RU2799081C2 - Prebiotic for the treatment of disorders associated with disturbed composition or functionality of the intestinal microbiome - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: group of inventions can be used for therapeutic or prophylactic treatment of a disorder associated with a violation of the intestinal microbiome in a patient. The following is proposed: the use of rhamnogalacturonan-I (RG-I) polysaccharides in the preparation of a prebiotic composition for the therapeutic or prophylactic treatment of diseases selected from the following: overweight, obesity, insulin resistance and dysfunction of the intestinal barrier, where the composition includes at least 0.1 wt.% dry matter of RG-I polysaccharides derived from fruit, carrot, pea, chicory, sugar beet, okra, or combinations thereof, wherein said RG-I polysaccharides have a molecular weight greater than 15 kDa and have a backbone consisting of galacturonic acid residues and rhamnose residues, the said residues rhamnose is contained in the residues of alpha(1→4)-galacturonic-alpha(1→2)-rhamnose, where the molar ratio of galacturonic acid residues and rhamnose residues in RG-I polysaccharides is in the range of 20:1–1:1. A synbiotic composition is also proposed, comprising at least 0.1 wt.% of the dry matter of the indicated rhamnogalacturonan I (RG-I) polysaccharides and 10⁴-10¹⁰ CFU/g Akkermansia muciniophila and/or 10⁶-10¹⁰ CFU/g Bifidobacterium spp.

EFFECT: enteric fermentation of RG-I polysaccharides produces beneficial short-chain fatty acids, reduces gas production, and stimulates the growth or activity of beneficial bacteria.

12 cl, 2 dwg, 11 tbl, 7 ex

Description

FIELD OF THE INVENTION

The present invention relates to a method for the therapeutic or prophylactic treatment of disorders associated with a disruption in the composition or functionality of the gut microbiome in a patient, the disorder is selected from a metabolic disorder and gut barrier dysfunction, said method comprising oral administration of a prebiotic composition to a patient, said prebiotic composition comprising the polysaccharide rhamnogalacturonan I (RG -I) derived from fruits, carrots, peas, chicory or sugar beets.

The invention further relates to a prebiotic composition and a synbiotic composition suitable for use in the aforementioned treatment method.

BACKGROUND OF THE INVENTION

The relationship between the gut microbiota and human health is being progressively studied. It is now established that a healthy gut microbiota is largely responsible for the overall health of the host organism.

The human host provides habitat and nutrition for a large and diverse ecosystem of microbial communities that play a key role in digestion, metabolism, and modulation of immune function and have a significant impact on the gastrointestinal tract. Changes in the diversity and function of these communities are associated with far-reaching consequences for host health and are associated with a number of disorders, including functional bowel disorders, inflammatory bowel disease and other immune-mediated disorders (celiac disease, allergies) and metabolic disorders (type 2 diabetes, NASH) ).

Dysbiosis (also called dysbacteriosis) is a term for a microbial imbalance or maladjustment on or within the body, such as a disturbed microbiota. For example, bacterial communities occupying certain areas of the host surface, such as the gut microbiota, skin microbiota, or vaginal microbiota, may become modified/altered with insufficient presence of normally dominant species, and normally competitive or contained species increase to undesirable levels. The set of bacteria in such communities is collectively referred to as the microbiota. The local microbiota, together with microorganisms including yeasts, fungi, viruses and parasites remaining in such niches, is called the microbiome. Dysbiosis is not limited to imbalances in the microbiota, but can also include other microorganisms in the microbiome (e.g., viruses, archaebacteria, and fungi).

When the microbiome is well balanced, called normobiosis, microorganisms occupying a specific niche form a more or less stable community that is well adapted to local conditions, has the metabolic ability to live on the available substrate, effectively interacts with stressors and changes in substrate availability, and has regulatory mechanisms. in situ, which contribute to the metastability of the community and are involved in maintaining the health of the host.

Dysbiosis is most commonly studied as a condition in the gastrointestinal tract, but it can affect any body cavity, mucosal surface, and skin that is inhabited by a microbial community.

Dysbiosis is associated with host diseases; intestinal dysbiosis, for example, is associated with inflammatory bowel disease, chronic fatigue syndrome, obesity, cancer, cardiometabolic conditions, insulin insensitivity, (pre)diabetes, bacterial vaginosis, and colitis.

The composition and stability of the microbiota is influenced by the host's genetic predisposition, environmental conditions or stressors, which include, for example, diet, lifestyle, drug use - such as antibiotics - and developmental stage (age) of the host. This translates into a constant and complex interaction between the host and the main components of the local microbial ecosystem. Such components include the microbiota, the host immune system, the local epithelial barrier and, in the case of the gut, the enteric nervous system.

The newborn microbiota is self-established from birth and changes markedly during the first weeks and months, becoming similar to the nuclear microbiota of an adult around 3-4 years of age. This progressive microbial colonization of the gut is key to the learning and maturation of host immunity and the enteric nervous system, intestinal barrier and function, and host metabolic programming, all of which influence short-term and later in life health status and disease risk. The microbiota of the newborn is affected by maternal diet and microbiota, type of delivery, infant nutrition (breastfeeding or formula), and environmental conditions.

Conversely, the nuclear microbiota of a healthy adult is more stable in the sense that when exposed to various stressors (e.g., antibiotics or drugs), it returns close to its original composition in healthy subjects, which is referred to as microbiota persistence. Loss of variability, lack or low enrichment of beneficial microorganisms, and loss of persistence are associated with diseases.

The microbiota of the elderly or aged differs from that of the healthy adult population in compositional changes resulting in less bacterial diversity, a decrease in beneficial microorganisms, and reduced persistence. All such changes are associated with changes in health status.

The microbiota consists of a wide variety of species that compete for space and resources/nutrients, but can also feed each other with their respective fermentation products, thus leading to a community of microorganisms that live in symbiosis with the mammalian host in a healthy state. High microbial diversity in the gut microbiota is considered beneficial to host health, as diversity makes the microbiota more resistant to factors that disrupt the gut microbiota (eg, antibiotics, dietary changes, invasion by new species). Also important is the association of microbes, i.e. metastable combinations of different microorganisms that feed each other with fermentation products and together form a more or less stable ecosystem that thrives in a specific niche using available substrate (e.g. mucus, cell debris and nutrition).

The typical microbial species found on or within the body are mostly beneficial or harmless. Pathobionts or even pathogens are also part of the “normal” microbiota as long as they remain below critical levels. The mammalian gut microbiota has a number of beneficial and indispensable functions such as aiding in digestion, providing energy from food, providing host-specific (micro)nutrients, producing key metabolites such as short chain fatty acids, and learning (newborns and infants) or maintenance ( adults) of the host immune system. They also help protect the body from invading pathogenic microbes or toxic compounds.

Microbial species also excrete many different types of by-product waste. With the use of various waste disposal mechanisms, under normal circumstances, the body efficiently handles such by-products with little or no problems. Unfortunately, excess microbial populations and inappropriate dominance of microbial species due to their increased abundance excrete increased amounts of such by-products. With an increase in microbial by-products, higher levels of by-products can overload the body's waste elimination mechanisms. An example of this is the production of ammonia during protein fermentation, which can be further fermented into compounds that are harmful to the host.

There is a relative dominance or underrepresentation of certain microbial species, low diversity and/or impaired production of microbial metabolites that cause many of the negative symptoms seen in patients suffering from dysbiosis.

Consumption of probiotics and/or prebiotics may have beneficial effects on the gut microbiota.

Probiotics are “live microorganisms which, when administered in adequate amounts, are beneficial to the health of the host” (definition by the World Health Organization).

Prebiotics are non-digestible food ingredients that are beneficial to the host by selectively stimulating the growth and/or activity of one or a limited number of microbial species in a community.

The best known prebiotics are oligomers of identical sugars (such as fructose, galactose, or arabinose) linked by glycosidic bonds. They stimulate the selective overgrowth of microbial species that have the metabolic ability to (rapidly) ferment such relatively simple substrates to produce beneficial metabolites such as short chain fatty acids. Typical side effects of these easily fermentable substrates include intestinal discomfort, bloating and regurgitation. These side effects are caused by rapid enzymatic gas production.

Pectin is a structural heteropolysaccharide that is present in the primary cell walls of land plants.

Pectic polysaccharides are a heterogeneous group of polysaccharides comprising varying amounts of the following polysaccharide components:

(i) homogalacturonan (HG),

(ii) xylogalacturonan (XG),

(iii) apiogalacturonan (AG)

(iv) rhamnogalacturonan-I (RG-I), and

(v) rhamnogalacturonan-II (RG-II).

In FIG. 1 is a schematic representation of the structure of pectin polysaccharides comprising the above 4 polysaccharide components. It is noted that the polysaccharide components HG, XG and RG-II usually represent only a minimal fraction of pectin polysaccharides.

The polysaccharide components HG, XG, and RG-II each comprise a backbone that consists of a linear chain of α-(1-4)-linked D-galacturonic acid monosaccharide units.

Only RG-I includes a backbone that consists of a linear chain of repeating disaccharide units: 4)-α-D-galacturonic acid-(1,2)-α-L-rhamnose-(1. A schematic representation of the structure of RG-I is shown in Fig. .2.

The composition and fine structure of pectic polysaccharides vary widely depending on the plant source and the extraction conditions used. The homogalacturonan domain can be up to about 100 consecutive D-GalA residues in length. The RG-I domain containing side chains is commonly referred to as the ‘branched region’ or ‘hairy region’, while the homogalacturonan domain (between two RG-I domains) is usually not substituted by oligosaccharides.

The GalA residues in RG-I are linked to the Rha residues through positions 1 and 4, while the Rha residue is linked to the GalA residue through the anomeric and 2-OH positions. In general, about 20-80% of Rha residues are branched at the 4-OH position (depending on the plant source and method of isolation), with neutral and acidic side chains. Such side chains consist primarily of Ara and Gal residues linked in various ways, constituting the polymers known as arabinogalactan I (AG-I) and/or AG-II. AG I consists of a backbone of beta-(1,4)-linked D-Gal with substitutions in the 3-OH alpha-L-arabinosyl groups; The Gal backbone may have alpha(1,5)-L-Ara fragments inserted. AG-II consists of a highly branched galactan with mostly internal beta(1,3)-linked D-Gal with short (1,6)-linked chain substitutions on the outside. The latter has additional attachments of (1,3)- and/or alpha(1,5)-linked L-Ara. The oligosaccharide side chains may be linear or branched and some of these side chains may terminate in alpha-L-fucosides, beta-D-glucuronides, and 4-O-methyl beta-D-glucuronyl residues.

Gomez et al. ( Prebiotic potential of pectins and pectic oligosaccharides derived from lemon peel wastes and sugar beet pulp: A comparative evaluation , Journal of Functional Foods, Volume 20, January 2016, Pages 108-121) describes the results of a study in which sugar beet pulp (SBP) and waste lemon peel (LPW) was used to prepare two mixtures of pectin oligosaccharides (designated as SBPOS and LPOS, respectively). Compliance of pectin oligosaccharides, pectins from SBP and LPW and commercial FOS with respect to development of prebiotic effects was compared by in vitro fermentation and in situ fluorescence hybridization using human feces inoculum and eight different bacterial assays. The total population of bifidobacteria and lactobacilli increased from 19% to 29%, 34% and 32% in cultures with LPOS, SBPOS and FOS, respectively. Faecalibacterium and Roseburia also increased with all substrates (especially LPOS). The highest concentrations of organic acids were observed in the medium containing oligosaccharides. According to the authors, this work confirms that pectin oligosaccharides exhibit better prebiotic properties than pectins and the same or better than FOS.

Chatterjee, et al. ( Effect of Fruit Pectin on Growth of Lactic Acid Bacteria , J Prob Health 2016, 4:2) report a study testing the effect of pectin extracted from various types of fruit waste ( Musa sp . and Citrus limetta and Citrullus lanatus rind and peeled fruits of Solanum lycopersicum and Psidium guajava ) on the growth of lactic acid bacteria (LAB) and bifidobacteria ( Lactobacillus casei, L. acidophilus and Bifidobacterium bifidum ). It was observed that pectin was able to significantly enhance bacterial growth and titratable acidity. The authors concluded that pectin, which is extracted from fruit waste, can be used to enhance the growth of lactobacilli and bifidobacteria.

Babbar, et al. ( Pectic oligosaccharides from agricultural by-products: production, characterization and health benefits , Crit. Rev. Biotech. 2016; 36(4) 594-606) mention that pectin-containing agricultural by-products are potential sources of a new class of prebiotics known as pectin oligosaccharides (POS). Controlled hydrolysis of pectin-containing agricultural by-products such as sugar beets, apples, olives and citrus fruits through chemical, enzymatic and hydrothermal treatment can be used to obtain oligo-galacturonides, galacto-oligosaccharides, rhamnogalacturonan oligosaccharides, etc.

Hoon Kim et al. ( Effect of arabinoxylan- and rhamnogalacturonan I-rich polysaccharides isolated from young barley leaf on intestinal immunostimulatory activity , Journal of Functional Foods, 2017; 35, 384-390) obtained four polysaccharide fractions from barley leaves and compared in vitro intestinal immunostimulatory activity. Among the fractions, the high molecular weight fraction obtained by enzymatic extraction (BLE-P) has been shown to be effective in activating bone marrow cell proliferation via Peyer's patches (PP), and in stimulating the production of cytokines in vitro . BLE-P was identified as a mixture of hemicellulose glucuronoarabinoxylan and pectin rhamnogalacturonan I, comprising over 80%. Subsequently, BLE-P was orally administered to mice for 20 days to study the effect on intestinal immunostimulatory activity in vivo . The introduction of BLEP not only promoted the production of immunoglobulin A (IgA), but also increased the levels of IgA-related cytokines such as transforming growth factor-β and interleukin-10.

US 2014/275233 describes a method of treating gastrointestinal dysbiosis in a patient, comprising orally administering to the patient an effective amount of a composition comprising isolated plant tissue having a glyceollin content of at least 0.25 mg per gram of plant tissue. Three very similar phytoalexins, called glyceollin I, glyceollin II, and glyceollin III, were obtained from soybeans when the plant was exposed to soil microorganisms, ultraviolet light (UV), or heavy metals.

WO 2011/069781 describes a polysaccharide that is capable of modulating an immune response, said polysaccharide is obtained from a plant of the species Camellia sinensis, where the backbone of the polysaccharide comprises alternating domains of rhamnogalacturonan-1 and alpha(1,4)-linked polygalacturonic acid or alpha(1,4 )-linked oligogalacturonic acid, wherein the molar ratio of galacturonic acid residues to rhamnosyl residues in the backbone of the polysaccharide ranges from 2.5:1 to 1:1, and wherein the polysaccharide has a molecular weight of at least 70 kDa.

WO 2012/148277 describes a preparation having a dry matter content of at least 20 wt.%, the specified preparation contains at least 50 wt.% dry matter of a mixture of pectin polysaccharides, including at least 20%, calculated by weight of pectin polysaccharides, pectins rhamnogalacturonan-I having a molecular weight of more than 40 kDa, this mixture of pectin oligosaccharides is characterized by:

* the degree of methylation of galacturonic acid residues is not more than 20%;

* the degree of acetylation of galacturonic acid residues is not more than 20%;

where the drug does not form a gel when it is diluted with an aqueous solution of 50 mm ammonium bicarbonate to a solids content of 2.5 wt.%. Also described is the use of this drug as a drug for modulating the immune response.

SUMMARY OF THE INVENTION

The inventors surprisingly found that disorders associated with impaired gut microbiome composition or functionality, namely metabolic disorders or gut barrier dysfunction, can be treated therapeutically or prophylactically by oral administration of rhamnogalacturonan I (RG-I) polysaccharides derived from fruits, carrots, peas. , chicory, or sugar beet, said RG-I polysaccharides have a molecular weight greater than 15 kDa and have a backbone comprising rhamnogalacturonan-I domains and optionally alpha(l,4)-linked homo-galacturonic acid domains, where the molar ratio of galacturonic acid residues to residues the rhamnose in said base ranges from 20:1 to 1:1.

Although the inventors do not wish to be bound by theory, it is believed that RG-I polysaccharides act as a prebiotic, by providing a high microbial diversity in the gut microbiota, by stimulating the growth or activity of beneficial bacteria (e.g., Akkermansia muciniphila and Bifidobacterium spp. ), and/or by increase the resistance of the intestinal microbiota against troubles.

In addition, enteric fermentation of RG-I polysaccharides has been found to produce beneficial short chain fatty acids. Surprisingly, the enzymatic conversion of RG-I polysaccharides to short chain fatty acids was accompanied by substantially less gas formation than that observed with classical prebiotics such as, for example, inulin.

In one aspect, the present invention relates to a prebiotic composition for use in a method for the therapeutic or prophylactic treatment of diseases associated with impaired gut microbiome composition or functionality in a patient, the disease being selected from a metabolic disorder and gut barrier dysfunction, said use comprising orally administering the prebiotic composition to a patient where the composition contains at least 0.1 wt.% dry matter of RG-I polysaccharides derived from fruits, carrots, peas, chicory or sugar beets, these RG-I polysaccharides have a molecular weight of more than 15 kDa and have a backbone consisting of galacturonic acid residues and rhamnose residues, said rhamnose residues are found in alpha(1→4)-galacturonic-alpha(1→2)-rhamnose residues, where the molar ratio of galacturonic acid residues to rhamnose residues in RG-I polysaccharides ranges from 20:1 to 1:1.

The RG-I polysaccharides that are used in accordance with the present invention can be isolated from fruits, carrots, peas, chicory or sugar beets by aqueous extraction, optionally in combination with an enzymatic treatment (using, for example, polygalacturonase).

Another aspect of the present invention relates to a prebiotic composition comprising:

* at least 0.1 wt.% dry matter of the above RG-I polysaccharides; And

* at least 1 wt.% dry matter of one or more prebiotics selected from lactulose, inulin, fructooligosaccharides, galactooligosaccharides, milk oligosaccharides, guar gum, gum arabic and any combinations thereof.

Another aspect of the invention relates to a synbiotic composition comprising:

* at least 0.1 wt.% dry matter of the above RG-I polysaccharides; And

* one or more probiotic microbial strains in the form of live microorganisms, non-living microorganisms, and combinations thereof.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention relates to a prebiotic composition for use in a method of therapeutic or prophylactic treatment of diseases associated with an impaired composition or functionality of the intestinal microbiome in a patient, the disorder is selected from a metabolic disorder and dysfunction of the intestinal barrier, this use includes oral administration of a prebiotic composition to a patient, where the composition contains at least 0.1 wt.% dry matter of rhamnogalacturonan I (RG-I) polysaccharides derived from fruits, carrots, peas, chicory or sugar beets, said RG-I polysaccharides have a molecular weight of more than 15 kDa and have a backbone consisting of galacturonic acid residues and rhamnose residues, these rhamnose residues are found in alpha(1→4)-galacturonic-alpha(1→2)-rhamnose residues, where the molar ratio of galacturonic acid residues to rhamnose residues in RG-I polysaccharides is in the range of 20 :1 to 1:1.

The terminology "disorders associated with impaired gut microbiome composition or functionality" as used herein encompasses an intestinal dysbiotic condition (impaired composition) and disorders associated with deficient enzymatic production of essential gut microbiome metabolites (impaired functionality). Short chain fatty acids (acetate, propionate and butyrate) are an example of such essential metabolites.

The term "intestinal dysbiotic condition", as used herein, refers to a condition that undesirably affects the health of the patient and which is caused by a significant deviation from a balanced intestinal microbiome (normobiosis).

The term "branched polysaccharides" as used herein refers to polysaccharides comprising a linear backbone of monosaccharide units linked together by glycosidic bonds, wherein at least one of the monosaccharide units carries a side chain of one or more glycosidic linked monosaccharide units.

The terms “backbone” and “backbone” are synonymous.

The term “pectic polysaccharides”, as used herein, refers to optionally branched polysaccharides having a molecular weight greater than 15 kDa and a backbone that consists of galacturonic acid residues and rhamnose residues, said rhamnose residues are found in alpha(1→4) residues. -galacturonic-alpha(1→2)-rhamnose.

The term “chain”, as used herein, refers to a sequence of two or more glycosidic linked monosaccharides with a backbone of a polysaccharide, excluding the side chains that are attached to it.

The term "domain" as used herein refers to the chain plus any side chains that are attached to the side chain.

The term “rhamnogalacturonan-I chain” or “RG-I chain” refers to a chain consisting of pairs of galacturonic acid (GalA) and rhamnose (Rha), where GalA residues are linked to Rha residues through positions 1 and 4, while Rha residues are linked with a GalA residue via the anomeric and 2-OH positions, i.e. alternating residues of alpha(1→4)-galacturonic-alpha(1→2)-rhamnose. Carboxyl groups of galacturonic acid residues in the RG-I chain can be esterified. Esterified galacturonic acid may appear in the form of a methyl ester or an acetyl ester.

The RG-I domain may include side chains such as, for example, galactan, arabinan and arabinogalactan.

The term “rhamnogalacturonan-I polysaccharide” or “RG-I polysaccharide” refers to an optionally branched pectic polysaccharide that includes a backbone that contains one or more rhamnogalacturonan-I chains.

The term “alpha(l,4)-linked galacturonic acid chain” refers to a chain consisting of alpha(1→4)-galacturonic residues.

In addition to the RG-I, RG-I domains, the polysaccharides of the present invention may contain one or more of the following domains:

* homogalacturonan (HG),

* xylogalacturonan (XG),

*apiogalacturonan (AG)

* rhamnogalacturonan-II (RG-II).

Domains XG, AG and RG-II usually represent only a minimal fraction of RG-I polysaccharides.

HG domains, XG domains, AG and RG-II domains, which are optionally present in the RG-I polysaccharides of the present invention, include a backbone that consists of a linear chain or two or more α-(1-4)-linked D-galacturonic acids . Carboxyl groups of galacturonic acid residues at the base of these domains can be esterified. The esterified galacturonic acid may appear in the form of a methyl ester or an acetyl ester.

HG domains do not contain any side chains.

The backbone of the XG domains contains one or more side chains in the form of D-xylose.

The backbone of AG domains contains one or more side chains that are composed of one or more D-apioses.

The RG-II backbone contains one or more side chains that are not exclusively composed of D-xylose or D-apiose.

The term "fruit" as used herein refers to seed-bearing structures in flowering plants.

The term "prebiotic", as used in the present description, refers to substances that selectively induce an increase in the activity of microorganisms that are involved in the well-being of their host.

The term “probiotic”, as used herein, refers to microorganisms that, when administered orally in adequate amounts, confer health and well-being. Such microorganisms are selected from living microorganisms, non-living microorganisms, fragments of microorganisms, and combinations thereof.

The term "synbiotic" refers to a composition that contains a combination of (a) one or more prebiotics and (b) one or more probiotics.

The concentration of various polysaccharides and their monosaccharide composition can be obtained by analytical methods known to the person skilled in the art. After acid hydrolysis, the monosaccharide composition can suitably be determined by High Performance Anion Exchange Chromatography in combination with Amperometric Determination (HPAEC-PAD).

Molecular size distribution can be determined by High Performance Size Exclusion Chromatography using Refractive Index (RI) determination (concentration), light scattering determination (molecular weight determination), UV determination (indicative of the presence of proteins), and differential pressure determination (intrinsic viscosity determination).

The above analytical methods are described in: Analytical Biochemistry Vol. 207, Issue 1, 1992, pg 176 (for analysis of neutral sugars) and in Mol. Nutr. Food Res., Vol 61, Issue 1, 2017, 1600243 (for analysis of galacturonic acid and molecular size distribution).

All percentages mentioned in the present description, unless otherwise indicated, refer to percentages by weight.

The patient to whom the composition containing RG-I polysaccharides is orally administered according to the present invention is preferably a mammal, more preferably a human. According to a preferred embodiment of the invention, the human patient is an infant (<4 years old) still developing a nuclear adult microbiota, or an elderly person (>50 years old) at risk of losing the diversity and persistence of his nuclear adult microbiota.

Oral administration in the context of the present method of treatment encompasses self-administration.

In accordance with a preferred embodiment of the invention, a patient who receives oral administration of a composition comprising RG-I polysaccharides suffers from or is at risk of developing a metabolic disorder. Most preferably, the patient is suffering from a metabolic disorder.

Metabolic disorders that are successfully treated (therapeutically or prophylactically) with the present treatment include overweight, obesity, metabolic syndrome, insulin deficiency or disorders associated with insulin resistance, type 2 diabetes mellitus, impaired glucose tolerance, impaired lipid metabolism, hyperglycemia, hepatic steatosis, dyslipidemia, high cholesterol, elevated triglycerides. The present treatment is particularly suitable for the therapeutic or prophylactic treatment of overweight or obesity and insulin resistance.

In accordance with another preferred embodiment of the invention, the patient suffers from or is at risk of developing an intestinal barrier dysfunction. More preferably, the patient is suffering from intestinal barrier dysfunction.

The intestinal barrier or intestinal mucosal barrier refers to the ability of the intestinal mucosa to adequately contain unwanted lumen contents in the intestine while maintaining nutrient absorption capacity. The separation it provides between the organism and the contents of the intestinal lumen prevents uncontrolled translocation of the contents of the lumen into the organism. Its role in protecting the mucosal and circulatory system from exposure to pro-inflammatory pathogens, toxins and antigens is vital to maintaining health and well-being. Intestinal barrier dysfunction has been implicated in a variety of health conditions such as: food allergies, microbial infections, irritable bowel syndrome, inflammatory bowel disease, celiac disease, metabolic syndrome, non-alcoholic fatty liver disease, diabetes, and septic shock.

In accordance with a preferred embodiment of the present invention, the prebiotic composition is used for the therapeutic or prophylactic treatment of an intestinal dysbiotic condition.

The patient receiving treatment for an intestinal dysbiotic condition according to the present invention is preferably a patient suffering from or at risk of developing a pathogenic intestinal dysbiotic condition, most preferably a patient suffering from such a pathogenic intestinal dysbiotic condition. Here "pathogenic" means that the condition is capable of causing or worsening the disease.

In accordance with another preferred embodiment of the invention, the prebiotic composition is used to increase the intestinal enzymatic production of short chain fatty acids.

The RG-I polysaccharides that are used in accordance with the present invention are preferably derived from a plant source selected from apple, bell pepper, blueberry, carrot, citrus, grape, pea, chicory, sugar beet and olive, okra, and combinations thereof. Even more preferably, RG-I polysaccharides derived from a plant source are selected from apple (e.g. apple pomace), bell pepper, carrot, citrus peel, grape, chicory, sugar beet (e.g. beet pulp), olives (e.g. olive pomace). ), okra, and combinations thereof. Most preferably, the RG-I polysaccharides are derived from carrots or apples.

The RG-I polysaccharides are preferably included in the prebiotic composition in the form of a pectic polysaccharide isolate that is enriched in RG-I polysaccharides. Accordingly, in a particularly preferred embodiment of the invention, the RG-I polysaccharides comprise at least 20 wt.%, more preferably at least 40 wt.%, even more preferably at least 50 wt.% and most preferably at least 60 wt. % pectin polysaccharides present in the prebiotic composition.

RG-I polysaccharides have a backbone that includes rhamnogalacturonan chains and optionally alpha(1,4)-linked homo-galacturonic acid chains. The molar ratio of galacturonic acid residues to rhamnose residues in RG-I polysaccharides ranges from 20:1 to 1:1. Preferably, the molar ratio of galacturonic acid residues to rhamnose residues in RG-I polysaccharides ranges from 15:1 to 1:1, more preferably from 12:1 to 1:1, even more preferably from 10:1 to 1:1, most preferably from 9:1 to 1:1.

Preferably, the rhamnose residues are 3-50%, more preferably 5-50% and most preferably 10-50% of the monosaccharide residues in the backbone of the RG-I polysaccharides.

Rhamnose residues typically represent 3-50%, more preferably 3.5-40% and most preferably 4-35% of all monosaccharide residues in RG-I polysaccharides, i. including the monosaccharide residues found in the side chains.

Galacturonic acid residues typically comprise 50-97%, more preferably 50-95% and most preferably 50-90% of the monosaccharide residues in the RG-I backbone of the polysaccharides.

Galacturonic acid residues typically represent 10-80%, more preferably 15-70% and most preferably 20-65% of all monosaccharide residues contained in RG-I polysaccharides, i. including the monosaccharide residues found in the side chains.

RG-I polysaccharides typically have a molecular weight of at least 20 kDa. Preferably the RG-I polysaccharides have a molecular weight of 25 kD to 2000 kD, more preferably 30 kD to 1500 kD, even more preferably 35 kD to 1200 kD, most preferably 40 kD to 1000 kD.

The average molecular weight of the RG-I polysaccharides contained in the present composition is preferably greater than 30 kDa, more preferably greater than 40 kDa, and most preferably greater than 60 kDa.

Preferably, less than 85% of the galacturonic acid residues in the RG-I polysaccharides are esterified in the form of a methyl ester. More preferably, the RG-I polysaccharides have an esterification degree of 0% to 70%, more preferably 0% to 60%, even more preferably 0% to 55%, most preferably 0% to 50%.

Preferably, 0-95% of the galacturonic acid residues in the RG-I polysaccharides are esterified in the form of an acetyl ester. More preferably, the RG-I polysaccharides have an esterification degree of 5% to 90%, more preferably 7% to 50%, most preferably 8% to 30%.

The backbone of RG-I polysaccharides consists of galacturonic acid residues and rhamnose residues. If the RG-I polysaccharides include one or more side chains, the polysaccharide may additionally contain arabinose and/or galactose residues. Moreover, the side chains of the RG-I polysaccharide can provide minimal amounts of fucose, glucose, glucuronic acid, xylose and/or uronic acid monomer residues. The one or more side chains are preferably selected from galactan side chains, arabinan side chains and arabinogalactan side chains.

The arabinan side chain includes at least one or more alpha(1,5)-linked arabinose residues and is substituted at the 4-OH position by a rhamnose residue in the RG-I domain. The arabinan side chain may be linear or branched. In the case where the side chain is linear, the side chain consists of alpha(1,5)-linked arabinose residues. When the arabinan side chain is a branched side chain, one or more alpha-arabinose residues are linked to 2-OH and/or 3-OH alpha(1,5)-linked arabinoses. The length of the arabinan side chain (expressed as the number of monomer units) is preferably 1 to 100 monomer units, more preferably 1 to 50 units, even more preferably 1 to 30 units.

The galactan side chain includes at least one or more beta(1,4)-linked galactose residues and is substituted at the 4-OH position with a rhamnose residue in the RG-I domain.

The galactan side chain is preferably substantially linear (unbranched), ie. less than 10 mol% of galactose residues in the chain are beta(1,3)-linked or beta(1,6)-linked galactose residues, preferably less than 5 mol%, preferably less than 2 mol%, preferably less than 1 mol% . The length of the galactan side chain is preferably 1 to 100 monomer units, more preferably 1 to 50 units, even more preferably 1 to 30 units.

The arabinogalactan side chain is substituted at the 4-OH position by a rhamnose residue in the RG-I domain and can be type I arabinogalactan (AGI) or type II arabinogalactan AGII). AGI consists of a (1→4)-β-D-Galp backbone on which substitutions of monomeric Galp units at the O-6 or O-3 position can take place. AGI is further substituted with α-L-Araf-p residues and/or short (1→5)-α-L-Araf side chains. AGII consists of an α(1→3)-β-D-Galp backbone provided with (1→6)-β-D-Galp secondary chains that are arabinosylated.

Preferably, the molar ratio of arabinose residues to rhamnose residues in the RG-I polysaccharide does not exceed 30:1, more preferably does not exceed 15:1, even more preferably does not exceed 8:1, and most preferably does not exceed 5:1.

The molar ratio of galactose to rhamnose residues in the RG-I polysaccharide preferably does not exceed 30:1, more preferably does not exceed 15:1, even more preferably does not exceed 8:1, and most preferably does not exceed 5:1.

According to a preferred embodiment of the invention, at least 20% of the rhamnose residues in the RG-I chains are substituted in the 4-OH position. More preferably at least 30%, even more preferably at least 40%, most preferably at least 45% of the rhamnose residues are substituted at the 4-OH position. Preferably at most 90%, more preferably at most 80% of such rhamnose residues are substituted at the 4-OH position.

The prebiotic composition for use in the present method preferably contains at least 0.2%, more preferably 0.3-10% and most preferably 0.4-5 wt.% dry matter of RG-I polysaccharides as described herein.

Prebiotic composition for use in the present method preferably contains one or more prebiotics, in addition to RG-I polysaccharides. Preferably the composition contains at least 1 wt.% dry matter, more preferably at least 3 wt.% dry matter of one or more prebiotics selected from lactulose, inulin, fructooligosaccharides, galactooligosaccharides, milk oligosaccharides, guar gum and gum arabic.

RG-I polysaccharides are considered to be particularly effective in the present treatment method if they are no longer entangled in the matrix of the cell wall material. Accordingly, in a certain preferred embodiment of the invention, the RG-I polysaccharides containing the composition of the present invention contain at least 0.05 wt.% dry matter, more preferably at least 0.1 wt.% dry matter, even more preferably at least at least 0.2 wt.% dry matter and most preferably at least 0.3 wt.% dry matter of readily water-soluble RG-I polysaccharides. The concentration of readily water-soluble RG-I polysaccharides in the RG-I polysaccharide-containing composition can be determined by mixing 100 ml of demineralized water (20°C) with enough RG-I polysaccharide-containing composition to obtain 2.5 grams of dry matter followed by stirring for 5 minutes and filtering through a 100 µm filter. The RG-polysaccharides in the filtrate are readily water-soluble RG-I polysaccharides.

In accordance with a certain preferred embodiment of the present invention, the present composition is administered orally to a patient over a period of at least 2 days in an amount providing at least 1 mg of RG-I polysaccharides per kg of body weight per day. More preferably an amount of at least 4 mg RG-I polysaccharides per kg of body weight, even more preferably at least 15 mg/kg of body weight per day, most preferably at least 20-100 mg/kg of RG-I polysaccharides per kg of body weight bodies per day are provided for a period of at least 7 days, most preferably for a period of at least 14 days, by administering a composition containing RG-I polysaccharides.

In accordance with another preferred embodiment of the invention, a composition containing RG-I polysaccharides is administered orally to a patient over a period of at least 21 days to provide RG-I polysaccharides in an amount of at least 4 mg of RG-I polysaccharides per kg of body weight in day, more preferably 15-300 mg of RG-I polysaccharides per kg of body weight per day.

Found that the prebiotic composition of the present invention is able to induce the growth of intestinal microorganisms that are able to provide health benefits, namely Akkermansia muciniphila and Bifidobacterium spp . Accordingly, in another preferred embodiment of the invention, the composition comprising RG-I polysaccharides is capable of inducing the growth or activity of Akkermansia muciniphila and/or Bifidobacterium spp. in the patient's gut microbiota. Most preferably, the composition is capable of inducing the growth or activity of Akkermansia muciniphila in the gut microbiota of the patient.

It has been found that the prebiotic composition of the present invention is able to induce the production of short chain fatty acids by the intestinal microbiota, while this is accompanied by significantly less gas production compared to classical prebiotics such as inulin. Accordingly, in another preferred embodiment of the invention, a composition comprising RG-I polysaccharides is capable of enhancing intestinal enzymatic production of short chain fatty acids with fewer side effects such as intestinal discomfort, bloating and vomiting associated with rapid gas production.

According to a particularly preferred embodiment of the invention, the composition is selected from beverage, oral dosage form, powder, bar and spread.

The drink is usually a liquid. Preferably the beverage contains at least 80 wt.%, more preferably at least 85 wt.% water. The beverage preferably contains at least 1.5 g/l, more preferably at least 3 g/l and most preferably 5-200 g/l RG-I polysaccharides.

The oral dosage form is preferably a capsule or tablet. The oral dosage form preferably has a weight in the range of 50 to 1500 milligrams, more preferably 100 to 800 milligrams. The oral dosage form preferably contains at least 1 wt.%, more preferably at least 20 wt.% and most preferably 40-90 wt.% RG-I polysaccharides.

The composition in powder form is preferably a water soluble powder which can be used to make a beverage. Typically, the powder contains at least 0.5 wt.%, more preferably at least 5 wt.% and most preferably 10-75 wt.% RG-I polysaccharides.

The bar-shaped composition is preferably a bar, preferably the bar has a weight in the range of 10 to 200 grams, more preferably 25 to 100 grams. The bar usually contains at least 0.1 wt.%, more preferably at least 0.5 wt.% and most preferably 1-20 wt.% RG-I polysaccharides.

The spread composition is preferably a water-in-oil emulsion, preferably a water-in-oil emulsion comprising 20-90% by weight of a fat phase and 10-80% by weight of an aqueous phase. The spread preferably contains at least 0.3 wt.%, more preferably at least 1-10 wt.% and most preferably 1.5-16 wt.% RG-I polysaccharides.

Another aspect of the present invention relates to a prebiotic composition comprising:

* at least 0.1 wt.% dry matter RG-I-polysaccharides, as defined in the present description earlier; And

* at least 1%, preferably at least 3 wt.% dry matter of one or more prebiotics selected from lactulose, inulin, fructooligosaccharides, galactooligosaccharides, milk oligosaccharides, guar gum and gum arabic.

Even more preferably, the product contains at least 1 wt.% dry matter, more preferably at least 3 wt.% dry matter of one or more prebiotics selected from lactulose, inulin, fructooligosaccharides, galactooligosaccharides and milk oligosaccharides.

Preferred embodiments of the prebiotic composition are the same as previously described herein with respect to the prebiotic composition for use in the present method of treatment.

Another aspect of the invention relates to a synbiotic composition comprising:

* at least 0.1 wt.% dry matter RG-I-polysaccharides, as defined in the present description earlier; And

* one or more probiotic microbial strains in the form of live microorganisms, non-living microorganisms, fragments of microorganisms, and combinations thereof.

Preferred embodiments of the synbiotic composition are the same as previously described herein in relation to the prebiotic composition for use in the present method of treatment.

The one or more probiotic microbial strains are preferably live microbial strains, more preferably live bacterial strains.

One or more probiotic microbial strains may be selected from yeast strains, fungal strains, bacterial strains, and combinations thereof. Examples of suitable yeast strains include strains belonging to Saccharomyces, Debaromyces, Candida Pichia and combinations thereof. Examples of suitable fungal strains include strains belonging to Aspergillus, Rhizopus, Mucor, Penicillium and combinations thereof. Examples of suitable bacterial strains include strains belonging to Bifidobacterium, Bacteroides, Fusobacterium, Melissococus, Propionibacterium, Enterococcus, Lactococcus, Staphylococcus, Peptostreptococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Faecalibacterium, Akkermansia, Oenococcus, Lactob acillus, Allobaculum, Eubacterium and their combinations.

According to a particularly preferred embodiment of the invention, one or more probiotic microbial strains are selected from Saccharomyces cerevisiae, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Enterococcus faecium, Enterococcus (Streptococcus) faecalis, Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus casei subsp. Casei, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus delbruckii subsp. Lactis, Lactobacillus farciminis, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus reuteri ococcus salivarius, Streptococcus thermophilus, Staphylococcus carnosus, Staphylococcus xylosus, Staphylococcus epidermidis, Akkermansia muciniphila, Faecalibacterium prausnitzii, Roseburia hominis , and Eubacterium hallii .

The synbiotic composition preferably contains one or more probiotic microbial strains at a concentration of 10 ⁴ to 10 ¹⁰ CFU, or their equivalent in the case of probiotic microbial strains used in non-living form. More preferably, the symbiotic composition contains one or more probiotic microbial strains at a concentration of 10 ⁵ to 10 ⁹ cfu or equivalent.

Akkermansia muciniphila is preferably present in the synbiotic composition at a concentration of 10 ⁵ to 10 ¹⁰ cfu/g, more preferably 10 ⁶ to 10 ⁹ cfu/g.

Bifidobacterium spp . preferably contained in the synbiotic composition at a concentration of 10 ⁶ to 10 ¹⁰ CFU/gram, more preferably 10 ⁷ to 10 ⁹ CFU/gram.

The invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1

The RG-I polysaccharide fraction was isolated from dry bell pepper powder (Paprika Mild 80-100 Atsa Steamtr-Felix Reverte S.A.) in an experimental setup using the procedure described below.

The bell pepper material (100 kg) was washed three times with gentle agitation with 80% aqueous ethanol, i.e. twice at 80°C for 2 hours and then overnight at room temperature; each time using 12.5% (w/v) to remove soluble ethanol material. The insoluble ethanol residue was recovered each time by centrifugation (1000 G for 10 min). The insoluble ethanol residue obtained after 3 washing cycles was dried and 90 kg was extracted twice with 1000 L of hot water at 95° C. for 90 minutes. Each time, the supernatant was retained after centrifugation at 1000 G for 10 minutes. The collected supernatant was subsequently filtered through tissue and ultrafiltered using 2 kDa restrictive membranes to remove low molecular weight material. A dry extract enriched in RG-I was obtained by lyophilization of the ultraconcentrate, obtaining approximately 5 kg of dry extract enriched in RG-I polysaccharides.

Characteristics of the extract enriched with RG-I polysaccharides

Molecular weight distribution :

The molecular weight distribution of the polysaccharide samples was determined by high performance size exclusion chromatography using refractive index (RI) determination (concentration), light scattering determination (molecular weight determination), UV determination (indicative of the presence of proteins), and differential pressure determination (intrinsic viscosity determination). Pullulan molecular weight standards were used for calibration.

Composition of monosaccharides:

Polysaccharide samples were dissolved in 0.5 M aqueous trifluoroacetic acid and hydrolyzed at 120°C for 2 hours. The samples were then neutralized with NaOH and stored frozen until analyzed by high pH anion exchange chromatography (HPAEC) using pulse amperometric determination (PAD). Uronic acid in the samples was determined using an m-hydroxybiphenyl colorimetric assay (Achmed & Labavitch, J. Food Biochem, 1978, 361) automated on an autoanalyzer (Skalar)

Degree of esterification

Polysaccharide samples were treated with sodium hydroxide (0.25 M, 5 h, 20° C.) and then neutralized. The released methanol was measured as absorbance at 420 nm after incubation with ethanol oxidase in combination with developer (acetylacetone and acetic acid in 2M ammonium acetate). Released acetic acid was determined using the K-ACETAF acetic acid assay kit (Megazyme). Sugar beet pectin with a known degree of methylation and acetylation was used as a standard. The degree of esterification was expressed as the molar amount of methanol and acetic acid released as a percentage of the amount of uronic acid.

The molecular characteristics of the RG-I fraction of polysaccharides are shown in tables 1a and 1b.

Table 1a

Monosaccharides (% mol/mol) Red bell pepper Rha (rhamnose) 5.0 GalA (Galacturonic acid) 70.0 Ara (Arabinosa) 9.0 Gal (galactose) 9.0 Glc (Glucose) 3.0 Xyl (xylose) 2.0

Table 1b

Molecular ratios Red bell pepper GalA/Rha 14 Ara/Rha 1.8 Gal/Rha 1.8 Degree of methylation, % 39 Degree of acetylation, % 9.0

Example 2

Six-month-old female C57BL/6 mice free of specific pathogens were fed as needed sterilized drinking water and semi-synthetic irradiated diet AIN-93G (Research Diet Services, Wijk bij Duurstede, The Netherlands) with 30 mmol/kg calcium (uninfected and infected group) or the same nutrition supplemented with 1% (w/w) RG-I enriched bell pepper extract.

Mice were randomized into treatment groups and housed 3 mice per cage for 2 weeks and then separately for another week. The nutritional composition of AIN-93G is described by Reeves et al. ( AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet . J Nutr (1993)123: 1939-1951).

Feces were collected before dietary intervention at baseline and after 3 weeks of dietary intervention. Genomic DNA was extracted from faecal samples following the manufacturer's protocol (Zymo research). The isolated gDNA was subjected to microbial identification by sequencing the 16S rRNA gene. Specific primers were used for PCR amplification of the genomic region of interest, such as the V3-V4 or V4 hypervariable regions of the 16S rRNA gene. Paired end sequence reads were obtained using the Illumina MiSeq system. FASTQ sequence files were created using Illumina Casava pipeline version 1.8.3. The original quality score is based on Illumina Chastity filtering. Subsequently, reads containing the PhiX control signal were removed using an internal filtering protocol (Baseclear). In addition, reads containing (partial) adapters were clipped (to a minimum read length of 50 bp). The second quality score was based on the remaining reads using the FASTQC quality control tool version 0.10.0.

Sequencing of bacterial DNA

Illumina Miseq data were analyzed using a flowchart using the Quantitative Insights Into Microbial Ecology (QIIME, v8) backbone (Caporaso et al., QIIME allows analysis of high-throughput community sequencing data, Nature Methods (2010), 7(5), 335 -336 and Edgar, Search and clustering orders of magnitude faster than BLAST, Bioinformatics (Oxford, England), (2010), 26(19), 2460-2461 Data were extracted and filtered for mismatched barcodes and small fragments (>50 nt). OTU open frame isolation was performed in QIIME using the Silva 111 database and chimeras were identified by USEARCH and filtered from OTUs A biome file and a phylogenetic tree file were generated from the filtered OTUs using the Silva 111 database The following outcomes were obtained by QIIME such as , for example, filtered reads per specimen, whole tree PD diversity score, and taxonomic distribution of level 1-6 with relative enrichment.

Effect of an extract enriched with RG-I polysaccharides on microbial composition

Individual fecal samples from 12 mice per group were subjected to Illumina 16S rRNA sequencing to obtain additional data on microbial composition. 26×10 ³ to 79×10 ⁴ readings were obtained per sample. The results are shown in tables 2, 3 and 4.

table 2

Enrichment % Firmicutes/Bacteriodetes ratio Firmicutes Bacteriodetes Source control 0.459±0.184 0.436±0.144 1.336±1.059 RG-I initially 0.554±0.117 0.381±0.089 1.596±0.684 Control 3 weeks 0.489±0.225 0.361±0.235 1.350±1.459 RG-I initially 0.502±0.190 0.178±0.124* 4.239±2.648*

* Denotes significance relative to RG-I at baseline

Table 3

Enrichment % Actinobacteria Verrucomicrobia Source control 0.083±0.080 0.000±0.000 RG-I initially 0.036±0.044 0.002±0.006 Control 3 weeks 0.071±0.084 0.002±0.006 RG-I initially 0.231±0.159* 0.036±0.022*

* Denotes significance relative to RG-I at baseline

Table 4

Enrichment % Bifidobacterium Akkermansia Source control 0.003±0.001 0.000±0.000 RG-I initially 0.001±0.001 0.002±0.006 Control 3 weeks 0.028±0.060 0.002±0.006 RG-I initially 0.134±0.110* 0.041±0.027*

* Denotes significance relative to RG-I at baseline

At the type level, supplementation of the extract enriched in RG-I polysaccharides in the diet significantly increased the enrichment of Actinobacteria and Verrucomicrobia , while the enrichment of Bacteroidetes and Proteobacteria decreased (>0.5% enrichment; Wilcoxon, P<0.05) leading to an increased Firmicutes/ Bacteroidetes .

At the genus level, excess analysis showed that the addition of the RG-I polysaccharide enriched extract to the diet had a significant effect on microbial composition (P<0.05; Monte Carlo conversion). The inclusion of an extract enriched in RG-I polysaccharides in the diet results in a higher enrichment of Bifidobacterium (Phylum Actinobacteria) , Allobaculum (Phylum Firmicutes), Akkermansia (Phylum Verrucomicrobia) and groups of unspecified bacteria.

Example 3

Indigestible polysaccharides are primarily digested in the human large intestine by the intestinal microbiota. This can lead to the growth of beneficial bacteria, a decrease in pH, an increase in resistance to enteric pathogens, and a modulation of the metabolic activity of the microbiota. Beneficial (prebiotic) functional carbohydrates will ensure the production of healthy metabolites such as short chain fatty acids (SCFAs, i.e. acetate, propionate and butyrate) while reducing the production of undesirable protein metabolism metabolites like branched chain SCFAs and ammonia.

Plant extracts of polysaccharides were tested for their effect on the metabolic activity of the microbiota using an established short-term colonic incubation model.

At the start of the incubation, sugar-free basic colonic medium containing nutrients that are present in the colon (e.g. host glycans such as mucin) was injected into 70 ml penicillin bottles already containing test extracts (5 g/l final concentration). The bottles were closed with rubber stoppers and anaerobiosis was obtained by passing N ₂ . Subsequently, an inoculum of human feces was obtained by mixing a freshly collected fecal sample with anaerobic phosphate buffer. After homogenization and removal of particles by centrifugation (2 min, 500 g), the fecal inoculum was added to various bottles. At this point, incubation was started for a period of 48 hours during which the temperature was set to 37° C. and continuous stirring was ensured with a mixer (90 rpm). Samples were obtained after 6, 24 and 48 h of incubation for analysis of pH (Senseline F410; ProSense, Oosterhout, The Netherlands), gas pressure (Hand-held pressure indicator CPH6200; Wika, Echt, The Netherlands) and SCFA. SCFA being acetate, propionate, butyrate and branched SCFA (isobutyrate, isovalerate and isocaproate) were measured as described by De Weirdt et al ( Human faecal microbiota display variable patterns of glycerol metabolism , FEMS Microbiol. Ecol. 2010, 74, 601-611 .).

Colon medium without sugar and inulin, a well known prebiotic (Beneo, DP ≥23, ~100% inulin), were used as negative and positive controls, respectively.

Sample A was prepared from bell pepper powder (Paprika poeder, Natural Spices Mijdrecht, the Netherlands) by aqueous extraction (10% w/w, 2h 90°C), centrifugation to remove insoluble residues, filtration (40 kDa cutoff) to remove small molecules and drying to obtain a powder.

Sample B was prepared from dried carrot pomace, residue from carrot juice production (carrot fiber powder, GreendFields, Poland). Sample B was obtained by aqueous extraction (10% w/w 2 h 45°C) using pectinase (Pectinex® Ultra Mash, Novozymes), heat inactivation (90°C, 10 min), removal of insoluble residues by draining and ultrafiltration (cut-off 40 kDa) and finally drying.

Sample C was extracted from apple pomace powder (apple pomace, GreendFields, Poland) in the same manner as sample B.

Sample D was prepared from okra powder (Ground Okra, My Foods, Blue mountain peak, UK) using hot water extraction (10% w/w, 2 h at 90°C), dialysis against water for several hours to remove low molecular weight material . The dialyzed extract was then lyophilized.

The determination of the monosaccharide composition of the above samples was carried out as described for Example 1. The results are shown in Table 5.

Table 5

Source A IN WITH D bell pepper Carrot Apple Okra Rha (rhamnose) %mol/mol 8 17 8 31 GalA (galacturonic acid) %mol/mol 50 23 18 40 Ara (arabinose) %mol/mol 13 23 48 1 Gal (galactose) %mol/mol 14 33 9 22 Glc (glucose) %mol/mol 9 2 6 0 Xyl (xylose) %mol/mol 3 2 6 0 Total 98 100 99 100 GalA/Rha 6.0 1.4 2.2 1.3 Ara/Rha 1.5 1.4 5.7 0.0 Gal/Rha 1.7 1.9 1.1 0.7 % methylation 39 16 27 N.d.

The results of the incubation experiments are shown in Table 6.

Table 6

Wednesday Inulin A IN WITH D bell pepper carrot apple okra pH drop 0.01 0.66 0.39 0.63 0.64 0.52 SCFA Products mm 27.8 51.1 49.3 56.4 55.6 52.9 Gas production kPa 28.6 86.5 47.9 49.5 57.2 57.1 Branched SCFAs mm 2.8 1.2 1.9 1.3 1.1 1.7 Ammonia mg/l 373 169 346 221 214 287

The results show that all four plant extracts of RG-I polysaccharides were easily fermented by the gut microbiota.

All RG-I polysaccharide extracts increased SCFA production. Virtually all extracts increased SCFA production to levels similar to or greater than those observed with inulin.

All extracts of RG-I polysaccharides reduced the production of branched SCFAs and ammonia. Surprisingly, the gas production observed for all RG-I polysaccharide extracts was substantially lower than the gas production observed for inulin. Excess gas production can lead to abdominal discomfort and bloating, a well-known unwanted side effect of most classic prebiotics, including inulin.

Example 4

The pasteurized milk drink was prepared based on the recipe shown in Table 7.

Table 7

Wt% milk fat 1 alkalized cocoa 0.1 sucrose 3 RG-I polysaccharide isolate ¹ 2 Inulin 2 Carrageenan 0.01 High Acyl Gellan 0.05 hydroxypropyl starch 0.2 Carboxymethylcellulose 0.1 Skim milk rest

¹ isolated from dried carrot pomace (see example 3)

Consuming 200 ml of this milk drink per day as an adult improves gut health and provides extra protection against colds and flu.

Example 5

A nutrition bar (45 g) was prepared based on the recipe shown in Table 8.

Table 8

Wt% Maltodextrin 12.9 milk protein isolate 9.0 soy protein isolate 0.5 Rice flour 5.3 Rye bran 14.4 Rice flakes 9.0 Crystalline fructose 6.8 Evaporated sugar cane syrup 27.7 salt 0.3 glycerol 1.0 Almond oil 3.3 Isolate RG-I polysaccharides ¹ 4.5 Inulin 1 Vitamin/Mineral Blend 3.1 vanilla 1.2

¹ isolated from dried carrot pomace (see example 3)

The bar was prepared as follows: all wet ingredients were mixed together (syrup, glycerin, almond oil and flavors) at 50°C.

Separately, the dry ingredients were mixed together, then the wet slurry was added to the dry mixture and the mass was mixed for 2-5 minutes at high shear. The dough was tiled and cut into bars before packaging.

Consuming 2 bars per day as an adult improves gut health and provides extra protection against the common cold and flu.

Example 6

The nutritional supplement was in the form of a capsule containing the dry mix shown in Table 9.

Table 9

mg Isolate RG-I polysaccharides ¹ 300 D-alpha tocopherol 10 Oligomeric proanthocyanidin 15 Vitamin B1 1.4 Vitamin B2 1.6 Niacin (Vitamin B3) 18.0 Pantothenic Acid (Vitamin B5) 6.0 Vitamin B6 2.0 Vitamin B12 1.0 Folic acid (vitamin B9) 0.2 Biotin 0.15 Microcrystalline cellulose thirty

¹ isolated from dried carrot pomace (see example 3)

Example 7

Fractions of RG-I polysaccharides were extracted from various source materials for testing in the short-term colonic incubation model described in Example 3. Colonic medium without sugar and inulin, a well known prebiotic (Beneo, DP ≥23, ~100% inulin), was used in as negative and positive controls, respectively.

Sample A was prepared from dried and ground pea skins (ex Cosucra, Warcoing, Belgium). The powder was dispersed in demineralized water (100 g/l) and subjected to enzymatic pre-hydrolysis with thermostable alpha-amylase (Megazyme) at 90°C for 30 min and further hydrolysis with pectinase (2 h 45°C, 0.2 vol /vol% Pectinex® Ultra Mash, Novozymes). Fermentation was terminated by heating at 100° C. for 10 min, followed by centrifugation (18,000 g, 10 min) and extensive dialysis of the supernatant using a 12-14 kD cutoff membrane (Visking, London, UK). The material was then lyophilized.

Sample B was prepared using the same method from dried and ground sugar beet cake (ex Suiker Unie, Dinteloord, NL), except for the α-amylase pre-incubation step.

Sample C was prepared using the same method from dried and ground chicory pomace (ex Cosucra, Warcoing, Belgium), except for the α-amylase pre-incubation step.

The monosaccharide composition of the above samples was determined using the method described in Example 1. The results are shown in Table 10.

Table 10

Source A IN WITH Peas Sugar beet Chicory Rha (rhamnose) % mol/mol 8 7 8 GalA (galacturonic acid) % mol/mol 57 40 35 Ara (Arabinosa) % mol/mol 14 45 48 Gal (galactose) % mol/mol 6 7 8 Glc (Glucose) % mol/mol 8 1 1 Xyl (Xylose) % mol/mol 6 0 0 Total GalA/Rha 7.1 5.7 4.4 Ara/Rha 1.8 6.4 6.0 Gal/Rha 0.8 1.0 1.0 % methylation 33 26 27

The results of the incubation experiment are shown in Table 11.

Table 11

Wednesday Inulin A IN WITH Peas Sugar beet Chicory pH drop 0.00 0.61 0.47 0.62 0.66 SCFA products mm 27.5 59.7 61.3 66.4 61.7 Gas production kPa 31.9 87.6 62.6 70.0 74.6 Branched SCFAs mm 3.4 0.5 3.0 3.0 1.9 Ammonia mg/l 362 181 316 293 228

The results obtained show that such extracts of RG-I polysaccharides obtained from various plants were easily fermented by the intestinal microbiota.

All RG-I polysaccharide extracts increased SCFA production. In fact, all extracts were found to increase SCFA production to a level similar to or greater than that obtained for inulin.

All extracts of RG-I polysaccharides reduced the production of branched SCFA and ammonia compared to the control medium.

Claims (14)

1. The use of rhamnogalacturonan-I (RG-I) polysaccharides in the preparation of a prebiotic composition for the therapeutic or prophylactic treatment of diseases associated with impaired composition or functionality of the intestinal microbiome in a patient, the disease is selected from overweight, obesity, insulin resistance and intestinal barrier dysfunction, where the composition includes at least 0.1 wt.% dry matter of RG-I polysaccharides derived from fruits, carrots, peas, chicory, sugar beets, okra, or combinations thereof, where these RG-I polysaccharides have a molecular weight of more than 15 kDa and have a basis , consisting of galacturonic acid residues and rhamnose residues, these rhamnose residues are contained in alpha(1→4)-galacturonic-alpha(1→2)-rhamnose residues, where the molar ratio of galacturonic acid residues and rhamnose residues in RG-I polysaccharides is in range 20:1-1:1. 2. The use of RG-I polysaccharides according to claim 1, wherein the RG-I polysaccharides constitute at least 20% by weight of the pectic polysaccharides present in the prebiotic composition. 3. The use of RG-I polysaccharides according to claim 1 or 2, wherein the molar ratio of galacturonic acid residues to rhamnose residues in the RG-I polysaccharides does not exceed 15:1, preferably does not exceed 12:1, more preferably does not exceed 10:1. 4. The use of RG-I polysaccharides according to any one of the preceding claims, wherein the molar ratio of arabinose residues to rhamnose residues in the RG-I polysaccharides does not exceed 30:1. 5. The use of RG-I polysaccharides according to any one of the preceding claims, wherein the molar ratio of galactose to rhamnose residues in the RG-I polysaccharides does not exceed 30:1. 6. The use of RG-I polysaccharides according to any one of the preceding claims, wherein less than 85% of the galacturonic acid residues in the RG-I polysaccharides are esterified in the form of methyl esters. 7. The use of RG-I polysaccharides according to any one of the preceding claims, wherein the RG-I polysaccharides are derived from a plant source selected from carrot, apple, bell pepper, citrus fruits, blueberries, grapes, peas, chicory, sugar beets, olives, okra and their combinations. 8. The use of RG-I polysaccharides according to any one of the preceding claims, wherein the patient suffers from or is at risk of developing a metabolic disorder. 9. The use of RG-I polysaccharides according to any one of paragraphs. 1-7, where the patient suffers from or is at risk of developing intestinal barrier dysfunction. 10. The use of RG-I polysaccharides according to any one of the preceding claims, wherein the composition is selected from beverage, capsule, tablet, powder, bar and spread. 11. Synbiotic composition comprising: * at least 0.1 wt.% dry matter of rhamnogalacturonan I (RG-I) polysaccharides derived from carrots or apples, said RG-I polysaccharides have a molecular weight greater than 15 kDa and have a backbone consisting of galacturonic acid residues and rhamnose residues , said rhamnose residues are contained in alpha(1→4)-galacturonic-alpha(1→2)-rhamnose residues, where the molar ratio of galacturonic acid residues and rhamnose residues in RG-I polysaccharides is in the range of 20:1-1:1; And * 10 ⁴ -10 ¹⁰ CFU/g Akkermansia muciniophila and/or 10 ⁶ -10 ¹⁰ CFU/g Bifidobacterium spp. 12. The composition of claim 11, wherein the composition is selected from beverage, capsule, tablet, powder, bar and spread.

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