “comprises” or “comprising” may also be understood to mean comprising or including “consists essentially of” or “consisting essentially of,” and may also be understood to include “consisting of” or “consists of.”

[0344] For the avoidance of doubt, when the amount of a component in a composition is described in wt%, this means the weight percentage of the specific component relative to the entire composition. For example, "wherein the oligosaccharide compound comprises up to 35 wt% of disaccharides" means that 35 wt% of the oligosaccharide compound in the composition is provided by disaccharides.

[0345] Where appropriate, the optional features set forth herein may be used alone or in combination with one another, particularly in the combinations set forth in the appended claims. Where appropriate, the optional features of each aspect or exemplary embodiment of the invention set forth herein should also be understood to be applicable to any other aspect or exemplary embodiment of the invention. That is, a technician reading this specification should consider the optional features of each exemplary embodiment of the invention to be interchangeable and combinable between different exemplary embodiments.

[0346] Please note that all papers and documents filed concurrently with or prior to this specification and related to this application are open to public inspection together with this specification, and the contents of all these papers and documents are incorporated herein by reference.

[0347] All features disclosed in this specification (including any accompanying claims and drawings), and / or all 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.

[0348] Unless expressly stated otherwise, each feature disclosed in this specification (including any attached claims and drawings) may be replaced by alternative features for the same, equivalent or similar purpose. Therefore, unless expressly stated otherwise, each feature disclosed is only an example of a series of equivalent or similar features.

[0349] The invention is not limited to the details of the foregoing embodiments. The invention extends to any novel one or any novel combination of the features disclosed in this specification (including any accompanying claims and drawings), or to any novel one or any novel combination of the steps of any method or process disclosed in this specification (including any accompanying claims and drawings).

Claims

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

2. The composition according to claim 1, wherein Based on the total weight of the oligosaccharide compounds present in the composition, the oligosaccharide compounds comprise: (d) at least 3 wt% of Gal-(β1-3)-Gal-(β1-3)-Gal-(β1-4)-X d ; Where X d Selected from monosaccharides.

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

4. A composition according to any one of the preceding claims, wherein The ratio of wt% of compound (a) to compound (b) is 1:1 to 3:

1.

5. A composition according to any one of the preceding claims, wherein The ratio of wt% of compound (a) to compound (c) is 1:1 to 3:

1.

6. A composition according to any one of the preceding claims, wherein The ratio of wt% of compound (b) to compound (c) is 2:1 to 1:

2.

7. A composition according to any one of the preceding claims, wherein Based on the total weight of the oligosaccharide compounds present in the composition, the oligosaccharide compounds comprise: (e) at least 5 wt% of Gal-(β1-4)-Gal-(β1-4)-X e ; Where X e Selected from monosaccharides.

8. A composition according to any one of the preceding claims, wherein X a , X b , X c , X d and X e Each is independently selected from glucose, fucose, arabinose, xylose, rhamnose, mannose, galactose, ribose, lyxose, allose, altrose, gulose, idose, talose, psicose, fructose, sorbose, tagatose, galactosamine, glucosamine and N-acetylglucosamine or a mixture thereof.

9. The composition according to any one of claims 1 to 7, wherein X a , X b , X c , X d and X e Each is Glc.

10. The composition according to any one of claims 1 to 7, wherein X a , X b , X c , X d and X e Each contains Fuc.

11. A composition according to any one of the preceding claims, wherein 0% to 55% of the Gal-Gal bonds in the oligosaccharide compounds are 1-3 bonds.

12. A composition according to any one of the preceding claims, comprising at least 50 wt% of the oligosaccharide compound, preferably wherein the composition is in the form of a syrup.

13. A composition according to any one of the preceding claims, comprising at least 75 wt% of the oligosaccharide compound, preferably wherein the composition is in the form of a powder.

14. A composition according to any one of the preceding claims, wherein The oligosaccharide compound comprises at least 25 wt% of trisaccharides.

15. A composition according to any one of the preceding claims, wherein The oligosaccharide compound comprises at least 10 wt % of tetrasaccharides.

16. A composition according to any preceding claim comprising 80 wt% trisaccharides, tetrasaccharides and higher oligosaccharides.

17. A composition according to any preceding claim comprising 80 wt% trisaccharide.

18. A pharmaceutical or nutraceutical composition comprising a composition according to any one of the preceding claims and at least one carrier, excipient or diluent.

19. Use of a composition according to any one of claims 1 to 17 as a dietary supplement.

20. The use according to claim 19, wherein: The use increases butyrate production in the intestine of a consumer of the dietary supplement.

21. Use of a composition according to any one of claims 1 to 17 as a medicament.

22. A method for preparing a composition comprising an oligosaccharide compound according to any one of claims 1 to 17, the method comprising the following steps: (i) providing a source of sugar compounds; (ii) treating the sugar compound source with one or more β-galactosidases to at least partially convert the sugar compound source into oligosaccharide compounds.

23. The method according to claim 22, wherein: The sugar compound source comprises lactose, lactulose or epi-lactose.

24. The method according to claim 22 or 23, wherein: The sugar compound source comprises additional sugars selected from the group consisting of glucose, fucose, arabinose, xylose, rhamnose, mannose, galactose, ribose, lyxose, allose, altrose, gulose, idose, talose, psicose, fructose, sorbose, tagatose, galactosamine, glucosamine and N-acetylglucosamine or a combination thereof.

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