WO2024079471A1 - Composition d'oligosaccharide destinée à être utilisée dans la réduction du niveau d'ammoniac - Google Patents

Composition d'oligosaccharide destinée à être utilisée dans la réduction du niveau d'ammoniac Download PDF

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Publication number
WO2024079471A1
WO2024079471A1 PCT/GB2023/052652 GB2023052652W WO2024079471A1 WO 2024079471 A1 WO2024079471 A1 WO 2024079471A1 GB 2023052652 W GB2023052652 W GB 2023052652W WO 2024079471 A1 WO2024079471 A1 WO 2024079471A1
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composition
gal
suitably
use according
oligosaccharide
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PCT/GB2023/052652
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Gediminas BALTULIONIS
Kelly LITHERLAND
Leunis Forrinus Harthoorn
Aleksandra Elzbieta MARUSZAK
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Clasado Research Services Limited
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Publication of WO2024079471A1 publication Critical patent/WO2024079471A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to galactooligosaccharides (GOS) compositions for use in reducing levels of ammonia in an individual in need thereof.
  • the present invention also relates to methods of preparing said compositions and the formulation of said compositions as dietary supplements and/or medicaments.
  • the present invention relates to an oligosaccharide composition with a relatively high amount of certain beneficial oligosaccharides.
  • Prebiotics are defined by the International Scientific Association for Probiotics and Prebiotics as “a substrate that is selectively utilized by host microorganisms conferring a health benefit”.
  • Prebiotics are non-digestible dietary fibers that resist digestion and absorption until they reach the large intestine where they are fermented by members of the gut microbiome. They can modify the composition and function of the gut microbiome.
  • a diet rich in dietary fiber/prebiotics has been shown to increase bacterial abundance and gut microbiome gene richness, as well as increasing the abundance of beneficial bacteria such as Bifidobacterium and Lactobacillus.
  • Prebiotics also play multiple roles in suppressing gut pathogens, as an example, members of the Bifidobacterium and Lactobacillus genera produce lactic acid during prebiotic fermentation.
  • Beneficial gut microbes utilize dietary fibers as an energy source.
  • SCFAs short-chain fatty acids
  • SCFAs are known to have several beneficial effects on human health including maintenance of the colonic epithelium; a potential role in regulating glucose homeostasis, lipid metabolism, and appetite regulation; and a role in regulating the immune system and inflammatory response.
  • GOS Galactooligosaccharides
  • GOS prebiotic products contain a mixture of many oligosaccharide compounds in varying proportions, not all of which have the desired beneficial effects on the intestinal microbiota of the consumer. Although such products have shown some beneficial results in improving the health of patients, there remains a need for further improved oligosaccharide compositions to fully realise the potential benefits to consumers of such prebiotics and to provide a long-lasting and inexpensive treatments for a number of conditions and/or diseases.
  • composition for reducing levels of ammonia wherein the oligosaccharide compounds comprise:
  • a method of preventing, ameliorating or treating conditions and/or diseases associated with the elevated level of ammonia which comprises the administration of a composition comprising oligosaccharide compounds to a subject in need of such prevention, amelioration or treatment, wherein the oligosaccharide compounds comprise:
  • a method of preventing, ameliorating or treating conditions and/or diseases related to elevated levels of ammonia in a subject in need of such prevention, amelioration or treatment comprising: i) determining whether the subject has elevated levels of 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 a therapeutically effective amount of a composition comprising oligosaccharide compounds to the subject, wherein oligosaccharide compounds comprise:
  • compositions for the manufacture of a medicament for the prevention, amelioration or treatment of a condition and/or disease associated with elevated levels of ammonia wherein the composition comprises oligosaccharide compounds, said oligosaccharide compounds comprising:
  • composition for use in the prevention and/or treatment of fatigue in an athlete or sports person comprising oligosaccharide compounds, wherein the oligosaccharide compounds comprise:
  • Plasma concentrations of ammonia during exercise often achieve or exceed those measured in liver disease patients, resulting in increased cerebral uptake which can result in disturbances in brain function, potentially through similar mechanisms underpinning pathology, which may impact on performance as fatigue or reduced function, especially during extreme exercise.
  • 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 extreme exercise. Therefore, it follows that the composition of the present invention, would be suitable for use in the prevention and/or treatment of fatigue in an athlete or sports person, especially as it would target the gut and in turn reduce the amount of available ammonia which could be absorbed into the blood.
  • compositions for use in the modulating the level of ammonia in an individual wherein the composition comprises oligosaccharide compounds, and the oligosaccharide compounds comprise:
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or can be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting or slowing its development; and (c) relieving the disease and/or symptoms of the disease, i.e., causing regression of the disease.
  • subject used herein includes any human or nonhuman animal.
  • nonhuman animal includes all mammals, such as nonhuman primates, sheep, dogs, cats, cows, horses.
  • a “therapeutically effective amount” refers to the amount of compounds, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease.
  • the “therapeutically effective amount” will vary depending on the compound(s) used, the disease and its severity and the age, weight, etc., of the subject to be treated.
  • condition and/or disease associated with elevated levels of ammonia refers to a range of conditions and/or diseases resulting from the elevated levels of ammonia in the body with identifiable symptoms.
  • the elevated levels could be in the blood, gut, gut and/or intestinal lining.
  • 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 related to (or may be) any one of the following conditions and/or diseases: hepatic encephalopathy, Reye syndrome, urea cycle disorders, kidney failure, encephalopathy and liver failure.
  • the condition and/or disease associated with elevated levels of levels of ammonia may be in an individual who is an athlete or sports person who is undergoing (or about to undergo) physical exercise or training.
  • the composition may be administered prior to, during, or after, undergoing exercise or physical activity. Alternatively, the composition may be administered according to a continuous dosage regime, such as once or twice daily.
  • the therapeutically effective amount may be a standardised dose or a dose determined by individual factors such as age, body mass index (BMI) or the overall weight of the individual.
  • performance refers to the ability of an individual to perform certain physical tasks associated with sports and exercise, including, but not limited to reducing fatigue and improving endurance.
  • the term “athlete” or “sports person” refers to an individual actively engaged in sports or exercise, including individuals who are professionals or amateurs.
  • the present inventors have advantageously, and unexpectedly, found that supplementation using the composition of the invention reduced the levels of ammonia in trials.
  • the composition acts systemically and studies have shown that the composition of the present invention can modulate levels of ammonia, which can result in a number of improvements such as the reduction of toxic byproducts such (as p-cresol) due to proteolytic microbial activity in the gut. This is particularly relevant for individuals who are on a high protein diet or feeding regimen as they would have a higher proteolytic microbial activity due to the higher protein levels in the gut environment.
  • the individuals may be on a high protein diet or feeding regimen for athletic purposes (e.g. to increase muscle mass and/or counteract muscle fatigue) or for medical purposes (e.g. to prevent or assist in the treatment of cachexia or sarcopenia).
  • the composition may be for use in the prevention, amelioration or treatment of one or more of the following conditions: malnutrition, sarcopenia, cachexia and frailty.
  • compositions of this first aspect provide advantages to the consumer in the establishment and maintenance of human gut microbiota when used as prebiotics.
  • the weight percentages of the specific oligosaccharide compounds discussed herein are based on the weight of all of the oligosaccharide compounds present in the whole composition. Therefore only the fraction of the composition which is provided by oligosaccharides, either components (a), (b), (c) or any other oligosaccharide present, is taken into account when determining the specified weight percentages. For this determination of oligosaccharide content, disaccharides are included, apart from lactose.
  • the composition of this first aspect may contain other non-oligosaccharide components, including monosaccharides and lactose. These components are not taken into account when determining the specified weight percentages of the oligosaccharide compounds discussed herein.
  • the specified amounts of oligosaccharide compounds in the composition can be referred to as a weight percentage of the oligosaccharide fraction of the composition of this first aspect.
  • Oligosaccharide compounds (a), (b) and (c) comprise X a , X b and X c respectively, which are each independently selected from saccharides.
  • the X a , X b and X c groups may be considered to be saccharide units. Therefore the groups X a , X b and X c can be considered terminal sugars of the oligosaccharide compounds.
  • the groups X a , X b and X c are suitably independently selected from monosaccharides.
  • Any suitable monosaccharide unit which can form oligosaccharides with the galactose units of compounds (a), (b) and (c) can provide X a , X b and X c .
  • X a , X b and X c are each independently selected from the following monosaccharides.
  • Suitable monosaccharide units are selected from glucose (Glc), fucose (Fuc), arabinose (Ara), xylose (Xyl), rhamnose (Rha), mannose (Man), galactose (Gal), ribose (Rib), lyxose (Lyx), allose (All), altrose (Alt), gulose (Gul), idose (Ido), talose (Tai), psicose (Psi), fructose (Fru), sorbose (Sor), tagatose (Tag), galactosamine (GalN), glucosamine (GlcN) and N-Acetylglucosamine (GIcNAc) or a mixture thereof. Therefore each of compounds (a), (b) and (c) may comprise a mixture of oligosaccharide compounds having different respective X groups, for example either Glc or Fuc X groups.
  • groups X a , X b and X c are independently selected from the monosaccharides listed above, suitably independently selected from Glc, Fuc, Ara, Xyl, Rha and Man or a mixture thereof.
  • the saccharide units of the oligosaccharides in the composition of this first aspect may have either the D or the L enantiomeric form.
  • the Gal saccharide units in components (a), (b) and (c) all have the D enantiomeric form.
  • the components (a), (b) and (c) may therefore be as follows:
  • the Gal and the X a , X b and X c saccharide units all have the D enantiomeric form.
  • the components (a), (b) and (c) may therefore be as follows:
  • each of X a , X b and X c are Glc. Therefore 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 the stated oligosaccharides with a suitable galactosidase enzyme.
  • the components (a), (b) and (c) are suitably as follows:
  • the Gal and the Glc saccharide units all have the D enantiomeric form.
  • the components (a), (b) and (c) may therefore be as follows: (a) D-Gal-(
  • each ofX a , X b and X c are a mixture of Glc and one or more of other saccharide units described above, for example Fuc, Ara, Xyl, Rha and Man. Therefore each of (a), (b) and (c) may comprise a mixture of oligosaccharide compounds having either Glc or one of the other saccharide units described above as the X group.
  • components (a), (b) and (c) may be formed by converting a mixture of lactose and an appropriate additional sugar, for example a monosaccharide selected from fucose, arabinose, xylose, rhamnose and mannose, to the oligosaccharides.
  • each of X a , X b and X c are a mixture of Glc and Fuc.
  • oligosaccharides in the composition comprising oligosaccharide compounds of this first aspect may also comprise the saccharide unit “X” groups referred to above.
  • composition of this first aspect comprises (a) at least 8 wt% Gal-(p1-3)-Gal-(p1-4)-X a .
  • composition comprises at least 9 wt% of component (a) or at least 10 wt% of component (a).
  • composition comprises up to 35 wt% of component (a), up to 30 wt% of component (a) or up to 25 wt% of component (a).
  • composition comprises from 8 to 35 wt% of component (a), from 8 to 25 wt% of component (a) or from 10 to 20 wt% of component (a).
  • composition of this first aspect comprises (b) at least 3 wt% Gal-(p1-3)-Gal-(p1-3)-X b .
  • composition comprises at least 4 wt% of component (b) or at least 5 wt% of component (b).
  • composition comprises up to 25 wt% of component (b), up to 20 wt% of component (b) or up to 15 wt% of component (b).
  • composition comprises from 3 to 25 wt% of component (b), from 4 to 20 wt% of component (b) or from 4 to 10 wt% of component (b).
  • composition of this first aspect comprises (c) at least 5 wt% Gal-(p1-3)-Gal-(p1-2)-X c .
  • composition comprises at least 6 wt% of component (c).
  • the composition comprises up to 25 wt% of component (c), up to 20 wt% of component (c) or up to 15 wt% of component (c).
  • the composition comprises from 5 to 25 wt% of component (c), from 5 to 20 wt% of component (c) or from 6 to 10 wt% of component (c).
  • the above amounts are based on the total weight of oligosaccharide compounds present in the composition.
  • component (a) is present in an amount up to 35 wt%; component (b) is present in an amount up to 25 wt%; and component (c) is present in an amount up to 25 wt%; based on the total weight of oligosaccharide compounds present in the composition.
  • component (a) is present in an amount from 8 to 25 wt%; component (b) is present in an amount from 3 to 25 wt%; and component (c) is present in an amount from 5 to 20 wt%; based on the total weight of oligosaccharide compounds present in the composition.
  • composition of this first aspect comprises:
  • the ratio of the wt% of compound (a) to compound (b) is from 1 :1 to 3:1 , suitably from 1 .5:1 to 2.5:1.
  • the ratio of the wt% of compound (a) to compound (c) is from 1 :1 to 3:1 , suitably from 1 .5:1 to 2.5:1.
  • the ratio of the wt% of compound (b) to compound (c) is from 2:1 to 1 :2, suitably from 1 .5:1 to 1 :1.5.
  • the oligosaccharide compounds comprise:
  • X d may be selected from the same monosaccharide units described above forX a , X b and X c .
  • X d is Glc.
  • the Gal saccharide units of component (d) have the D enantiomeric form.
  • the Gal and the X d saccharide units all have the D enantiomeric form.
  • the components (d) may therefore be as follows: D-Gal-(p1-3)-D-Gal-(p1-3)-D-Gal-(p1-4)-D-X d .
  • X d is a mixture of Glc and one or more of other monosaccharide units, for example Fuc, Ara, Xyl, Rha and Man, suitably Fuc.
  • composition comprises at least 4 wt% of component (d) or at least 5 wt% of component (d).
  • composition comprises up to 25 wt% of component (d), up to 20 wt% of component (d) or up to 15 wt% of component (d).
  • composition comprises from 3 to 25 wt% of component (d), from 4 to 20 wt% of component (d) or from 4 to 10 wt% of component (d).
  • component (a) is present in an amount from 8 to 25 wt%; component (b) is present in an amount from 3 to 25 wt%; component (c) is present in an amount from 5 to 20 wt%; and component (d) is present in an amount from 3 to 25 wt%; based on the total weight of oligosaccharide compounds present in the composition.
  • composition of this first aspect comprises:
  • the oligosaccharide compounds comprise:
  • X e may be selected from the same monosaccharide units described above forX a , X b and X c .
  • the Gal saccharide units of component (e) have the D enantiomeric form.
  • the Gal and the X e saccharide units all have the D enantiomeric form.
  • the components (e) may therefore be as follows: D-Gal-(p1-4)-D-Gal-(p1-4)-D-X e .
  • X e is Glc.
  • X e is a mixture of Glc and one or more of other monosaccharide units, for example Fuc, Ara, Xyl, Rha and Man, suitably Fuc.
  • composition comprises at least 6 wt% of component (e) or at least 7 wt% of component (e).
  • composition comprises up to 25 wt% of component (e), up to 20 wt% of component (e) or up to 15 wt% of component (e).
  • composition comprises from 5 to 25 wt% of component (e), from 5 to 20 wt% of component (e) or from 6 to 10 wt% of component (e).
  • component (a) is present in an amount from 8 to 25 wt%; component (b) is present in an amount from 3 to 25 wt%; component (c) is present in an amount from 5 to 20 wt%; component (d) is present in an amount from 3 to 25 wt%; and component (e) is present in an amount from 5 to 25 wt%; based on the total weight of oligosaccharide compounds present in the composition.
  • composition of this first aspect comprises:
  • X a , X b , X c , X d and X e are each independently selected from the monosaccharides described above.
  • X a , X b , X c , X d and X e may each be independently selected from Glc, Fuc, Ara, Xyl, Rha and Man or a mixture thereof.
  • X a , X b , X c , X d and X e are each Glc.
  • X a , X b , X c , X d and X e each comprise Fuc.
  • X a , X b , X c , X d and X e are each a mixture of Glc and Fuc.
  • the oligosaccharide compounds of the compositions of this first aspect suitably have a relatively high proportion of p1 -3 Gal-Gal linkages - mainly due to the presence of the components (a), (b), (c) and optionally (d) in the proportions discussed herein.
  • the inventors have found that such relatively high proportions of p1-3 Gal-Gal linkages may be particularly advantageous for the uses of the composition discussed herein.
  • the Gal-Gal linkages in the oligosaccharide compounds are 1-3 linkages, suitably from 40 to 55%, suitably from 40 to 50%.
  • the Gal-Gal linkages in the oligosaccharide compounds are 1-4 linkages, suitably from 45 to 55%, suitably from 45 to 52%.
  • Suitably from 8 to 20% of the Gal-X linkages in the oligosaccharide compounds are 1-3 linkages, suitably from 10 to 18%, suitably from 12 to 16%, suitably wherein X is Glc.
  • Gal-X linkages in the oligosaccharide compounds are 1-4 linkages, suitably from 16 to 24%, suitably from 17 to 22%, suitably wherein X is Glc.
  • Gal-X linkages in the oligosaccharide compounds are 1-2 linkages, suitably from 32 to 43%, suitably from 34 to 41 %, suitably wherein X is Glc.
  • Gal-X linkages in the oligosaccharide compounds are 1-6 linkages, suitably from 22 to 34%, suitably from 25 to 32%, suitably wherein X is Glc.
  • the Gal-X, linkages referred to above are p linkages, i.e p-glycosidic bonds.
  • the composition comprises at least 25 wt% of trisaccharides, based on the total weight of in the composition.
  • the composition comprises at least 28 wt% of trisaccharides or at least 30 wt% of trisaccharides.
  • the composition comprises up to 70 wt% of trisaccharides, suitably up to 60 wt% trisaccharides or up to 50 wt% trisaccharides.
  • the composition comprises from 25 to 70 wt% of trisaccharides, suitably from 30 to 60 wt% trisaccharides or from 30 to 50 wt% trisaccharides.
  • the composition comprises at least 10 wt% of tetrasaccharides, based on the total weight of in the composition.
  • the composition comprises at least 12 wt% of tetrasaccharides or at least 15 wt% of tetrasaccharides.
  • the composition comprises at least 20 wt% tetrasaccharides or at least 25 wt% tetrasaccharides.
  • the composition comprises up to 40 wt% of tetrasaccharides, suitably up to 25 wt% tetrasaccharides or up to 20 wt% tetrasaccharides.
  • the composition comprises from 10 to 40 wt% of tetrasaccharides, suitably from 10 to 30 wt% tetrasaccharides or from 12 to 25 wt% tetrasaccharides.
  • the composition comprises from 30 to 50 wt% trisaccharides and from 10 to 25 wt% tetrasaccharides, based on the total weight of the composition.
  • the determination of disaccharides present in the composition excludes lactose Therefore in some embodiments the composition suitably comprises up to 40 wt% of disaccharides, suitably up to 30 wt% disaccharides or up to 20 wt% disaccharides. Suitably the content of lactose in the composition of this first aspect is minimised. Suitably the composition is substantially free of lactose. Suitably the composition does not contain lactose.
  • the composition comprises from 10 to 40 wt% of disaccharides, suitably from 10 to 30 wt% disaccharides or from 10 to 20 wt% disaccharides.
  • the composition comprises from 10 to 40 wt% disaccharides, from 30 to 60 wt% trisaccharides and from 10 to 25 wt% tetrasaccharides, based on the total weight of the composition.
  • the oligosaccharide compounds comprise up to 40 wt% of disaccharides, suitably up to 30 wt% disaccharides or up to 20 wt% disaccharides.
  • the oligosaccharide compounds comprise from 0 to 40 wt% of disaccharides, suitably from 10 to 30 wt% disaccharides or from 10 to 20 wt% disaccharides.
  • the oligosaccharide compounds comprise at least 25 wt% of trisaccharides, based on the total weight of oligosaccharide compounds present in the composition.
  • the oligosaccharide compounds comprise at least 30 wt% of trisaccharides or at least 33 wt% of trisaccharides.
  • the oligosaccharide compounds comprise up to 75 wt% of trisaccharides, suitably up to 65 wt% trisaccharides or up to 55 wt% trisaccharides.
  • the oligosaccharide compounds comprise from 25 to 75 wt% of trisaccharides, suitably from 30 to 65 wt% trisaccharides or from 34 to 55 wt% trisaccharides.
  • the oligosaccharide compounds comprise at least 10 wt% of tetrasaccharides, based on the total weight of oligosaccharide compounds present in the composition.
  • the oligosaccharide compounds comprise at least 12 wt% of tetrasaccharides or at least 15 wt% of tetrasaccharides.
  • the oligosaccharide compounds comprise up to 45 wt% of tetrasaccharides, suitably up to 35 wt% tetrasaccharides or up to 30 wt% tetrasaccharides.
  • the oligosaccharide compounds comprise from 10 to 45 wt% of tetrasaccharides, suitably from 10 to 35 wt% tetrasaccharides or from 15 to 30 wt% tetrasaccharides.
  • the oligosaccharide compounds comprise from 0 to 40 wt% disaccharides and from 30 to 75 wt% trisaccharides, based on the total weight of oligosaccharide compounds present in the composition.
  • the oligosaccharide compounds comprise from 0 to 40 wt% disaccharides, from 30 to 75 wt% trisaccharides and from 10 to 45 wt% tetrasaccharides, based on the total weight of oligosaccharide compounds present in the composition.
  • composition of this first aspect suitably comprises at least 50 wt% of oligosaccharide compounds, including components (a), (b), (c) and optionally (d) and (e).
  • composition comprises at least 55 wt% of oligosaccharide compounds, suitably at least 60 wt%, based on the total weight of the composition.
  • the composition comprises up to 100 wt% oligosaccharide compounds, suitably up to 95 wt%, up to 90 wt% or up to 85 wt% oligosaccharide compounds.
  • the composition comprises from 50 to 100 wt% oligosaccharide compounds, suitably from 55 to 95 wt%, or from 60 to 85 wt% oligosaccharide compounds.
  • the composition is in the form of a syrup.
  • the syrup suitably comprises at least 50 wt% of oligosaccharide compounds, at least 55 wt% of oligosaccharide compounds, or at least 60 wt%.
  • the syrup comprises from 50 to 75 wt% of oligosaccharide compounds, suitably from 55 to 70 wt% or from 60 to 70 wt% oligosaccharide compounds.
  • the syrup may comprise a significant amount of monosaccharides, for example glucose and/or galactose.
  • the syrup may comprise from 15 to 30 wt% monosaccharides, suitably from 20 to 28 wt% monosaccharides or from 21 to 25 wt% monosaccharides, for example glucose and/or galactose.
  • the syrup suitably comprises from 20 to 30 wt% water, suitably from 22 to 28 wt% water.
  • the syrup may also contain lactose, for example 4 to 14 wt% lactose.
  • the composition is in the form of a powder.
  • the powder suitably comprises at least 60 wt% of oligosaccharide compounds, suitably at least 70 wt% or at least 75 wt% of oligosaccharide compounds.
  • the powder comprises from 60 to 100 wt% of oligosaccharide compounds, suitably from 70 to 95 wt% or from 75 to 90 wt% oligosaccharide compounds.
  • the powder suitably comprises a reduced amount of monosaccharides, for example glucose and/or galactose, compared to the syrup discussed above.
  • the powder may comprise from 1 to 10 wt% monosaccharides, suitably from 2 to 8 wt% monosaccharides or from 3 to 7 wt% monosaccharides, suitably approximately 5 wt%, for example of glucose and/or galactose.
  • the powder suitably comprises from 1 to 10 wt% water, suitably from 3 to 6 wt% water.
  • the composition of this first aspect may have been purified to remove monosaccharides and optionally disaccharides from the composition.
  • the composition of this first aspect may have been fractionated to separate the oligosaccharide components of the composition according to their molecular weight, for example to remove disaccharides from the composition or to isolate trisaccharides from 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 a fractionation may be referred to as an oligosaccharide fraction or a GOS fraction.
  • the composition suitably comprises at least 70 wt% of trisaccharides, tetrasaccharides and higher oligosaccharides, suitably at least 80 wt% or at least 90 wt%.
  • Such higher oligosaccharides have a degree of polymerisation of 5 and above.
  • the composition comprises at least 95 wt% of trisaccharides, tetrasaccharides and higher oligosaccharides.
  • the composition consists or consists essentially of trisaccharides, tetrasaccharides and higher oligosaccharides.
  • the composition suitably comprises from 40 to 70 wt% of trisaccharides, suitably from 45 to 70 wt% trisaccharides or from 50 to 70 wt% trisaccharides.
  • the oligosaccharide compounds comprise from 15 to 50 wt% of tetrasaccharides, suitably from 15 to 40 wt% tetrasaccharides or from 20 to 40 wt% tetrasaccharides.
  • the oligosaccharide compounds comprise from 5 to 25 wt% of higher oligosaccharides, suitably from 5 to 20 wt% higher oligosaccharides or from 10 to 20 wt% higher oligosaccharides.
  • the composition comprises from 40 to 70 wt% trisaccharides, from 15 to 40 wt% tetrasaccharides and 5 to 25 wt% of higher oligosaccharides, based on the total weight of the composition.
  • the composition comprises from 50 to 70 wt% trisaccharides, from 20 to 40 wt% tetrasaccharides and from 10 to 20 wt% higher oligosaccharides, based on the total weight of the composition.
  • composition suitably comprises the components (a), (b) and (c) as described above in the following amounts:
  • composition (or oligosaccharide fraction) comprises the components (a), (b) and (c) as described above in the following amounts:
  • composition may comprise components (d) and/or component (e) as described above.
  • composition suitably comprises said components in the ratios discussed above.
  • the composition of this first aspect is a trisaccharide and tetrasaccharide fractionated product (which may be referred to as a DP3/DP4 fraction).
  • the composition (or DP3/DP4 fraction) suitably comprises at least 70 wt% of trisaccharides and tetrasaccharides, suitably at least 80 wt% or at least 90 wt%.
  • the composition consists or consists essentially of trisaccharides and tetrasaccharides.
  • the composition suitably comprises from 50 to 80 wt% of trisaccharides, suitably from 55 to 75 wt% trisaccharides or from 60 to 75 wt% trisaccharides.
  • the oligosaccharide compounds comprise from 20 to 50 wt% of tetrasaccharides, suitably from 25 to 45 wt% tetrasaccharides or from 25 to 40 wt% tetrasaccharides.
  • the composition comprises from 50 to 80 wt% trisaccharides and from 20 to 50 wt% tetrasaccharides, based on the total weight of the composition.
  • the composition comprises from 60 to 75 wt% trisaccharides and from 25 to 40 wt% tetrasaccharides, based on the total weight of the composition.
  • composition suitably comprises the components (a), (b) and (c) as described above in the following amounts:
  • composition (or oligosaccharide fraction) comprises the components (a), (b) and (c) as described above in the following amounts:
  • composition may comprise components (d) and/or component (e) as described above.
  • composition suitably comprises said components in the ratios discussed above.
  • the composition of this first aspect is a trisaccharide fractionated product (which may be referred to as a DP3 fraction).
  • a trisaccharide fractionated product may be obtained by the known fractionation methods referred to above.
  • Such a composition suitably comprises at least 70 wt% of trisaccharides, suitably at least 80 wt% or at least 90 wt%.
  • the composition comprises at least 95 wt% of trisaccharides.
  • composition suitably comprises the components (a), (b) and (c) as described above in the following amounts:
  • composition or oligosaccharide fraction
  • the composition comprises the components (a), (b) and (c) as described above in the following amounts:
  • composition may comprise component (e) as described above.
  • composition suitably comprises said components in the ratios discussed above.
  • composition of this first aspect may be for use as, and incorporated into, a food supplement product for ingestion by a consumer
  • a food supplement product for ingestion by a consumer
  • a product may be selected from the group consisting of dairy products (for example, liquid milk, dried milk powder such as whole milk powder, skimmed milk powder, fat filled milk powders, whey powders, infant formula, ice cream, yoghurt, cheese, fermented dairy products), beverages, sport drinks, infant foods, cereals, bread, biscuits, confectionary, cakes, food supplements, dietary supplements, medical food/nutrition, food for specific medical purposes, animal feeds, poultry feeds or indeed any other food or beverage.
  • dairy products for example, liquid milk, dried milk powder such as whole milk powder, skimmed milk powder, fat filled milk powders, whey powders, infant formula, ice cream, yoghurt, cheese, fermented dairy products
  • beverages sport drinks, infant foods, cereals, bread, biscuits, confectionary, cakes, food supplements, dietary supplements, medical food
  • composition of this first aspect may be incorporated into a synbiotic composition.
  • a synbiotic composition is suitably a mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefit on the host, i.e. a probiotic and a prebiotic.
  • composition of this first aspect may be, for use as, and in the form of, a pharmaceutical or nutraceutical composition comprising at least one carrier, excipient, or diluent.
  • the composition may be administered in a single dose or in multiple doses.
  • a suitable frequency of administration may be at least once per day, every other day, once per week, once every two, three, or four weeks, once every month, two months, or once every three to six months.
  • the composition may be administered over a period of at least a week, at least a month, at least three to six months, at least one, two, three, four, or five years, or over the course of the disease, or the lifetime of the subject.
  • compositions of the invention can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers, pharmaceutically acceptable diluents, or pharmaceutically acceptable excipients, and can be formulated into preparations in solid, semi-solid, or liquid forms, such as tablets, capsules, powders, granules and solutions.
  • Pharmaceutically acceptable carriers, excipients, or diluents may include, for example: water, saline, dextrose, maltodextrin, glycerol, ethanol, a salt, e.g., NaCI, MgCh, KCI, MgSC , etc.; a buffering agent, e.g., a phosphate buffer, a citrate buffer, a 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), etc.; a solubilizing agent; a detergent,
  • Pharmaceutically acceptable carriers, excipients and diluents are nontoxic to recipients at the dosages and concentrations employed, and can for example include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor- m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, 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)
  • the composition of the invention may include appropriate additives to make tablets, powders, granules or capsules, for example, with 30 conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
  • lubricants such as talc or magnesium stearate
  • the pharmaceutical composition can be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration.
  • the standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equivalent to the volume removed during lyophilization).
  • a tonicity agent can be included in the formulation to modulate the tonicity of the formulation.
  • exemplary tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof.
  • the aqueous formulation is isotonic, although hypertonic or hypotonic solutions can be suitable.
  • isotonic denotes a solution having the same tonicity as some other solution with which it is compared, such as a physiological salt solution or serum.
  • the composition may modulate the abundance of a bacterial genus present in the gut. In some embodiments, the composition modulates the abundance of a bacterial genus present in one or both of the small intestine or large intestine. In some embodiments, the composition modulates the abundance of a bacterial genus predominant in the small intestine selected from the group of genus Achromobacter, Agrobacterium, Blautia, Burkholderia, Coprococcus, Cryocola, Enterococcus, Eubacterium, Holdemania, Lactococcus, Mycobacterium, Pseudoramibacter, Ralstonia, Sphingomonas, Streptococcus, and Turicibacter.
  • a composition comprising oligosaccharide compounds according to the first aspect, the method comprising the steps of:
  • step (i) the steps of the method of this second aspect are carried out in the order step (i) followed by step (ii).
  • the source of saccharide compounds comprises lactose, lactulose or epilactose.
  • the source of saccharide compounds comprises lactose.
  • the source of saccharide may be lactose, for example a lactose syrup which may be derived from cow’s milk.
  • the lactose may be heat treated.
  • no further saccharides such as monosaccharides or disaccharides are added to the source of saccharide.
  • the method produces galactooligosaccharide compounds.
  • the source of saccharide compounds comprises at least one additional saccharide.
  • the at least one additional saccharide provides the oligosaccharide compounds with an alternative terminal monosaccharide unit, as discussed above.
  • the at least one additional saccharide is a source of such a monosaccharide unit.
  • the at least one additional saccharide which is a source of such a monosaccharide unit may be a monosaccharide or may be a higher sugar, such as a disaccharide.
  • the at least one additional saccharide may 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 (Tai), psicose (Psi), fructose (Fru), sorbose (Sor), tagatose (Tag), galactosamine (GalN), glucosamine (GlcN) and N-Acetylglucosamine (GIcNAc) or a mixture thereof.
  • the at least one additional saccharide may be one or more of the monosaccharides listed above.
  • the method produces oligosaccharides having one or more of the above monosaccharides as the terminal sugar unit.
  • the at least one additional saccharide may be selected from Fucose, Arabinose, Xylose, Rhamnose, Mannose or mixtures thereof.
  • the source of saccharides may comprise lactose and one or more sources of said monosaccharides.
  • the method produces oligosaccharides having as a terminal sugar unit selected from Glc, Fuc, Ara, Xyl, Rha and Man, or mixtures thereof.
  • Step (ii) of the method involves treating the source of saccharide compounds with at least one galactosidase enzyme.
  • the galactosidase enzyme may be an alpha- or beta-galactosidase enzyme, depending on whether alpha or beta linkages between the saccharide units of the oligosaccharide compounds is required.
  • the enzyme exhibits galactosyltransferase (transgalactosidic) activity and forms alpha- or beta- linkages between sugar units in the source of saccharide compounds. This results in the synthesis of oligosaccharide compounds with two or more galactose units derived from lactose.
  • step (ii) is carried out until conversion of the source of saccharides to the oligosaccharide compounds is complete.
  • Step (ii) may involve treating the source of saccharide compounds with one or more additional enzymes which are not galactosidase enzymes.
  • the method comprises a step (iii) of separating the galactosidase enzyme from the composition comprising oligosaccharide compounds.
  • Step (iii) may involve removing the enzyme by filtration, for example by nanofiltration.
  • composition comprising oligosaccharide compounds produced in step (iii) may be heat treated.
  • the composition is evaporated to reduce the water content to provide the final composition comprising oligosaccharide compounds as a syrup, as discussed above in relation to the first aspect.
  • glucose is removed from the composition produced by step (iii) before evaporation. This suitably lowers the glucose content of the composition from 20-30 wt% to below 10 wt%, suitably approximately 5 wt% or lower.
  • the water content of the composition is then reduced by evaporation and the product dried to provide the final composition comprising oligosaccharide compounds as a powder, as discussed above in relation to the first aspect.
  • composition comprising oligosaccharide compounds according to the first aspect, i.e. containing the specified amounts of particular oligosaccharide compounds, by combining said oligosaccharides obtained and isolated from different sources in the required amounts.
  • Figure 1 is a graph illustrating the results of the butyrate analysis across three donors after being administered the oligosaccharide of the present invention alongside a comparative oligosaccharide in Comparative Example 1 .
  • Figure 2 are graphs showing the overall microbial community activity (acidification and gas production) shown as (A) pH and (B) gas pressure. Measurements were collected in triplicate in Example 4. Data for average values were derived using data from Donors A/B/C. Error bars represent standard deviation.
  • GOS Bimuno® galactooligosaccharides.
  • Figure 3 are graphs showing the individual microbial community activity (acidification and gas production) shown as (A) pH and (B) gas pressure across all donors in Example 4.
  • Figure 7 are plots showing the Principal Coordinate Analysis of species data using Bray-Curtis distance for each donor at (A) 6h after treatment (relative data) and (B) 24h after treatment (absolute data) in Example 4. Each dot represents one replicate.
  • Figure 8 are graphs showing changes in the microbial community composition in Example 4 (A) relative abundance (family level) 6h after treatment, (B) linear discriminant analysis effect size of relative abundances (species level) 6h after treatment, (C) boxplots for the most important enrichments (species level) 6h after treatment, (D) absolute abundances (family level) 24h after treatment, (E) linear discriminant analysis effect size of absolute abundances (species level) 24h after treatment, (F) boxplots for the most important enrichments (species level) 24h after treatment.
  • LDA linear discriminant analysis.
  • Figure 9 are graphs showing microbial metabolic activity on different moments of the incubation in Example 5 (error bars reflect STDEV for individual donors and SEM for averages across donors)
  • A acetate,
  • B propionate,
  • C butyrate,
  • D total SCFA,
  • E lactate and
  • F ammonium, where two conditions (B-GOS and a commercially available GOS (denoted “GOS-2”)) were tested and a negative control (blank) was included.
  • GOS commercially available GOS
  • a composition comprising oligosaccharide compounds according to the present invention, in the form of a syrup, was obtained by the following procedure. Lactose was rehydrated with potable water to give a working solution of between 35-65 wt% solids. The lactose solution was heat treated then cooled to 40-65°C. The pH of the solution was adjusted to pH 5.5-7.5. A beta-galactosidase enzyme was then added to the solution in a closed vessel and subsequently allowed to react with the lactose to catalyse the transfer of galactose molecules to produce oligosaccharide compounds. The progress of the reaction was monitored by measuring the generation of glucose. The reaction was allowed to proceed for between 8 and 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 22-28 wt% to provide the product as a syrup.
  • a composition comprising oligosaccharide compounds according to the present invention, in the form of a powder, was obtained by a modification of the procedure described above. After removal of the enzyme, the mixture was further filtered to remove a significant portion of the glucose and other monosaccharides, reducing the monosaccharide content from around 23 wt% to around 5 wt%. The water content of the composition was then reduced by evaporation and the product dried to provide a powder having a water content to approximately 3-6 wt%.
  • a commercially available composition comprising oligosaccharide compounds was obtained in powder form.
  • Example 2 of the present invention Samples of Example 2 of the present invention and Comparative Example 1 were analysed to determine their oligosaccharide content by the following procedure.
  • Samples of dry powder of each of Example 2 and Comparative Example 1 were dissolved in water to provide solutions having a concentration of 40 g/l for analysis.
  • HPAEC-PAD high performance anion exchange chromatography
  • Table 1 shows DP (degree of polymerisation) composition results for the samples using Rezex-RSO system, based on Rl calibration with glucose (values expressed as g/L in the samples). All material eluting in the >DP5 window was combined. Table 1
  • Table 2 shows information on HPAEC-PAD peak areas of all annotated peaks together with information on incubation conditions, sample concentration, dilution and injection volume as shown.
  • the peak areas were assumed to approximately correspond to the amount of each oligosaccharide compound present in the composition. Where a particular oligosaccharide compound was not identified then “unknown” and a number is entered in the table. The identified compounds are either identified by name or by a number which corresponds to the number assigned to particular galactooligosaccharides in van Leeuwen et al., Carbohydrate Research 2016, 425, 48-58. A list of these galactooligosaccharides and their corresponding numbers is provided below.
  • butyrate is produced by gut microbiota which convert acetate and/or lactate (along with other substrates) to butyrate. As butyrate can be a secondary metabolite, it is often produced during late stages of fermentation as butyrate can be directly produced by certain bacterial butyrate producers.
  • Example 2 of the present invention Comparative Example 1 and a control blank sample were subjected to dialysis using a 0.5 kDa membrane to provide 5 g/l samples which were then mixed with faecal matter obtained from three healthy human adult subjects (donors A, B and C).
  • the mixtures were shaken under anaerobic conditions and monitored over a 48 hour period for colonic fermentation products including butyrate (with 6, 24 and 48 h collection points).
  • the distribution of oligosaccharides in the mixtures was also monitored over this time period using the method described above in relation to Table 2.
  • Example 2 was well fermented by all donors, mainly during the time period 0-24 hours and that butyrate production increased compared to the Comparative Example 1 and the control at the 6 and 48 hour time points.
  • the results of the butyrate analysis are shown in Figure 1.
  • the results of the oligosaccharides analysis for the samples at the different time points are shown in Table 3. These results show that the oligosaccharides in the samples were actively consumed by the microbiota present in the faecal samples during the experiments.
  • the present invention provides a composition comprising oligosaccharide compounds, for example galactooligosaccharide compounds, which includes: (a) at least 8 wt% Gal-(p1-3)-Gal- (p1-4)-X a ; (b) at least 3 wt% Gal-(p1-3)-Gal-(p1-3)-X b ; and (c) at least 5 wt% Gal-(p1-3)-Gal-(p1-2)- X c , based on the total weight of oligosaccharide compounds present in the composition; wherein X a , X b and X c are each independently selected from monosaccharides.
  • oligosaccharide compounds for example galactooligosaccharide compounds, which includes: (a) at least 8 wt% Gal-(p1-3)-Gal- (p1-4)-X a ; (b) at least 3 wt% Gal-(p1-3)-Gal-(p1-3)
  • compositions contain relatively high amounts of the oligosaccharide compounds (a), (b) and (c) and a relatively high amount of p1-3 Gal-Gal linkages, compared to known oligosaccharide compositions.
  • the compounds present in the composition showed a belated effect of butyrate increase over time which is believed to be due to the cross-feeding of other bacteria which are acetate-producers and this acetate in turn stimulates butyrate producers to growth and secrete butyrate.
  • These particular features of the composition are believed to provide benefits to the gut health of a consumer, for example due to these compositions providing an increased production of butyrate in the gut of a consumer compared to known compositions.
  • B-GOS GOS of the present invention
  • Evaluation was based on the effects on overall microbial fermentation (pH, gas production), microbial metabolic activity (production of SOFA, lactate, and ammonium), and community composition using shallow shotgun sequencing.
  • B-GOS utilization by gut microbiota was analyzed to understand the dynamics of this process.
  • fecal suspensions were prepared and mixed with a cryoprotectant. Suspensions were then aliquoted, flash frozen, and stored at -80°C until needed.
  • the GOS composition of the present invention (herein also referred to as B-GOS”) was dialyzed prior to colonic incubation to simulate absorption during small intestinal passage.
  • Stock solutions of B- GOS were prepared in water at 40 g/L which were then added inside dialysis membranes (0.5 kDa pore size) to allow for monosaccharides and disaccharides to pass through the membrane. Membranes were sealed and the stock solution was dialyzed in a solution of NaHCOs (3.75 g/L, pH 7.0) for 24h at a low temperature to prevent microbial growth.
  • Short-term colonic incubations were performed as previously described in Van den Abbeele, et. al. 2018 (Different oat ingredients stimulate specific microbial metabolites in the gut microbiome of three human individuals in vitro. ACS Omega 2018, 3, 12446-12456). Briefly, individual reactors were filled with sugar-depleted nutritional medium containing basal colonic nutrients. Next, dialyzed B-GOS (5 g/L final concentration) or blank medium was added followed by fecal inoculum. Incubations were performed in triplicate for B-GOS and blank (media control) and for each donor (six incubations per donor, three with B-GOS and three blank). Samples were collected at Oh, 6h, 24h, and 48h. Shallow shotgun sequencing and flow cytometry (cell counts) were performed on samples collected at 6h and 24h.
  • Lactate levels were monitored using a commercially available enzymatic assay kit (R-Biopharm, Darmstadt, Germany) according to the manufacturer’s instructions. Ammonium was analyzed as described by Van de Wiele et al. 2004 (Prebiotic effects of chicory inulin in the simulator of the human intestinal microbial ecosystem. FEMS Microbiol Ecol 2004, 51, 143-153).
  • DNA libraries were prepared using the Illumina Nextera XT library preparation kit, with a modified protocol. Library quantity was assessed with Qubit (ThermoFisher). Libraries were then sequenced on an Illumina HiSeq platform 2 x 150bp. Unassembled sequencing reads were directly analyzed according to 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.
  • Hasan et al. 2014 Microbial community profiling of human saliva using shotgun metagenomic sequencing. PLoS One 2014, 9, e97699), and Lax et al. 2014 (Longitudinal analysis of microbial interaction between humans and the indoor environment. Science 2014, 345, 1048- 1052) for multi-kingdom microbiome analysis and quantification of relative abundances. Briefly, curated genome databases were utilized in combination with a high-performance data-mining algorithm that rapidly disambiguates hundreds of millions of metagenomic sequence reads into the discrete microorganisms engendering the sequences.
  • the total number of bacterial cells was determined using a BD FACSVerse Cell Analyzer (BD Biosciences, Franklin Lakes, NJ, USA) on the high flow rate setting with a threshold of 200 on the SYTO channel. Proportional values obtained using shotgun sequencing were converted to absolute quantities by multiplying relative abundances of each population in a sample with the total cell count obtained by flow cytometry.
  • GOS chain length distribution analysis was performed using undialed B-GOS, dialyzed B-GOS and samples obtained at all fermentation timepoints (6, 24 and 48 hours).
  • B-GOS chain length distribution was performed using gel permeation chromatography (GPC).
  • a HPLC equipped with a Rezex RSO and a Rl detector and in-line desalting (for removal of salts and charged material like proteins) was applied for the aqueous GPC separation.
  • the separation was performed at elevated temperature (80°C).
  • the separation range of the Rezex RSO column was ranging from DP1 (monosaccharide) up to about DP10.
  • the FDR was set at 0.10, meaning that the lowest p-value should be below 0.033 to be significantly different, the second lowest below 0.066, etc.
  • Comparisons of the absolute and relative abundances of specific members of the microbial community were conducted using analysis of variance (ANOVA). Linear discriminant analysis (LDA) effect size analysis (LEfSe) was conducted to detect between group differences in bacterial abundances. A p-value of ⁇ 0.05 was considered statistically significant.
  • PCoA Principle coordinates analysis
  • FIG 8A Average relative abundances for the microbial community composition at the family level at 6h are shown in Figure 8A.
  • Treatment with B-GOS resulted in a significant decrease in the relative abundance for several families.
  • LEfSe revealed a high LDA score of >4 for Bifidobacterium longum with B-GOS treatment at 6h (relative abundance) ( Figure 8B).
  • B-GOS Using short-term colonic incubations, the effects of B-GOS on the overall microbial fermentation was assessed along with microbial metabolic activity, and microbial community composition of colonic bacteria isolated from healthy donors. B-GOS had notable effects on these parameters, including increased SOFA production and increased abundance of beneficial bacteria. It has been also noted that almost 95% B-GOS was taken up by gut microbiota already by 24 hours.
  • B-GOS increased the production of acetate, propionate, butyrate, and lactate.
  • This result agrees with previous in vitro studies using fecal batch cultures that reported an increased production of acetate with B-GOS supplementation, an increase in acetate and lactate with B-GOS supplementation, and an increased production of lactate and all SCFAs, particularly butyrate, with B-GOS supplementation.
  • the increased butyrate production was quite marked in our study and could be observed as early as 6h to 24h, demonstrating a maximum increase at 24h to 48h.
  • the marked increase in butyrate production was likely largely driven by lactate-to-butyrate conversion, which would explain the consumption of lactic acid after 6h.
  • Acetate- to-butyrate conversion may also have contributed to the increase in butyrate.
  • Butyrate is preferentially consumed by colonocytes, making it an important component in gut membrane health.
  • butyrate plays an important role in regulating the integrity of the epithelial barrier via coordinated regulation of tight junction proteins. The importance of this function is highlighted by the fact that loss of barrier integrity is thought to contribute to metabolic disorders, inflammatory bowel disease, and obesity.
  • B-GOS supplementation was shown to improve intestinal barrier function.
  • B-GOS stimulated health-related microbial metabolites, confirming its usefulness as a prebiotic, and, unexpectedly, demonstrating its beneficial effects on unfavorable metabolites such as ammonium.
  • ammonium is a marker of proteolytic fermentation
  • a reduction indicates that the level of proteolytic fermentation was low with B-GOS, indicating a shift in the fermentation pattern to one more beneficial for the host.
  • This may be considered beneficial, as some metabolic derivatives of proteolytic fermentation are implicated in disease, including colorectal cancer. Avoiding proteolytic fermentation is considered beneficial, as highly toxic compounds may be produced during this process. This is especially important for individuals on a high protein diet due to increased protein loads and increased proteolytic fermentation.
  • B-GOS supplementation increases the growth of bifidobacteria and lactobacilli and studies in humans have demonstrated that B-GOS supplementation results increased bifidobacteria and lactobacilli in healthy adults and elderly.
  • Bifidobacteria and lactobacilli are well known for their role in human health. The increase in both was likely responsible for augmenting the observed increase in butyrate production via lactate production. Thus, the observed increase in bifidobacteria and Lactobacillaceae support the prebiotic effects of B-GOS.
  • a role for Megamonas in health or disease has not been clearly defined, so it is difficult to speculate on the implications of this finding.
  • Donor A produced very little propionate and there was no difference in propionate production between blank or B-GOS supplementation. However, this donor produced a much higher amount of butyrate with B-GOS supplementation than the other two donors. In addition, lactate production and consumption occurred to a greater degree in Donor A than the others. We observed an enrichment of Streptococcaceae for Donor A upon B-GOS supplementation. This may have contributed to the early strong increase in lactate production (0-6h) and subsequent conversion into butyrate (24-48h).
  • B-GOS demonstrated prebiotic activity in short-term colonic incubations using the colonic microbiota of three healthy adult donors. Supplementation resulted in an increase in lactic acid and SOFA production, including butyrate, and increased growth of the beneficial bacterial families, Bifidobacteriaceae and Lactobacillaceae, while for the first time it was demonstrated that B- GOS reduced unfavorable metabolites such as ammonium.
  • Example 5 Comparative analysis of the effect of B-GOS with commercially available GOS for the reduction of ammonium and pH in a short-term colonic fermentation model
  • Figure 9B shows that both products yielded significantly higher propionate levels than the blank (strongest effects in donors B and C).
  • GOS-2 yielded slightly higher propionate concentrations than B-GOS. Fermentation in donor C was characterized by strong propionate production between 0-6h, in donor B by strong propionate production between 6-24h.
  • the aim of the experiments 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).
  • GOS-2 commercially available GOS Powder
  • effects on microbial metabolic activity were studied, using the gut microbiota of three adult donors.
  • compositions consisting essentially of a set of components will comprise less than 5% by weight, typically less than 3% by weight, more typically less than 1 % by weight of non-specified components.
  • oligosaccharide compounds comprise up to 35 wt% of disaccharides
  • 35 wt% of the oligosaccharide compounds in the composition is provided by disaccharides.

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Abstract

L'invention concerne une composition d'oligosaccharide prébiotique destinée à être utilisée pour réduire le niveau d'ammoniac. La composition comprend des composés du type oligosaccharide, par exemple des composés du type galacto-oligosaccharide, comprenant : (a) au moins 8 % en poids de Gal-(β1-3)-Gal-(β1-4)-Xa ; (b) au moins 3 % en poids de Gal-(β1-3)-Gal-(β1-3)-Xb ; et (c) au moins 5 % en poids de Gal-(β1-3)-Gal-(β1-2)-Xc, par rapport au poids total des composés du type oligosaccharide présents dans la composition ; Xa, Xb et Xc étant chacun indépendamment choisis parmi des monosaccharides. Lesdites compositions contiennent des quantités relativement élevées des composés du type oligosaccharide (a), (b) et (c) et une quantité relativement élevée de liaisons Gal-Gal β1-3, par comparaison avec les compositions d'oligosaccharide connues. La présente invention concerne également une composition comprenant des composés oligosaccharidiques destinés à être administrés aux athlètes et/ou aux personnes sportives qui pratiquent et/ou sont sur le point de pratiquer un entraînement physique, et en particulier les athlètes et/ou les sportifs qui suivent un régime riche en protéines. Une méthode de préparation de ladite composition et l'utilisation de ladite composition en tant que complément alimentaire ou médicament sont également divulgués.
PCT/GB2023/052652 2022-10-12 2023-10-12 Composition d'oligosaccharide destinée à être utilisée dans la réduction du niveau d'ammoniac WO2024079471A1 (fr)

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