Oligosaccharide compositions for use in reducing ammonia levels - Patents.com

Abstract

A prebiotic oligosaccharide composition for use in reducing ammonia levels, the composition comprising oligosaccharide compounds, e.g., galactooligosaccharide compounds, that comprise, based on the total weight of the oligosaccharide compounds present in the composition: (a) at least 8 wt% Gal-(β1-3)-Gal-(β1-4)-X a (b) at least 3 wt% Gal-(β1-3)-Gal-(β1-3)-X b and (c) at least 5 wt% Gal-(β1-3)-Gal-(β1-2)-X c ;where X a , X b , and X c 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. The present invention also relates to compositions comprising oligosaccharide compounds for administration to athletes and / or sportspeople undergoing and / or about to undergo physical training, and particularly athletes and / or sportspeople on a high-protein diet. Methods for preparing the compositions and the use of the compositions as dietary supplements or pharmaceuticals are also disclosed.

Description

[Technical Field]

[0001] The present invention relates to a galactooligosaccharide (GOS) composition for use in reducing ammonia levels in individuals in need thereof. The present invention also relates to a method for preparing the composition, and to formulating the composition as a dietary supplement and / or a pharmaceutical. In particular, the present invention relates to an oligosaccharide composition containing relatively high amounts of specific beneficial oligosaccharides. [Background technology]

[0002] Prebiotics are defined by the International Society for Probiotics and Prebiotics Science as "substrates selectively utilized by host microorganisms to confer health benefits." Prebiotics are indigestible dietary fibers that are difficult to digest and absorb until they reach the large intestine, where they are fermented by members of the gut microbiota. Prebiotics can alter the composition and function of the gut microbiota. Diets rich in dietary fiber / prebiotics have been shown to increase bacterial abundance and the richness of gut microbial genes, as well as the abundance of beneficial bacteria such as Bifidobacterium and Lactobacillus. Prebiotics also play a multifaceted role in suppressing enteric pathogens. For example, members of the genera Bifidobacterium and Lactobacillus produce lactic acid during prebiotic fermentation. Beneficial gut microbes utilize dietary fiber as an energy source. As a by-product of this fermentation, gut microbes produce lactic acid and short-chain fatty acids (SCFAs) such as acetate, butyrate, and propionate. SCFAs are known to have several beneficial effects on human health, such as maintaining the colonic epithelium; potential roles in regulating glucose homeostasis, lipid metabolism, and appetite regulation; and roles in regulating the immune system and inflammatory responses.

[0003] Galactooligosaccharides (GOS) are an important and well-studied class of prebiotics. GOS have been shown to strongly stimulate the growth of bifidobacteria in the intestine, and to a lesser extent, lactic acid bacteria and bacteroidetes. Bifidobacteria are thought to be highly beneficial to the human host, primarily due to their SCFA-producing capacity. In addition, bifidobacteria are strongly associated with improved intestinal epithelial barrier and intestinal permeability, and play a beneficial role in host immune modulation.

[0004] Known GOS prebiotic products contain a mixture of many oligosaccharide compounds in various proportions, not all of which have the desired beneficial effect on the consumer's gut microbiota. Although such products have shown some beneficial results in improving patient health, there remains a need for further improvements in oligosaccharide compositions to fully realize the potential benefits of such prebiotics to consumers and to provide long-lasting and inexpensive treatment for many conditions and / or diseases. Summary of the Invention

[0005] Among other things, one object of the present invention is to provide a composition comprising an oligosaccharide compound that overcomes at least one of the shortcomings of the prior art, whether identified herein or elsewhere, or to provide an alternative to an existing composition. 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 a particular beneficial oligosaccharide than known oligosaccharide compositions.

[0006] According to an aspect of the present invention, there is provided a method and a composition as defined in the appended claims. Uses are provided. Other features of the invention are apparent from the dependent claims and the following description.

[0007] According to a first aspect of the present invention, there is provided a composition for reducing the level of ammonia, wherein the oligosaccharide compound comprises, based on the total weight of the oligosaccharide compounds present in the composition: (a) at least 8 wt% Gal-(β1-3)-Gal-(β1-4)-X a ; (b) at least 3 wt% Gal-(β1-3)-Gal-(β1-3)-X b ; and (c) at least 5 wt% Gal-(β1-3)-Gal-(β1-2)-X c Including, where X a , X b , and X c are each independently selected from monosaccharides.

[0008] In an embodiment related to the first aspect of the present invention, there is also provided a method of preventing, ameliorating, or treating conditions and / or diseases associated with elevated levels of ammonia, the method comprising administering to a subject in need of such prevention, amelioration, or treatment a composition comprising an oligosaccharide compound, wherein the oligosaccharide compound comprises: (a) at least 8 wt% Gal-(β1-3)-Gal-(β1-4)-X a ; (b) at least 3 wt% Gal-(β1-3)-Gal-(β1-3)-X b ; and (c) at least 5 wt% Gal-(β1-3)-Gal-(β1-2)-X c Including, where X a , X b , and X c are each independently selected from monosaccharides.

[0009] In a further embodiment related to the first aspect of the present invention, there is further provided a method of preventing, ameliorating or treating a condition and / or disease associated with elevated levels of ammonia in a subject in need thereof, said method comprising: i) determining whether the subject has elevated levels of ammonia and / or whether the subject is suffering from a condition and / or disease associated with elevated levels of ammonia; and ii) administering to the subject a composition comprising a therapeutically effective amount of oligosaccharide compounds, wherein the oligosaccharide compounds are present in the composition in an amount based on the total weight of the oligosaccharide compounds present in the composition. (a) at least 8 wt% Gal-(β1-3)-Gal-(β1-4)-X a ; (b) at least 3 wt% Gal-(β1-3)-Gal-(β1-3)-X b ; and (c) at least 5 wt% Gal-(β1-3)-Gal-(β1-2)-X c Including, where X a , X b , and X c are each independently selected from monosaccharides.

[0010] In yet another embodiment of the present invention, there is provided a use of a composition for the manufacture of a medicament for the prevention, amelioration, or treatment of conditions and / or diseases associated with elevated levels of ammonia, wherein said composition comprises an oligosaccharide compound, the oligosaccharide compound comprising: (a) at least 8 wt% Gal-(β1-3)-Gal-(β1-4)-Xa; (b) at least 3 wt% Gal-(β1-3)-Gal-(β1-3)-Xb; and (c) containing at least 5 wt% Gal-(β1-3)-Gal-(β1-2)-Xc; wherein Xa, Xb, and Xc are each independently selected from monosaccharides.

[0011] In a further embodiment of the present invention there is provided a composition comprising an oligosaccharide compound for use in the prevention and / or treatment of fatigue in an athlete or sportsperson, wherein said oligosaccharide compound comprises, based on the total weight of oligosaccharide compounds present in said composition: (a) at least 8 wt% Gal-(β1-3)-Gal-(β1-4)-X a ; (b) at least 3 wt% Gal-(β1-3)-Gal-(β1-3)-X b ; and (c) at least 5 wt% Gal-(β1-3)-Gal-(β1-2)-X c Including, where X a , X b , and X c are each independently selected from monosaccharides.

[0012] Many studies have emphasized the detrimental effects of elevated ammonia levels. Wilkinson, DJ et al. (2010) (Ammonia metabolism, the brain, and fatigue: revisiting the link, Progress in Neurobiology, 91, (3), 200-219) discusses how hyperammonemia occurs during exercise as a result of increased production by muscle contraction through the deamination of adenosine monophosphate (AMP) (purine nucleotide cycle) and the deamination of branched-chain amino acids (BCAAs) prior to their oxidation. Plasma ammonia concentrations during exercise often reach or exceed those measured in patients with liver disease, leading to increased brain uptake that may result in impaired brain function and may affect performance as fatigue or impaired function, particularly during extreme exercise, possibly through mechanisms similar to those underlying pathology. Atbone, M. et al., (2021) (The effect of acute moderate-intensity exercise on the serum and fecal metabolomes and the gut microbiota of cross-country endurance athletes, Nature Scientific Reports, 11, 3558) describes how exercise significantly increases fecal ammonia content, especially in individuals undergoing extreme exercise. Therefore, the compositions of the present invention are suitable for use in the prevention and / or treatment of fatigue in athletes or sportspeople, particularly when they target the intestine and reduce the amount of available ammonia that can then be absorbed into the blood.

[0013] In a further embodiment, there is also provided a composition for use in regulating the level of ammonia in an individual, wherein said composition comprises an oligosaccharide compound, and said oligosaccharide compound comprises, based on the total weight of oligosaccharide compounds present in said composition: (a) at least 8 wt% Gal-(β1-3)-Gal-(β1-4)-Xa ; (b) at least 3 wt% Gal-(β1-3)-Gal-(β1-3)-X b ; and (c) at least 5 wt% Gal-(β1-3)-Gal-(β1-2)-X c Including, where X a , X b , and X c are each independently selected from monosaccharides.

[0014] As used herein, the terms "treatment," "treating," "treat," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents the disease or condition, and / or the effect may be therapeutic, in that it partially or completely cures the disease and / or the adverse effects caused by the disease. As used herein, "treatment" covers any treatment of a disease in a mammal, particularly a human, and includes (a) treatment of a patient who may be predisposed to the disease but has not yet been diagnosed with the disease. (b) preventing the occurrence of said disease in a subject; (b) inhibiting said disease, i.e., arresting or slowing the progression of said disease; and (c) alleviating said disease and / or symptoms of said disease, i.e., causing regression of said disease.

[0015] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all mammals, such as non-human primates, sheep, dogs, cats, cows, horses, etc.

[0016] "Therapeutically effective amount" refers to the amount of a compound that, when administered to a subject for treating a disease, is sufficient to effectively treat the disease. The "therapeutically effective amount" varies depending on the compound used, the disease and its severity, and the age, weight, etc. of the subject to be treated.

[0017] The term "conditions and / or diseases associated with elevated levels of ammonia" refers to various conditions and / or diseases resulting from elevated levels of ammonia in the body, with identifiable symptoms. The elevated levels may occur in the blood, intestines, gut and / or intestinal lining.

[0018] Preferably, the condition and / or disease associated with elevated levels of ammonia is hyperammonemia. The condition and / or disease associated with elevated levels of ammonia may be (or may be) associated with any one of the following conditions and / or diseases: hepatic encephalopathy, Reye's syndrome, urea cycle disorders, renal failure, encephalopathy, and liver failure.

[0019] Conditions and / or diseases associated with elevated ammonia levels may occur in individuals who are athletes or sportspeople undergoing (or about to undergo) physical exercise or training. The composition may be administered before, during, or after exercise or physical activity. Alternatively, the composition may be administered according to a continuous dosing schedule, such as once or twice daily. The therapeutically effective amount may be a standardized dose, or a dose determined by individual factors such as the individual's age, body mass index (BMI), or total body weight.

[0020] The term "performance" refers to an individual's ability to perform specific physical tasks associated with sports and exercise, including, but not limited to, reducing fatigue and improving endurance.

[0021] The term "athlete" or "sportsperson" refers to an individual who actively engages in a sport or athletic activity, and includes professional or amateur individuals.

[0022] The inventors have advantageously and unexpectedly found that supplementation with the composition of the present invention reduces ammonia levels in tests. The composition acts systemically, and studies have shown that the composition of the present invention can regulate ammonia levels and result in many improvements, such as a reduction in toxic by-products (such as p-cresol) caused by proteolytic microbial activity in the intestine. This is particularly relevant for people on high-protein diets or feeding plans, because they have higher proteolytic microbial activity due to higher protein levels in the intestinal environment. Such people may be on high-protein diets or feeding plans for athletic purposes (e.g., to increase muscle mass and / or combat muscle wasting) or medical purposes (e.g., to aid in the prevention or treatment of cachexia or sarcopenia). In certain embodiments, the composition is used to treat one or more of malnutrition, sarcopenia, cachexia, and frailty. The compounds may be for use in the prevention, amelioration, or treatment of a number of conditions.

[0023] These types of oligosaccharides, which have a specific bond between galactose unit (represented as Gal) and terminal monosaccharide, are considered to be particularly beneficial for maintaining healthy human intestinal microflora, and therefore are considered to be beneficial to the health of subjects.Known oligosaccharide compositions either do not contain these types of oligosaccharides, or have a low content of these oligosaccharides.Therefore, when used as prebiotics, this first aspect of the composition provides consumers with the advantage of establishing and maintaining human intestinal microflora.

[0024] The weight percentage of a particular oligosaccharide compound discussed herein is based on the weight of all oligosaccharide compounds present in the entire composition. Therefore, only the fraction of the composition provided by oligosaccharides, either components (a), (b), (c), or any other present oligosaccharides, is considered in determining the specific weight percentage. This determination of oligosaccharide content includes disaccharides, apart from lactose. The composition of this first embodiment may contain monosaccharides and other non-oligosaccharide components, including lactose. These components are not considered in determining the specific weight percentage of the oligosaccharide compounds discussed herein. The specific amount of oligosaccharide compounds in the composition may be referred to as the weight percentage of the oligosaccharide fraction of the composition of this first embodiment.

[0025] Oligosaccharide compounds (a), (b), and (c) are each X a , X b , and X c and each of the X's is independently selected from a sugar. a , X b and X c The X group can be considered to be a sugar unit. a , X b , and X c The X group can be considered as the terminal sugar of the oligosaccharide compound. a , X b , and X c The groups are preferably independently selected from monosaccharides. Any suitable monosaccharide unit capable of forming an oligosaccharide with said galactose unit of compounds (a), (b), and (c) may be selected from X a , X b , and X c Preferably, X a , X b , and X care each independently selected from the following monosaccharides. Suitable monosaccharide units are 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) can comprise a mixture of oligosaccharide compounds, each having a different X group, e.g., either a Glc or Fuc X group.

[0026] In some embodiments, X a , X b , and X c The groups are independently selected from the monosaccharides listed above, preferably independently selected from Glc, Fuc, Ara, Xyl, Rha, and Man, or mixtures thereof.

[0027] 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. Thus, components (a), (b) and (c) are (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 It could be.

[0028] Preferably, Gal and X a , X b , and X cAll of the sugar units have the D enantiomeric form. Thus, components (a), (b), and (c) are (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 It could be.

[0029] In some embodiments, X a , X b , and X c is Glc. Thus, compounds (a), (b), and (c) may be galactooligosaccharide compounds (GOS), and the composition of this first aspect may be referred to as a galactooligosaccharide composition. Such galactooligosaccharides may be formed by converting lactose to said oligosaccharides with a suitable galactosidase enzyme. In such an embodiment, said components (a), (b), and (c) are preferably (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.

[0030] Preferably, the Gal and the Glc sugar units are all in the D enantiomeric form. Thus, components (a), (b), and (c) are (a)D-Gal-(β1-3)-D-Gal-(β1-4)-D-Glc; (b) D-Gal-(β1-3)-D-Gal-(β1-3)-D-Glc; and (c) It can be D-Gal-(β1-3)-D-Gal-(β1-2)-D-Glc.

[0031] In some embodiments, X a , X b , and Xc Each of (a), (b), and (c) is a mixture of Glc and one or more of the 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 having Glc as the X group and one of the other sugar units described above. In such embodiments, components (a), (b), and (c) may be formed by converting a mixture of lactose and a suitable additional sugar, such as a monosaccharide selected from fucose, arabinose, xylose, rhamnose, and mannose, into the oligosaccharide. Preferably, X a , X b , and X c Each of these is a mixture of Glc and Fuc.

[0032] The other oligosaccharides in the composition comprising the oligosaccharide compound of this first aspect may also comprise the sugar unit "X" groups described above, in addition to components (a), (b), and (c) discussed above.

[0033] The composition of this first embodiment comprises at least 8 wt % of (a) Gal-(β1-3)-Gal-(β1-4)-X a Suitably, the composition comprises at least 9 wt% of component (a), or at least 10 wt% of component (a).

[0034] Suitably, the composition comprises at most 35 wt% of component (a), at most 30 wt% of component (a), or at most 25 wt% of component (a).

[0035] Suitably, the composition comprises 8 wt% to 35 wt% of component (a), 8 wt% to 25 wt% of component (a), or 10 wt% to 20 wt% of component (a).

[0036] The composition of this first aspect contains at least 3 wt % of (b) 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).

[0037] Suitably, the composition comprises up to 25 wt% of component (b), up to 20 wt% of component (b), or up to 15 wt% of component (b).

[0038] Suitably, the composition comprises 3 wt% to 25 wt% of component (b), 4 wt% to 20 wt% of component (b), or 4 wt% to 10 wt% of component (b).

[0039] The composition of this first embodiment contains at least 5 wt% of (c)Gal-(β1-3)-Gal-(β1-2)-X c Preferably, the composition comprises at least 6 wt% of component (c).

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

[0041] Suitably, the composition comprises 5 wt% to 25 wt% of component (c), 5 wt% to 20 wt% of component (c), or 6 wt% to 10 wt% of component (c).

[0042] The above amounts are based on the total weight of the oligosaccharide compounds present in the composition.

[0043] Suitably, in the composition of this first aspect, based on the total weight of the oligosaccharide compounds present in said 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%.

[0044] Suitably, in the composition of this first aspect, based on the total weight of the oligosaccharide compounds present in said composition: component (a) is present in an amount of 8 wt% to 25 wt%; component (b) is present in an amount of 3 wt% to 25 wt%; and Component (c) is present in an amount of 5 wt% to 20 wt%.

[0045] In some embodiments, the composition of this first aspect comprises, based on the total weight of oligosaccharide compounds present in the composition: (a) 8 wt% to 25 wt% Gal-(β1-3)-Gal-(β1-4)-Glc; (b) 3 wt% to 25 wt% Gal-(β1-3)-Gal-(β1-3)-Glc; and (c) containing 5 wt% to 20 wt% Gal-(β1-3)-Gal-(β1-2)-Glc.

[0046] Preferably, the ratio of compound (a) to compound (b) by weight percent is from 1:1 to 3:1, preferably from 1.5:1 to 2.5:1.

[0047] Preferably, the ratio of wt% of compound (a) to compound (c) is from 1:1 to 3:1, preferably from 1.5:1 to 2.5:1.

[0048] Preferably, the ratio of compound (b) to compound (c) by weight is from 2:1 to 1:2, preferably from 1.5:1 to 1:1.5.

[0049] Suitably, in the composition of this first aspect, the oligosaccharide compounds comprise, based on the total weight of oligosaccharide compounds present in the composition: (d) at least 3 wt% Gal-(β1-3)-Gal-(β1-3)-Gal-(β1-4)-X d Including, where X d is selected from sugars.

[0050] X d is X a , X b , and X c The monosaccharide units may be selected from the same monosaccharide units described above for

[0051] In some embodiments, X d is Glc.

[0052] Preferably, the Gal sugar unit of component (d) has the D enantiomeric form. Preferably, the Gal and the X d All sugar units have the D enantiomeric form. Thus, component (d) can be: D-Gal-(β1-3)-D-Gal-(β1-3)-D-Gal-(β1-4)-DX d .

[0053] In some embodiments, X d is a mixture of Glc with one or more other monosaccharide units such as Fuc, Ara, Xyl, Rha, and Man, preferably Fuc.

[0054] Suitably, the composition comprises at least 4 wt% of component (d), or at least 5 wt% of component (d).

[0055] Suitably, the composition comprises up to 25 wt% of component (d), up to 20 wt% of component (d), or up to 15 wt% of component (d).

[0056] 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).

[0057] Suitably, in the composition of this first aspect, based on the total weight of the oligosaccharide compounds present in said composition: component (a) is present in an amount of 8 wt% to 25 wt%; component (b) is present in an amount of 3 wt% to 25 wt%; Component (c) is present in an amount of 5 wt% to 20 wt%; and Component (d) is present in an amount of 3 wt% to 25 wt%.

[0058] In some embodiments, the composition of this first aspect comprises, based on the total weight of oligosaccharide compounds present in the composition: (a) 8 wt% to 25 wt% Gal-(β1-3)-Gal-(β1-4)-Glc; (b) 3 wt% to 25 wt% Gal-(β1-3)-Gal-(β1-3)-Glc; (c) 5 wt% to 20 wt% Gal-(β1-3)-Gal-(β1-2)-Glc, and (d) containing 3 wt% to 25 wt% Gal-(β1-3)-Gal-(β1-3)-Gal-(β1-4)-Glc.

[0059] Suitably, in the composition of this first aspect, the oligosaccharide compounds comprise, based on the total weight of oligosaccharide compounds present in the composition: (e) at least 5 wt% Gal-(β1-4)-Gal-(β1-4)-X e Including, where X e is selected from monosaccharides.

[0060] X e is X a , X b , and X c The monosaccharide units are selected from the same monosaccharide units as those described above for obtain.

[0061] Preferably, the Gal sugar unit of component (e) has the D enantiomeric form. Preferably, the Gal and the X e All sugar units have the D enantiomeric form. Thus, component (e) can be: D-Gal-(β1-4)-D-Gal-(β1-4)-DX e .

[0062] In some embodiments, X e is Glc.

[0063] In some embodiments, X e is a mixture of Glc with one or more other monosaccharide units such as Fuc, Ara, Xyl, Rha, and Man, preferably Fuc.

[0064] Suitably, the composition comprises at least 6 wt% of component (e), or at least 7 wt% of component (e).

[0065] Suitably, the composition comprises at most 25 wt% of component (e), at most 20 wt% of component (e), or at most 15 wt% of component (e).

[0066] Suitably, the composition comprises 5 wt% to 25 wt% of component (e), 5 wt% to 20 wt% of component (e), or 6 wt% to 10 wt% of component (e).

[0067] Suitably, in the composition of this first aspect, based on the total weight of the oligosaccharide compounds present in said composition: component (a) is present in an amount of 8 wt% to 25 wt%; component (b) is present in an amount of 3 wt% to 25 wt%; component (c) is present in an amount of 5 wt% to 20 wt%; Component (d) is present in an amount of 3 wt% to 25 wt%; and Component (e) is present in an amount of 5 wt% to 25 wt%.

[0068] In some embodiments, the composition of this first aspect comprises, based on the total weight of oligosaccharide compounds present in the composition: (a) 8 wt% to 25 wt% Gal-(β1-3)-Gal-(β1-4)-Glc; (b) 3 wt% to 25 wt% Gal-(β1-3)-Gal-(β1-3)-Glc; (c) 5 wt% to 20 wt% Gal-(β1-3)-Gal-(β1-2)-Glc, and (d) 3 wt% to 25 wt% Gal-(β1-3)-Gal-(β1-3)-Gal-(β1-4)-Glc, and (e) Contains 5 wt% to 25 wt% Gal-(β1-4)-Gal-(β1-4)-Glc.

[0069] In the composition of this first aspect, X a , Xb , X c , X d , and X e are each independently selected from the monosaccharides described above.

[0070] 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.

[0071] In some embodiments, X a , X b , X c , X d , and X e are Glc, respectively.

[0072] 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.

[0073] The oligosaccharide compounds of the composition of this first aspect preferably have a relatively high proportion of β1-3 Gal-Gal linkages—due primarily to the presence of components (a), (b), (c), and optionally (d) in the proportions discussed herein—and 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.

[0074] 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%.

[0075] Preferably, 40% to 60% of the Gal-Gal bonds in the oligosaccharide compound are 1-4 bonds, preferably 45% to 55%, preferably 45% to 52%.

[0076] Preferably, 8% to 20% of the Gal-X linkages in the oligosaccharide compound are 1-3 linkages, preferably 10% to 18%, preferably 12% to 16%, and preferably X is Glc.

[0077] Preferably, 15% to 25% of the Gal-X linkages in the oligosaccharide compound are 1-4 linkages, preferably 16% to 24%, preferably 17% to 22%, and preferably X is Glc.

[0078] Preferably, 30% to 45% of the Gal-X linkages in the oligosaccharide compound are 1-2 linkages, preferably 32% to 43%, preferably 34% to 41%, and preferably X is Glc.

[0079] Preferably, 20% to 36% of the Gal-X linkages in the oligosaccharide compound are 1-6 linkages, preferably 22% to 34%, preferably 25% to 32%, and preferably X is Glc.

[0080] Preferably, the Gal-X bond referred to above, such as the Gal-Glu bond, is a β bond, i.e. a β-glycosidic bond.

[0081] Preferably, the composition comprises at least 25 wt% trisaccharides, based on the total weight of the composition, preferably at least 28 wt% trisaccharides, or at least 30 wt% trisaccharides.

[0082] Preferably, the composition comprises at most 70 wt% trisaccharides, preferably at most 60 wt% trisaccharides, or at most 50 wt% trisaccharides.

[0083] Preferably, the composition comprises 25wt% to 70wt% trisaccharides, preferably 30wt% to 60wt% trisaccharides, or 30wt% to 50wt% trisaccharides.

[0084] Preferably, the composition comprises at least 10 wt% tetrasaccharides, based on the total weight of the composition. Preferably, the composition comprises at least 12 wt% tetrasaccharides, or at least Preferably, the composition comprises at least 20 wt% tetrasaccharides, or at least 25 wt% tetrasaccharides.

[0085] Preferably, the composition comprises at most 40 wt% tetrasaccharides, preferably at most 25 wt% tetrasaccharides, or at most 20 wt% tetrasaccharides.

[0086] Preferably, the composition comprises 10wt% to 40wt% tetrasaccharide, preferably 10wt% to 30wt% tetrasaccharide, or 12wt% to 25wt% tetrasaccharide.

[0087] Preferably, the composition comprises 30 wt% to 50 wt% trisaccharides and 10 wt% to 25 wt% tetrasaccharides, based on the total weight of the composition.

[0088] As mentioned above, the definition of disaccharides present in the composition excludes lactose. Thus, in some embodiments, the composition preferably contains up to 40 wt% disaccharides, preferably up to 30 wt% disaccharides, or up to 20 wt% disaccharides. Preferably, the lactose content in the composition of this first aspect is minimized. Preferably, the composition is substantially lactose-free. Preferably, the composition does not contain lactose.

[0089] Preferably, the composition comprises from 10wt% to 40wt% disaccharides, preferably from 10wt% to 30wt% disaccharides, or from 10wt% to 20wt% disaccharides.

[0090] Preferably, the composition comprises 10 wt% to 40 wt% disaccharides, 30 wt% to 60 wt% trisaccharides, and 10 wt% to 25 wt% tetrasaccharides, based on the total weight of the composition.

[0091] The following description relates to the content of disaccharides in the oligosaccharide compounds: Preferably, the oligosaccharide compounds contain at most 40 wt% disaccharides, preferably at most 30 wt% disaccharides, or at most 20 wt% disaccharides.

[0092] Preferably, said oligosaccharide compound comprises 0 wt% to 40 wt% disaccharides, preferably 10 wt% to 30 wt% disaccharides, or 10 wt% to 20 wt% disaccharides.

[0093] Preferably, the oligosaccharide compounds comprise at least 25 wt% trisaccharides, based on the total weight of the oligosaccharide compounds present in the composition, and preferably at least 30 wt% trisaccharides, or at least 33 wt% trisaccharides.

[0094] Preferably, said oligosaccharide compound comprises at most 75 wt% trisaccharides, preferably at most 65 wt% trisaccharides, or at most 55 wt% trisaccharides.

[0095] Preferably, the oligosaccharide compound comprises 25wt% to 75wt% trisaccharides, preferably 30wt% to 65wt% trisaccharides, or 34wt% to 55wt% trisaccharides.

[0096] Preferably, the oligosaccharide compound comprises at least 10 wt% tetrasaccharides, based on the total weight of the oligosaccharide compounds present in the composition, preferably at least 12 wt% tetrasaccharides, or at least 15 wt% tetrasaccharides.

[0097] Preferably, said oligosaccharide compound comprises at most 45 wt% tetrasaccharides, preferably at most 35 wt% tetrasaccharides, or at most 30 wt% tetrasaccharides.

[0098] Preferably, the oligosaccharide compound contains 10 wt% to 45 wt% of tetrasaccharides, preferably 10 wt% to 45 wt% of tetrasaccharides. It contains between 15wt% and 30wt% of tetrasaccharides.

[0099] Preferably, the oligosaccharide compounds comprise from 0 wt% to 40 wt% disaccharides and from 30 wt% to 75 wt% trisaccharides, based on the total weight of the oligosaccharide compounds present in the composition.

[0100] Preferably, the oligosaccharide compounds comprise 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.

[0101] The composition of this first aspect preferably comprises at least 50 wt% of an oligosaccharide compound comprising components (a), (b), (c), and optionally (d) and (e). Preferably, the composition comprises at least 55 wt%, preferably at least 60 wt%, of the oligosaccharide compound, based on the total weight of the composition.

[0102] Preferably, the composition comprises up to 100 wt% of oligosaccharide compounds, preferably up to 95 wt%, up to 90 wt%, or up to 85 wt% of oligosaccharide compounds.

[0103] Preferably, the composition comprises between 50wt% and 100wt% of oligosaccharide compounds, preferably between 55wt% and 95wt%, or between 60wt% and 85wt% of oligosaccharide compounds.

[0104] In some embodiments, the composition is in the form of a syrup. The syrup preferably contains at least 50 wt% of oligosaccharide compounds, at least 55 wt% of oligosaccharide compounds, or at least 60 wt% of oligosaccharide compounds. Preferably, the syrup contains 50 wt% to 75 wt% of oligosaccharide compounds, preferably 55 wt% to 70 wt%, or 60 wt% to 70 wt% of oligosaccharide compounds.

[0105] The syrup may contain significant amounts of monosaccharides, such as glucose and / or galactose, for example, 15 to 30 wt %, preferably 20 to 28 wt %, or 21 to 25 wt % of monosaccharides, such as glucose and / or galactose.

[0106] The syrup preferably contains 20wt% to 30wt% water, preferably 22wt% to 28wt% water.

[0107] The syrup may also contain lactose, for example from 4% to 14% by weight.

[0108] In some embodiments, the composition is in the form of a powder. The powder preferably comprises at least 60 wt% of the oligosaccharide compounds, preferably at least 70 wt%, or at least 75 wt% of the oligosaccharide compounds. Preferably, the powder comprises 60 wt% to 100 wt% of the oligosaccharide compounds, preferably 70 wt% to 95 wt%, or 75 wt% to 90 wt% of the oligosaccharide compounds.

[0109] The powder preferably contains a reduced amount of monosaccharides, such as glucose and / or galactose, compared to the syrups discussed above. The powder may contain 1 wt% to 10 wt%, preferably 2 wt% to 8 wt%, or 3 wt% to 7 wt%, preferably about 5 wt% of monosaccharides, such as glucose and / or galactose.

[0110] The powder preferably contains 1 wt% to 10 wt% water, preferably 3 wt% to 6 wt% Contains water.

[0111] The composition of this first aspect may be purified to remove monosaccharides and optionally disaccharides from the composition.

[0112] The composition of this first aspect may be fractionated according to their molecular weight to separate the oligosaccharide components of the composition, for example to remove disaccharides from the composition or to isolate trisaccharides from the other oligosaccharide components. This may be carried out 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 GOS fraction.

[0113] In such embodiments, the composition (or oligosaccharide fraction) preferably comprises at least 70 wt% trisaccharides, tetrasaccharides, and higher oligosaccharides, preferably at least 80 wt% or at least 90 wt%. Such higher oligosaccharides have a degree of polymerization of 5 or greater. Preferably, the composition comprises at least 95 wt% trisaccharides, tetrasaccharides, and higher oligosaccharides. Preferably, the composition consists of, or consists essentially of, trisaccharides, tetrasaccharides, and higher oligosaccharides.

[0114] In such embodiments, the composition preferably comprises 40wt% to 70wt% trisaccharides, preferably 45wt% to 70wt% trisaccharides, or 50wt% to 70wt% trisaccharides.

[0115] Preferably, said oligosaccharide compound comprises 15wt% to 50wt% tetrasaccharides, preferably 15wt% to 40wt% tetrasaccharides, or 20wt% to 40wt% tetrasaccharides.

[0116] Preferably, the oligosaccharide compound comprises 5wt% to 25wt% higher oligosaccharides, preferably 5wt% to 20wt% higher oligosaccharides, or 10wt% to 20wt% higher oligosaccharides.

[0117] Preferably, the composition contains 40 to 70 wt% trisaccharides, 15 to 40 wt% tetrasaccharides, and 5 to 25 wt% higher oligosaccharides, based on the total weight of the composition. Preferably, the composition contains 50 to 70 wt% trisaccharides, 20 to 40 wt% tetrasaccharides, and 10 to 20 wt% higher oligosaccharides, based on the total weight of the composition.

[0118] In such embodiments, the composition (or oligosaccharide fraction) suitably comprises the components (a), (b), and (c) above in the following amounts, based on the total weight of oligosaccharide compounds present in the composition: (a) at least 8 wt% Gal-(β1-3)-Gal-(β1-4)-X a ; (b) at least 3 wt% Gal-(β1-3)-Gal-(β1-3)-X b ; and (c) at least 5 wt% Gal-(β1-3)-Gal-(β1-2)-X c Including, where X a , X b , and X c are each independently selected from monosaccharides.

[0119] Preferably, the composition (or oligosaccharide fraction) comprises the components (a), (b) and (c) described above in the following amounts: (a) at least 10 wt% Gal-(β1-3)-Gal-(β1-4)-X a ; (b) at least 5 wt% Gal-(β1-3)-Gal-(β1-3)-X b ; and (c) at least 7 wt% Gal-(β1-3)-Gal-(β1-2)-X c .

[0120] The composition may include component (d) and / or component (e) described above.

[0121] The composition preferably contains the ingredients in the proportions discussed above.

[0122] In some embodiments, the composition of this first aspect is a trisaccharide and tetrasaccharide fraction (which may be referred to as the DP3 / DP4 fraction).

[0123] In such embodiments, the composition (or DP3 / DP4 fraction) preferably comprises at least 70 wt% trisaccharides and tetrasaccharides, preferably at least 80 wt%, or at least 90 wt%. Preferably, the composition consists of or consists essentially of trisaccharides and tetrasaccharides.

[0124] In such embodiments, the composition preferably comprises 50wt% to 80wt% trisaccharides, preferably 55wt% to 75wt% trisaccharides, or 60wt% to 75wt% trisaccharides.

[0125] Preferably, said oligosaccharide compound comprises 20wt% to 50wt% tetrasaccharides, preferably 25wt% to 45wt% tetrasaccharides, or 25wt% to 40wt% tetrasaccharides.

[0126] Preferably, the composition comprises 50 to 80 wt% trisaccharides and 20 to 50 wt% tetrasaccharides, based on the total weight of the composition. Preferably, the composition comprises 60 to 75 wt% trisaccharides and 25 to 40 wt% tetrasaccharides, based on the total weight of the composition.

[0127] In such embodiments, the composition (or oligosaccharide fraction) suitably comprises the above-mentioned components (a), (b), and (c) in the following amounts, based on the total weight of oligosaccharide compounds present in the composition: (a) at least 8 wt% Gal-(β1-3)-Gal-(β1-4)-X a ; (b) at least 3 wt% Gal-(β1-3)-Gal-(β1-3)-X b ; and (c) at least 5 wt% Gal-(β1-3)-Gal-(β1-2)-X c Including, where X a , X b , and X c are each independently selected from monosaccharides.

[0128] Preferably, the composition (or oligosaccharide fraction) comprises the above components (a), (b), and (c) in the following amounts: (a) at least 10 wt% Gal-(β1-3)-Gal-(β1-4)-X a ; (b) at least 5 wt% Gal-(β1-3)-Gal-(β1-3)-X b ; and (c) at least 7 wt% Gal-(β1-3)-Gal-(β1-2)-X c .

[0129] The composition may include component (d) and / or component (e) described above.

[0130] The composition preferably contains the ingredients in the proportions discussed above.

[0131] In some embodiments, the composition of this first aspect is a trisaccharide fractionation product (which may be referred to as a DP3 fraction). Such a trisaccharide fractionation product may be obtained by the known fractionation methods mentioned above. Such a composition preferably contains at least 70 wt% trisaccharides, and preferably at least 70 wt% trisaccharides. Preferably, the composition comprises at least 95 wt% of trisaccharides.

[0132] In such embodiments, the composition (or oligosaccharide fraction) suitably comprises the above-mentioned components (a), (b), and (c) in the following amounts, based on the total weight of oligosaccharide compounds present in the composition: (a) at least 8 wt% Gal-(β1-3)-Gal-(β1-4)-X a ; (b) at least 3 wt% Gal-(β1-3)-Gal-(β1-3)-X b ; and (c) at least 5 wt% Gal-(β1-3)-Gal-(β1-2)-X c Including, where X a , X b , and X c are each independently selected from monosaccharides.

[0133] Preferably, the composition (or oligosaccharide fraction) comprises the above components (a), (b), and (c) in the following amounts: (a) at least 10 wt% Gal-(β1-3)-Gal-(β1-4)-X a ; (b) at least 5 wt% Gal-(β1-3)-Gal-(β1-3)-X b ; and (c) at least 7 wt% Gal-(β1-3)-Gal-(β1-2)-X c .

[0134] The composition may include component (e) described above.

[0135] The composition preferably contains the ingredients in the proportions discussed above.

[0136] The composition of this first aspect can be used as and incorporated into a food supplement product intended for consumption by consumers. Such products can be selected from the group consisting of dairy products (e.g., liquid milk, whole milk powder, skim milk powder, fat-filled milk powder, whey powder, dry milk powder such as infant formula, ice cream, yogurt, cheese, fermented milk products), beverages, sports drinks, baby foods, cereals, breads, biscuits, confectionery, cakes, food supplements, dietary supplements, medical foods / nutritional foods, foods for specific medical uses, animal feed, poultry feed, or indeed any other food or beverage.

[0137] The composition of this first aspect may be incorporated into a synbiotic composition, which is preferably a mixture of live microorganisms and substrates that are selectively utilized by the host microorganisms to confer a health benefit to the host, i.e. a mixture of probiotics and prebiotics.

[0138] The composition of this first aspect may be used as, and may be in the form of, a pharmaceutical or nutraceutical composition comprising at least one carrier, excipient, or diluent.

[0139] Suitable additional ingredients for pharmaceutical or nutraceutical compositions, and methods for preparing such pharmaceutical or nutraceutical compositions, are known in the art.

[0140] The composition can be administered once or multiple times. Suitable administration frequency can be at least once a day, once every other day, once a week, once every two, three, or four weeks, once a month, once every two months, or once every three to six months. The composition can be administered for at least one week, at least one month, at least three to six months, at least one year, two years, three years, four years, or five years, or throughout the course of the disease or the lifetime of the subject.

[0141] It will be apparent to one skilled in the art that the administration of the composition may be optimized during clinical trials.

[0142] The compositions of the present invention can be formulated into pharmaceutical compositions in combination with suitable pharmaceutically acceptable carriers, pharmaceutically acceptable diluents, or pharmaceutically acceptable excipients, and can be formulated into solid, semi-solid, or liquid preparations such as tablets, capsules, powders, granules, and solutions.

[0143] Pharmaceutically acceptable carriers, excipients, or diluents may include, for example, water, saline, dextrose, maltodextrin, glycerol, ethanol, salts such as NaCl, MgCl, KCl, MgSO; buffers such as phosphate buffer, citrate buffer, Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS); solubilizing agents; detergents such as non-ionic detergents like Tween-20; glycerol; and the like.

[0144] Pharmaceutically acceptable carriers, excipients, and diluents are nontoxic to recipients at the dosages and concentrations employed, and may include, for example, buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine, and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, or combinations thereof); arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, thiamin mononitrate ... The composition may include amino acids such as isine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and valine, and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0145] For oral formulations, the compositions of the present invention may contain, for example, conventional additives such as lactose, mannitol, corn starch, or potato starch; binders such as microcrystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatin; disintegrants such as corn starch, potato starch, or sodium carboxymethylcellulose; lubricants such as calcium stearate or magnesium stearate; and optionally diluents, buffers, wetting agents, preservatives, and flavoring agents as suitable additives for producing tablets, powders, granules, or capsules.

[0146] The pharmaceutical composition may be in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form, with the lyophilized formulation being reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of purified water (typically a volume equivalent to the volume removed during lyophilization).

[0147] Tonics may be included in the formulation to adjust the tonicity of the formulation. Exemplary tonics include sodium chloride, potassium chloride, glycerin, and any component from the group of amino acids, sugars, and combinations thereof. In some embodiments, The aqueous formulations are isotonic, although hypertonic or hypotonic solutions may also be suitable. The term "isotonic" refers to a solution that has the same tonicity as some other solution to which it is being compared, such as saline or serum.

[0148] The composition may modulate the abundance of bacterial genera present in the intestine. In some embodiments, the composition modulates the abundance of bacterial genera present in one or both of the small intestine and the large intestine. In some embodiments, the composition modulates the abundance of a bacterial genera predominant in the small intestine selected from the group consisting of Achromobacter, Agrobacterium, Blautia, Burkholderia, Coprococcus, Cryocola, Enterococcus, Eubacterium, Holdemania, Lactococcus, Mycobacterium, Pseudoramibacter, Ralstonia, Sphingomonas, Streptococcus, and Turicibacter.

[0149] According to a second aspect of the present invention there is provided a method for preparing a composition comprising an oligosaccharide compound according to the first aspect, said method comprising: (i) providing a source of sugar compounds; (ii) treating said source of sugar compounds with one or more galactosidase enzymes to at least partially convert said source of sugar compounds to said oligosaccharide compounds; Includes.

[0150] Preferably, the steps of the method of this second aspect are carried out in the order step (i) followed by step (ii).

[0151] 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, e.g., lactose syrup, which may be derived from cow's milk. The lactose may be heat-treated.

[0152] In some embodiments, no additional sugars, such as monosaccharides or disaccharides, are added to the source of sugars, hi such embodiments, the method produces a galactooligosaccharide compound.

[0153] In some embodiments, the source of sugar compounds includes at least one additional sugar. Preferably, the at least one additional sugar provides an alternative terminal monosaccharide unit to the oligosaccharide compound, as discussed above. Preferably, the at least one additional sugar is a source of such a monosaccharide unit. The at least one additional sugar that is a source of such a monosaccharide unit may be a monosaccharide or a higher sugar, such as a disaccharide. The at least one additional sugar can be a source of a 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 a mixture thereof. The at least one additional sugar can be one or more of the above monosaccharides.

[0154] In such embodiments, the method further comprises pre-treating one or more of the above monosaccharides. An oligosaccharide having the above as a terminal sugar unit is produced.

[0155] In some embodiments, the at least one additional sugar may be selected from fucose, arabinose, xylose, rhamnose, mannose, or a mixture thereof. The source of sugars may include lactose and a source of one or more of the monosaccharides. In such embodiments, the method produces oligosaccharides terminated with a sugar unit selected from Glc, Fuc, Ara, Xyl, Rha, and Man, or a mixture thereof.

[0156] Step (ii) of the method comprises treating the source of sugar compounds with at least one galactosidase enzyme. The galactosidase enzyme may be an α- or β-galactosidase enzyme, depending on whether the sugar units of the oligosaccharide compounds have α- or β-linkages. Preferably, the enzyme exhibits galactosyltransferase (transgalactosidase) activity and forms α- or β-linkages between the sugar units of the source of sugar compounds. This results in the synthesis of oligosaccharide compounds having two or more galactose units derived from lactose. Preferably, step (ii) is carried out until the conversion of the source of sugars to the oligosaccharide compounds is complete.

[0157] Step (ii) may involve treating said source of sugar compounds with one or more additional enzymes which are not galactosidase enzymes.

[0158] Suitably, the method comprises the step (iii) of separating the galactosidase enzyme from the composition comprising the oligosaccharide compound, which may comprise removing the enzyme by filtration, for example nanofiltration.

[0159] The composition comprising the oligosaccharide compound produced in step (iii) may be heat treated.

[0160] In some embodiments, the composition is evaporated to reduce the water content to provide a final composition comprising the oligosaccharide compound as a syrup, as discussed above in relation to the first aspect.

[0161] In some embodiments, prior to evaporation, glucose is removed from the composition produced by step (iii). This preferably reduces the glucose content of the composition from 20-30 wt% to less than 10 wt%, preferably about 5 wt% or less. The water content of the composition is then reduced by evaporation, and the product is dried to provide the final composition comprising oligosaccharide compounds as a powder, as discussed above in relation to the first aspect.

[0162] It is also understood that a person skilled in the relevant art can prepare a composition comprising an oligosaccharide compound according to the first aspect, i.e., a composition comprising a defined amount of a particular oligosaccharide compound, by combining the required amounts of said oligosaccharides isolated and obtained from different sources.

[0163] The invention is described below, by way of example, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0164] [Figure 1] FIG. 1 is a graph showing the results of butyric acid analysis of three donors after administration of the oligosaccharides of the present invention together with comparative oligosaccharides in Comparative Example 1. [Figure 2] Figure 2 is a graph showing total microbial community activity (acidification and gas production) expressed as (A) pH and (B) gas pressure. In Example 4, measurements were performed in triplicate. Mean data were derived using data from donors A / B / C. Error bars represent standard deviation. GOS = Bimuno® galactooligosaccharides. [Figure 3] FIG. 3 is a graph showing individual microbial community activity (acidification and gas production) across all donors in Example 4, expressed as (A) pH and (B) gas pressure. [Figure 4] Figure 4 is a graph showing microbial metabolic activity: (A) acetate, (B) propionate, (C) butyrate, (D) total SCFA, (E) lactate, and (F) ammonia. In Example 4, measurements were performed in triplicate. Mean data were derived using data from donors A / B / C. Error bars represent standard deviation. SCFA = short-chain fatty acids. [Figure 5] Figure 5 is a graph showing the microbial metabolic activity (A) acetate, (B) propionate, (C) butyrate, (D) total SCFA, (E) lactate, and (F) ammonia for individual donors in Example 4. Measurements were performed in triplicate. Error bars represent standard deviation. SCFA = short chain fatty acids. [Figure 6]Figure 6 is a graph showing the representation of the major phyla in the fecal microbiota of each donor at time 0 in Example 4. Samples were analyzed in triplicate. FM = feces; LOQ = limit of quantification. [Figure 7] Figure 7 is a plot showing principal coordinate analysis of seed data using Bray-Curtis distances for (A) 6 hours post-treatment (relative data) and (B) 24 hours post-treatment (absolute data) for each donor in Example 4. Each dot represents one replicate. [Figure 8] 8 is a graph showing changes in microbial community composition in Example 4. (A) Relative abundance (family level) 6 hours after treatment, (B) effect size of linear discriminant analysis of relative abundance (species level) 6 hours after treatment, (C) box plot of most significant enrichment (species level) 6 hours after treatment, (D) absolute abundance (family level) 24 hours after treatment, (E) effect size of linear discriminant analysis of absolute abundance (species level) 24 hours after treatment, (F) box plot of most significant enrichment (species level) 24 hours after treatment. *: p<0.05, **: p<0.01, ***: p<0.001. LDA = linear discriminant analysis. [Figure 9] Figure 9 is a graph showing microbial metabolic activity at different time points of incubation in Example 5 (error bars reflect the STDEV of individual donors and the SEM of the mean across all donors) for (A) acetate, (B) propionate, (C) butyrate, (D) total SCFA, (E) lactate, and (F) ammonia, where two conditions (B-GOS and commercial GOS (denoted GOS-2)) were tested and a negative control (blank) was included. [Example]

[0165] Example 1 - Oligosaccharide Syrup A composition containing an oligosaccharide compound according to the present invention in the form of a syrup was obtained by the following procedure. Lactose was rehydrated with drinking water to obtain a working solution with a solids content of 35 to 65 wt %. The lactose solution was heated and then cooled to 40 to 65°C. The pH of the solution was adjusted to 5.5 to 7.5. Next, β-galactosidase enzyme was added to the solution in a sealed container, and then reacted with the lactose to catalyze the transfer of galactose molecules to produce oligosaccharide compounds. The progress of the reaction was monitored by measuring the production of glucose. The reaction was allowed to proceed for 8 to 26 hours. The reaction was then terminated 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 approximately 22 to 28 wt % to provide the product as a syrup.

[0166] Example 2 - Oligosaccharide Powder A composition containing the oligosaccharide compounds of the present invention in powder form was obtained by a modification of the above-described procedure. After removing the enzyme, the mixture was further filtered to remove most of the glucose and other monosaccharides, reducing the monosaccharide content from about 23 wt% to about 5 wt%. The composition was then The moisture content of the material was reduced by evaporation and the product was dried to provide a powder with a moisture content of about 3-6 wt%.

[0167] Example 3 - Fractionation Isolation of different fractions (DP2, DP3, DP4, or DP5) from the sample of Example 2 was performed using a 5 × 70 cm BioGel P2 column with water as the eluent. The column was operated at room temperature (21 °C) with a flow rate of 40 to 100 mL / h. A 0.5 g / mL solution of Example 2 in ultrapure water was loaded at 0.5, 0.75, 1.0, 1.5, or 2.0 mL depending on the run. This solution was loaded onto the column a total of 15 times to obtain a sufficient amount of the low-concentration fraction, i.e., DP5. After an excluded volume of approximately 725 mL, 5 mL fractions were collected. Analysis using thin-layer chromatography (TLC) and HPAEC-PAD was performed to confirm that molecules of the same DP were pooled.

[0168] The 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 experiments.

[0169] Comparative Example 1 A commercially available composition containing an oligosaccharide compound was obtained in powder form.

[0170] Oligosaccharide Analysis Samples of Inventive Example 2 and Comparative Example 1 were analyzed to determine their oligosaccharide content by the following procedure.

[0171] Materials and Methods A sample of each dry powder of Example 2 and Comparative Example 1 was dissolved in water to provide a solution having a concentration of 40 g / l for analysis.

[0172] Gel permeation chromatography Aqueous GPC separation of sample components was performed using an HPLC system equipped with a Rezex RSO and RI detector and in-line desalting (to remove salts and charged substances such as proteins). The 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.

[0173] GOS Fingerprinting To separate the monosaccharides and oligosaccharides of the different samples, HPAEC-PAD (High-Performance Anion Exchange Chromatography) with a PA-1 column was used. We strived to achieve the separation quality described in van Leeuwen et al., Carbohydrate Research 2016, 425, 48-58. A 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 many peaks. The samples used for GPC were diluted 100-fold with DMSO before injection.

[0174] result Gel permeation chromatography (GPC-RI) Table 1 shows the DP (degree of polymerization) composition of the samples using the Rezex-RSO system (values ​​expressed as g / L of the sample) based on RI calibration with glucose. All materials eluting at >DP5 are grouped in the >DP5 box.

[0175] [Table 1]

[0176] The concentration information can be used to calculate the relative weight percentages of different DP fractions of oligosaccharides contained in the sample, as shown in Table 1, where DP=2 refers to disaccharides, DP=3 refers to trisaccharides, etc.

[0177] GOS Fingerprinting To identify individual galactooligosaccharides in the samples (GOS fingerprinting), a gradient was developed that provided a separation comparable to that reported by 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 for the oligosaccharide compounds by van Leeuwen et al. A retention window of approximately 15 seconds was applied for peak annotation.

[0178] Table 2 thus presents information on the HPAEC-PAD peak areas of all annotated peaks, along with information relating to incubation conditions, sample concentration, dilution, and injection volume.

[0179] [Table 2]

[0180] The peak areas were assumed to correspond approximately to the amount of each oligosaccharide compound present in the composition. If a specific oligosaccharide compound was not identified, "unknown" and a number were entered in the table. Identified compounds were identified either by name or by a number corresponding to the number assigned to a specific galactooligosaccharide in van Leeuwen et al., Carbohydrate Research 2016, 425, 48-58. A list of these galactooligosaccharides and their corresponding numbers is provided below.

[0181] The oligosaccharide compositions (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.

[0182] Prebiotic effect - butyric acid production Butyrate is produced by the gut microbiota through the conversion of acetate and / or lactate (among other substrates) to butyrate. It is often produced during the later stages of fermentation, as it can be a secondary metabolite, but it can also be produced directly by certain butyrate-producing bacteria.

[0183] The purpose of these experiments was to evaluate the difference in butyrate production of the compositions of the present invention alongside comparative GOS compositions to determine whether they were more effective as prebiotics. The experiments used the following procedure.

[0184] The composition of Example 2 of the present invention, Comparative Example 1, and the control blank sample were dialyzed using a 0.5 kDa membrane to obtain a 5 g / L sample. This sample was 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 for colonic fermentation products, including butyrate, over a 48-hour period (at collection points of 6, 24, and 48 hours). The distribution of the oligosaccharides in the mixtures was also monitored over this period using the method described above in connection with Table 2. The results showed that Example 2 was well fermented by all donors, primarily during the 0-24 hour period, and that butyrate production was increased at 6 and 48 hours compared to Comparative Example 1 and the control. The results of butyrate analysis are shown in Figure 1. The results of oligosaccharide analysis of 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 microbiota present in the fecal samples during the experiment.

[0185] To assess whether the treatment effects on gut microbial activity were 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. The Benjamini-Hochberg false discovery rate (FDR) was also used in this analysis. If the obtained p-value was smaller than the criterion, the difference between the treatment effects was considered significant. Table 4 below shows the difference in average butyrate production over 48 hours for the compared samples, with an asterisk indicating whether the difference was deemed significant by the above analysis. These results indicate that the increase in butyrate production provided by Example 2 during the 0-48 hour period was statistically significant compared to the control and Comparative Example 1.

[0186] [Table 3-1]

[0187] [Table 3-2]

[0188] [Table 4]

[0189] In summary, the present invention provides a composition comprising an oligosaccharide compound, e.g., a galactooligosaccharide compound, wherein the oligosaccharide compound comprises, based on the total weight of the oligosaccharide compounds present in the composition: (a) at least 8 wt % Gal-(β1-3)-Gal-(β1-4)-X a (b) at least 3 wt% Gal-(β1-3)-Gal-(β1-3)-X b and (c) at least 5 wt% Gal-(β1-3)-Gal-(β1-2)-X c ;where X a , X b , and X care each independently selected from monosaccharides. These compositions contain relatively high amounts of the oligosaccharide compounds (a), (b), and (c) and relatively high amounts of β1-3 Gal-Gal linkages compared to known oligosaccharide compositions. Advantageously, the compounds present in the compositions exhibited a delayed increase in butyrate over time, believed to be due to symbiosis with other bacteria that are acetate producers, which in turn stimulate the growth and secretion of butyrate from the butyrate producers. These particular characteristics of the compositions are believed to benefit the intestinal health of consumers, for example, due to these compositions resulting in increased production of butyrate in the intestines of consumers compared to known compositions.

[0190] Example 4 - Evaluation of (GOS) for ammonia and pH reduction in a short-term colonic fermentation model Experiments were conducted to evaluate the effects of the GOS of the present invention (referred to as B-GOS) in the composition and the activity of the colonic microbiota using short-term colonic incubation. Evaluations were based on the effects on overall microbial fermentation (pH, gas production), microbial metabolic activity (SCFA, lactate, and ammonia production), and community composition using shallow shotgun sequencing. In addition, the utilization of B-GOS by the gut microbiota was analyzed to understand the kinetics of this process.

[0191] Fecal sample After collecting feces from three healthy adult donors, fecal suspensions were prepared and mixed with cryoprotectant, then aliquoted, flash-frozen, and stored at -80°C until needed.

[0192] Product dialysis Prior to colonic incubation to simulate absorption during small intestinal passage, the GOS composition of the present invention (also referred to herein as "B-GOS") was dialyzed. A stock solution of B-GOS was prepared in water at 40 g / L and then placed in a dialysis membrane (pore size 0.5 kDa) to allow mono- and disaccharides to pass through the membrane. The membrane was sealed, and the stock solution was dialyzed against NaHCO3 solution (3.75 g / L, pH 7.0) for 24 hours at low temperature to prevent microbial growth.

[0193] Short-term colonic incubation Short-term colonic incubation was previously reported by Van den Abbeele, et.al., 2018 (Different oat ingredients stimulate specific microbial metabolites in the The gut microbiome of three human individuals in vitro. ACS Omega 2018,3,12446-12456. Briefly, individual reactors were prepared using the basic large The cells were filled with sugar-deficient nutrient medium containing intestinal nutrients. Dialyzed B-GOS (final concentration 5 g / L) or blank medium was then added, followed by the fecal inoculum. Incubations were performed in triplicate for B-GOS and blank (medium control) and for each donor (six incubations per donor: three for B-GOS and three for blank). Samples were collected at 0, 6, 24, and 48 hours. Shallow shotgun sequencing and flow cytometry (cell counts) were performed on samples collected at 6 and 24 hours.

[0194] Microbial metabolic activity analysis Changes in pH, gas pressure, SCFA, lactate, and ammonia were measured at 0, 6, 24, and 48 hours. Changes in pH were measured using Senseline Measurements were made using an F410 pH meter (ProSense, Oosterhout, The Netherlands). Changes in gas pressure were measured using a portable manometer (CPH6200; Wika, Echt, The Netherlands). Acetate, propionate, and butyrate were measured as previously described by De Weirdt et al., 2010 (Human faecal microbiota display variable patterns of glycerol metabolism. FEMS Microbiol Ecol 2010, 74, 601-611). Lactate levels were monitored using a commercially available enzyme assay kit (R-Biopharm, Darmstadt, Germany) according to the manufacturer's instructions. Ammonia was measured using a portable manometer (CPH6200; Wika, Echt, The Netherlands) as previously described by Van de Wiele et al., 2004 (Prebiotic The effects of chicory insulin in the simulator of the human intestinal microbial ecosystem. FEMS Microbiol Ecol 2004, 51, 143-153) were analyzed.

[0195] Microbial community analysis DNA libraries were prepared using the Illumina Nextera XT Library Preparation Kit with a modified protocol. Library yield was assessed using Qubit (ThermoFisher). The libraries were then sequenced on an Illumina HiSeq platform with 2 × 150 bp sequencing. Unassembled sequence reads were analyzed using the following methods: Ottensen et al., 2016 (Enrichment dynamics of Listeria monocytogenes and the associated microbiome from naturally contaminated ice cream linked to a listeriosis outbreak. BMC Microbiol 2016, 16, 275); Ponnusamy et al., 2016 (Cross-talk among flesh-eating Aeromonas hydrophila strains in mixed infection leading to necrotizing fasciitis. Proc Natl Acad Sci USA 2016, 113, 722-727); Hasan et al., 2014 (Microbial community profiling of human saliva). Multi-kingdom microbiota analysis and relative abundance quantification were performed directly following Lax et al., 2014 (Longitudinal analysis of microbial interaction between humans and the indoor environment. Science 2014, 345, 1048-1052) using shotgun metagenomic sequencing. PLoS One 2014, 9, e97699). Briefly, curated genome databases were integrated into a high-performance database that rapidly disambiguates hundreds of millions of metagenomic sequence reads and links them to the individual microorganisms that generated them. This was combined with a data-mining algorithm. Total bacterial cell counts were measured using a BD FACSVerse Cell Analyzer (BD Biosciences, Franklin Lakes, NJ, USA) at a high flow rate and a threshold of 200 in the SYTO channel. Proportional values ​​obtained using shotgun sequencing were converted to absolute abundances by multiplying the relative abundance of each population in the sample by the total cell count obtained by flow cytometry.

[0196] Temporal utilization of GOS by the gut microbiota GOS chain length distribution analysis was performed using undialyzed B-GOS, dialyzed B-GOS, and samples obtained at all fermentation time points (6, 24, and 48 h) using gel permeation chromatography (GPC).

[0197] Gel permeation chromatography Aqueous GPC separation was performed using a HPLC equipped with a Rezex RSO and RI detector and in-line desalting (to remove salts and charged substances such as proteins). The separation was performed at elevated temperature (80 °C). The separation range of the Rezex RSO column was from DP1 (monosaccharides) to approximately DP10.

[0198] statistical methods A paired, two-tailed Student's t-test was used to calculate the study results between B-GOS treatments and the blank. To control the false discovery rate when performing multiple comparisons, the Benjamini-Hochberg false discovery rate (FDR) was applied. Differences between treatment effects were considered significant if the resulting p-value (obtained by a paired, two-tailed t-test) was smaller than a criterion value. The criterion value was obtained by ranking the resulting p-values ​​in ascending order within the donor group. The rank of the resulting p-values ​​was designated (i) and varied from 1 to the total number of p-values ​​(m = 3). The criterion value was calculated by multiplying the FDR by the rank of the p-value and dividing by the total number of comparisons performed (ref = FDR × i / m). To compare treatment effects in terms of changes in pH, gas pressure, and microbial metabolites (SCFAs, lactate, and ammonia), the FDR was set to 0.10. This means that a significant difference was detected if the lowest p-value was less than 0.033, the second lowest was less than 0.066, and so on. Comparisons of the absolute and relative abundance of specific members of the microbial community were performed using analysis of variance (ANOVA). Linear discriminant analysis (LDA) effect size analysis (LEfSe) was performed to detect differences in bacterial abundance between groups. A p-value of <0.05 was considered statistically significant. All statistical analyses were performed using Microsoft Excel version 2110 (Microsoft, Redmond, WA, USA). Principal coordinate analysis (PCoA) was performed using Analyze-it (v4.51) software. The increase or decrease in each parameter at 6 or 24 h of incubation was used to generate a joint PCoA biplot for each of the two time points.

[0199] Microbial metabolic activity The pH remained relatively stable in the blank (untreated) reactor at all time points and decreased with B-GOS treatment at 6, 24, and 48 hours compared to 0 hours (Figure 2A). In the blank samples, gas production was slightly higher between 6 and 24 hours than between 0 and 6 hours, and then decreased rather dramatically between 24 and 48 hours (Figure 2B). A similar pattern was observed for B-GOS, although gas production was generally higher. The pH and gas production for individual donors are shown in Figures 3A and 3B, respectively.

[0200] The mean changes in SCFA levels are shown in Figure 4D, and data for individual donors are shown in Figure 5D. In the blank reactor, acetate levels increased from 0 to 6 hours. The B-GOS-treated reactors showed increased acetate production between 0 and 6 h, and between 6 and 24 h, then remained stable between 24 and 48 h (Figure 4A). In reactors treated with B-GOS, the most dramatic acetate production occurred between 0 and 6 h, with relatively low levels between 6 and 24 h, and minimal production between 24 and 48 h. Data for individual donors are shown in Figure 4. Mean propionate production showed increased production in the presence of B-GOS compared to the blank, with production maximal between 0 and 6 h, secondary between 6 and 24 h, and minimal between 24 and 48 h (Figure 4B). Propionate production varied between donors (Figure 5B); little propionate was produced in donor A, and levels were comparable between treatments and the blank. Butyrate production showed a distinct pattern, being lowest between 0 and 6 h and highest between 24 and 48 h in the B-GOS treatment (Figure 4C). Donors B and C produced comparable total amounts of butyrate with B-GOS (5.7 mM and 6.6 mM, respectively), but the total amount produced by donor A was much higher (17.4 mM total) (Figure 5C). Total SCFA production is shown in Figure 4D. SCFA production was higher in the B-GOS-treated reactor compared to the blank reactor. In general, SCFA production was highest between 0 and 6 h, followed by higher between 6 and 24 h; the lowest production was observed between 24 and 48 h. Data for individual donors are shown in Figure 5D. Lactate levels were highest between 0 and 6 h, and the levels were higher in the B-GOS treatment compared to the blank (Figure 4E). Although there were some differences between donors (Figure 5E), lactate levels decreased between 6 and 24 hours and between 24 and 48 hours. The level of lactate reduction between 24 and 48 hours with B-GOS treatment was greater than that with the blank, but this was less pronounced for donor C. B-GOS treatment reduced ammonia production in all donors (Figure 4F); there were no obvious differences between donors (Figure 5F).

[0201] Microbial community composition The microbial community composition at the start of the study is shown for each donor in Figure 6. While the number of cells / g of feces varied between donors, Bacteroidetes was the most abundant phylum, followed by Firmicutes and Actinobacteria. Other phyla represented only minor proportions of the community composition. PCoA plots showing the relative data at 6 h (Figure 7A) and the absolute data at 24 h (Figure 7B) show clear shifts in microbial community composition between the B-GOS and blank reactors for each donor, along with differences between donors.

[0202] The mean relative abundance of the family-level microbial community composition at 6 h is shown in Figure 8A. B-GOS treatment resulted in a significant decrease in the relative abundance of several families. LEfSe revealed a high LDA score of >4 (relative abundance) for Bifidobacterium longum at 6 h with B-GOS treatment (Figure 8B). The relative abundance of B. longum and Megamonas (unidentified) significantly increased with B-GOS treatment compared to blank at 6 h (p = 0.006 and p = 0.03, respectively [ANOVA]) (Figure 8C). Changes in absolute abundance at the family level at 24 h are shown in Figure 8D. Compared to blank, B-GOS treatment resulted in significant changes in several families, most notably a significant increase in the absolute abundance of Bifidobacteriaceae (p < 0.001). Lactobacillaceae was also significantly increased (p<0.05), and Clostridiales (p<0.001), Erysipelotrichaceae (p<0.05), Odoribacteraceae (p<0.01), and Oscillospiraceae (p<0.001) were significantly decreased by B-GOS treatment relative to the blank. The absolute family-level abundances at 24 hours are shown in Table 5 below (by donor and overall). (In Table 5, the intensity of the shading is an indication of the abundance of families in the different conditions per donor / average. Values ​​in bold indicate the abundance of families in the different conditions per donor / average.) , represent statistically significant differences in abundance between blank and B-GOS. For intradonor comparisons, unpaired t-tests were used for technical replicates (n=3), and for interdonor comparisons, paired t-tests were used.)

[0203] [Table 5-1]

[0204] [Table 5-2]

[0205] The absolute abundance of Streptococcaceae was significantly increased in B-GOS compared to the blank for donor A, but was below the limit of quantification for the other two donors. LEfSe revealed that B. adolescentis, Collinsella spp., and Collinsella aerofaciens had high LDA scores of >4 at 24 h with B-GOS treatment, and Ruminococcus torques, Bifidobacterium kashiwanohense, and Bifidobacterium ruminantium had LDA scores of >3 (but <4) (absolute abundance) (Figure 8E). The absolute abundances of B. adolescentis and B. ruminantium were significantly increased with B-GOS treatment compared to blank at 24 h (p < 0.001 and p = 0.01, respectively [ANOVA]) (Figure 8F).

[0206] Utilization of GOS by the gut microbiota Using short-term colonic incubation, the effects of B-GOS on total microbial fermentation, along with the microbial metabolic activity and microbial community composition of colonic bacteria isolated from healthy donors, were evaluated. B-GOS had significant effects on these parameters, including increased SCFA production and increased abundance of beneficial bacteria. It was also shown that nearly 95% of B-GOS was already taken up by the gut microbiota by 24 h.

[0207] During the experiment, B-GOS was found to increase the production of acetate, propionate, butyrate, and lactate. This result is consistent with previous in vitro studies using fecal batch cultures that reported increased production of acetate, acetate, and lactate with B-GOS supplementation, as well as increased production of lactate and all SCFAs (especially butyrate) with B-GOS supplementation. The increase in butyrate production was highly significant in our study, observable as early as 6 to 24 hours, with the greatest increase occurring between 24 and 48 hours. The significant increase in butyrate production was likely caused by the conversion of lactate to butyrate, which explains the consumption of lactate from 6 hours onward. The conversion of acetate to butyrate may also have contributed to the increase in butyrate. Butyrate is a key component in the health of the intestinal membrane because it is preferentially consumed by colonocytes. In addition, butyrate plays an important role in regulating epithelial barrier integrity through the coordinate regulation of tight junction proteins. The importance of this function is underscored by the fact that loss of barrier integrity is thought to contribute to metabolic disorders, inflammatory bowel disease, and obesity. B-GOS supplementation has been shown to improve intestinal barrier function in obese adults. During the study, B-GOS stimulated health-related microbial metabolites, confirming its prebiotic value, and unexpectedly demonstrated beneficial effects on undesirable metabolites such as ammonia.

[0208] B-GOS supplementation reduced ammonia production compared to the blank. Because ammonia is a marker of proteolytic fermentation, the reduction indicates that the level of proteolytic fermentation was lower with B-GOS, indicating a shift in the fermentation pattern to one more beneficial to the host. This may be beneficial because some metabolic derivatives of proteolytic fermentation are involved in diseases such as colon cancer. Avoiding proteolytic fermentation may be beneficial because highly toxic compounds can be produced during this process. This is particularly important for people on a high-protein diet due to the increased protein load and increased proteolytic fermentation.

[0209] Previous in vitro studies have reported that B. longum and other Bifidobacterium species, along with some Lactobacillus species, are directly involved in B-GOS fermentation. In our experiments, the relative abundance of B. longum and Megamonas (unspecified) significantly increased (relative to blank) at 6 hours after B-GOS supplementation. In addition, at 24 hours, the absolute abundance of B. adolescentis and B. ruminantium increased (relative to blank), as did the absolute abundance of Bifidobacteriaceae and Lactobacillaceae. ) significantly increased. Other in vitro fecal batch studies have shown that B-GOS supplementation increases the proliferation of bifidobacteria and lactobacilli, and human studies have shown that B-GOS supplementation leads to increases in bifidobacteria and lactobacilli in healthy adults and elderly people. Bifidobacteria and lactobacilli are well known for their role in human health. Increases in both species likely contributed to the observed increase in butyrate production via the production of lactate. Thus, the observed increases in bifidobacteria and lactobacillaceae support the prebiotic effect of B-GOS. Because the role of Megamonas in health or disease has not been clearly determined, it is difficult to speculate on the implications of this finding.

[0210] There were several donor-specific effects on propionate and butyrate production. Donor A produced almost no propionate, and there was no difference in propionate production between the blank and B-GOS supplementation. However, this donor produced significantly more butyrate with B-GOS supplementation than the other two donors. In addition, lactate production and consumption occurred to a greater extent in Donor A than in the other two donors. We observed an enrichment of Streptococcaceae in Donor A under B-GOS supplementation. This may have contributed to the strong increase in initial lactate production (0–6 h) and its subsequent conversion to butyrate (24–48 h). Interestingly, while Donors A and B had increases in the absolute abundance of both Bifidobacteriaceae and Lactobacillaceae, Donor C had an increase in Bifidobacteriaceae but not Lactobacillaceae. However, these differences in the effects of B-GOS on the three donors do not fully explain the inter-donor differences in butyrate or lactate production.

[0211] In conclusion, B-GOS demonstrated prebiotic activity in short-term colonic incubations with the colonic microbiota of three healthy adult donors. Supplementation led to increased production of lactic acid and SCFAs, including butyrate, and increased proliferation of the beneficial bacterial families Bifidobacteriaceae and Lactobacillaceae. This study demonstrates for the first time that B-GOS reduces undesirable metabolites, such as ammonia.

[0212] Example 5 - Comparative analysis of the effects of B-GOS and commercial GOS on reducing ammonia and pH in a short-term colonic fermentation model Experiments were conducted to evaluate the effects of a powdered version of the GOS of the present invention (designated "B-GOS") and a commercially available GOS powder (designated "GOS-2") on the colonic microbiota of three healthy adult humans. The evaluation was based on the effects on total microbial carbohydrate fermentation (acetic acid, propionic acid, and butyric acid) and microbial metabolic activity (production of SCFAs, lactate, and ammonia). To this end, short-term colonic incubations were performed in a similar manner as outlined in Example 4 above, and the results were subjected to the same statistical analyses.

[0213] Carbohydrate fermentation As shown in Figure 9A, the stimulatory effect began between 0 and 6 hours, and after 6 hours, both products produced similar acetate concentrations. By the end of the incubation, both products produced similar acetate concentrations, significantly higher than the blank. The consumption of acetate during the incubation (24-48 hours) with Donor A may have been due to its conversion to butyrate, explaining the stimulatory effect on butyrate and gas production during the same time frame.

[0214] Figure 9B shows that both products produced significantly higher propionic acid levels than the blank (the strongest effect in Donor B and Donor C). It resulted in slightly higher propionic acid concentrations than OS. Fermentation in donor C was characterized by strong propionic acid production between 0 and 6 h, and in donor B by strong propionic acid production between 6 and 24 h.

[0215] In Figure 9C, it can be seen that both products significantly stimulated butyrate production in three donors by the end of the incubation, with Bimuno resulting in higher butyrate concentrations than GOS-2. The butyrate production between 24 and 48 hours in donor A could be attributed to the conversion of acetate (and lactate, see below) during the same time frame.

[0216] microbial metabolic activity Total SCFA levels reflect the overall fermentation of the test ingredients. Both products yielded significantly higher SCFA levels than the blank (Figure 9D). The stimulatory effect on SCFAs began between 0 and 6 hours, and after 6 hours, both products yielded comparable SCFA concentrations, reflecting efficient product fermentation by the gut microbiota. By the end of the incubation, both products yielded comparable SCFA concentrations, significantly higher than the blank.

[0217] As shown in Figure 9E, lactate production and consumption were low in the blank incubations. Each product significantly stimulated lactate production between 0 and 6 h, providing another confirmation of efficient product fermentation. The strongest stimulatory effect was obtained with Bimuno. Lactate accumulation (observed after 6 h) suggests that the lactate production rate was higher than the lactate consumption rate. Overall, the lactate produced during the first 6 h was efficiently consumed by the end of the incubation in all conditions. This indicates efficient lactate conversion (to propionate / butyrate).

[0218] As shown in Figure 9F, both products significantly reduced ammonia production over the incubation period, a reduction that was already observed after 24 hours. The strongest reduction was obtained with B-GOS, which resulted in significantly lower ammonia levels than GOS-2 after 48 hours.

[0219] conclusion The purpose of the study was to investigate the potential health-promoting effects of the B-GOS composition of the present invention, alongside a commercially available GOS powder (GOS-2), by examining the effects on microbial metabolic activity in the gut microbiota of three adult donors.

[0220] Short-term colonic incubation showed that both products were fermented by the gut microbiota of the three donors, stimulating the production of acetate, propionate, butyrate, and lactate and significantly reducing ammonia production. Both products produced comparable levels of acetate. B-GOS produced higher butyrate and lactate levels; GOS-2 produced higher propionate levels.

[0221] Ammonia production is due to proteolytic bacterial activity and is associated with the formation of toxic by-products such as p-cresol. Therefore, high ammonia production in the large intestine has been associated with adverse health effects. As a result, products that reduce ammonia production are beneficial to health, and the GOS of the present invention reduces the formation of toxic by-products and provides superior results over currently available GOS products, making it a useful pharmaceutical or dietary supplement.

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

[0223] Throughout this specification, the terms "comprising" or "comprises" mean including the specified component but not excluding the presence of other components. The terms "of" or "consists essentially of" mean including the specified components, but excluding other components, except for impurities, unavoidable materials present as a result of the process used to provide said components, and materials present as components added for purposes other than achieving the technical effects 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 components.

[0224] The terms "consisting of" or "consists of" mean the inclusion of specified ingredients and the exclusion of the addition of other ingredients.

[0225] Wherever appropriate and depending on the context, use of the words "comprise" or "comprising" may be taken to include or include the meaning of "consist essentially of" or "consist essentially of" and may be taken to include the meaning of "consisting of" or "consisting of."

[0226] For the avoidance of doubt, when the amount of a component in a composition is stated in wt%, this refers to the weight percent of the particular component relative to the total composition referred to. For example, "oligosaccharide compounds comprising up to 35 wt% of disaccharides" means that 35 wt% of the oligosaccharide compounds in said composition are provided by disaccharides.

[0227] The optional features defined herein may be used either individually or, where appropriate, in combination with one another, particularly in combinations as defined in the appended claims. The optional features for each aspect or exemplary embodiment of the invention defined herein should also be read as applicable to any other aspect or exemplary embodiment of the invention, where appropriate. In other words, those skilled in the art reading this specification should consider the optional features for each exemplary embodiment of the invention to be interchangeable and combinable between different exemplary embodiments.

[0228] Attention is directed to all papers and documents filed contemporaneously or prior to this application in connection with this application and released to the public herewith, and the contents of all such papers and documents are incorporated herein by reference.

[0229] 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.

[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. Thus, 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 foregoing embodiments. The invention is not limited to any novel features disclosed in this specification (including any accompanying claims and drawings). This invention extends to any novel object or any novel combination of steps of any method or process so disclosed.

Claims

1. 1. A composition for use in reducing the level of ammonia 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 wt % Gal-(β1-3)-Gal-(β1-4)-X ａ ; (b) at least 3 wt % Gal-(β1-3)-Gal-(β1-3)-X ｂ and (c) at least 5 wt % of Gal-(β1-3)-Gal-(β1-2)-X ｃ Including, Here, X ａ , X ｂ , and X ｃ are each independently selected from monosaccharides.

2. 2. A composition for use according to claim 1, wherein the oligosaccharide compound comprises, based on the total weight of the oligosaccharide compounds present in the composition: (d) at least 3 wt % of Gal-(β1-3)-Gal-(β1-3)-Gal-(β1-4)-X ｄ Including, Here, X ｄ is selected from monosaccharides.

3. 3. A composition for use according to claim 1 or claim 2, comprising, based on the total weight of oligosaccharide compounds present in said 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 %; composition.

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

1.

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

1.

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

2.

7. 10. A composition for use according to any one of the preceding claims, wherein the oligosaccharide compounds comprise, based on the total weight of the oligosaccharide compounds present in the composition: (e) at least 5 wt % of Gal-(β1-4)-Gal-(β1-4)-X ｅ Including, Here, X ｅ is selected from monosaccharides.

8. X ａ , X ｂ , X ｃ , X ｄ , and X ｅ are each 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 mixtures thereof.

9. X ａ , X ｂ , X ｃ , X ｄ , and X ｅ 8. The composition for use according to claim 1, wherein each of

10. X ａ , X ｂ , X ｃ , X ｄ , and X ｅ each comprises Fuc. A composition for use according to any one of claims 1 to 4.

11. 10. The composition for use according to any one of the preceding claims, wherein 40% to 55% of the Gal-Gal linkages in said oligosaccharide compound are 1-3 linkages.

12. 10. A composition for use according to any one of the preceding claims, comprising at least 50 wt% of said oligosaccharide compound, preferably said composition being in the form of a syrup.

13. 10. A composition for use according to any one of the preceding claims, comprising at least 75 wt% of said oligosaccharide compound, preferably said composition being in the form of a powder.

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

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

16. 10. A composition for use according to any one of the preceding claims, comprising 80 wt% trisaccharides, tetrasaccharides and higher oligosaccharides.

17. 10. A composition for use according to any one of the preceding claims, comprising 80 wt% trisaccharides.

18. 10. The composition for use according to any one of the preceding claims, wherein the composition is in the form of one or more of a dietary supplement, a medical food and / or nutritional product, and a specific medical food.

19. 18. The composition for use according to any one of claims 1 to 17, wherein the composition is in the form of a pharmaceutical or dietary supplement.

20. 20. The composition for use of claim 19, wherein the pharmaceutical or dietary supplement further comprises at least one carrier, excipient, or diluent.

21. 10. The composition of any one of the preceding claims, wherein the composition is for use in the prevention, amelioration, or treatment of diseases and / or conditions associated with elevated levels of ammonia.

22. 10. The composition of any one of the preceding claims, wherein the composition is for use in the prevention, amelioration, or treatment of hyperammonemia.

23. 21. The composition of any one of claims 1 to 20, wherein the composition is for use in the prevention, amelioration, or treatment of one or more of the following conditions: hepatic encephalopathy, Reye's syndrome, urea cycle disorders, renal failure, encephalopathy, and liver failure.

24. 10. A composition for use according to the preceding claims, for administration to athletes or sportspeople undergoing and / or about to undergo physical training.

25. 25. The composition for use according to claim 24, wherein the athlete or sportsperson is on a high protein diet.

26. The composition is intended to treat one or more of malnutrition, sarcopenia, cachexia, and frailty.

21. A composition for use according to any one of claims 1 to 20, which is for use in the prevention, amelioration or treatment of a number of conditions.

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