Prebiotics for modulating the gut microbiota

Abstract

The present invention relates to a prebiotic composition for modulating the gut microbiota of a subject, said use comprising orally administering said prebiotic composition to said subject, wherein said composition comprises at least 0.1% by weight of dry matter of RG-I polysaccharide derived from fruit, carrot, pea, chicory or beet, said RG-I polysaccharide having a molecular weight of more than 15 kDa and having a backbone composed of galacturonic acid residues and rhamnose residues, said rhamnose residues being contained in α(1→4)-galacturonic acid-α(1→2)-rhamnose residues, wherein the molar ratio of galacturonic acid residues to rhamnose residues in said RG-I polysaccharide is in the range of 20:1 to 1:1.

Description

[0001] Technical Field of the Invention

[0002] The present invention relates to a method for modulating the gut microbiota of a subject, the method comprising orally administering to the subject a prebiotic composition comprising rhamnogalacturonan I (RG-I) polysaccharide derived from fruit, carrot, pea, chicory or beet.

[0003] The present invention also relates to prebiotic compositions and synbiotic compositions suitable for the above method. Background of the Invention

[0005] There is a growing recognition of the relationship between the gut microbiota and human health. It is now well established that a healthy gut microbiota is primarily responsible for the overall health of the host.

[0006] The human host provides a habitat and nutrients for a large and diverse ecosystem of microbial communities that play key roles in digestion, metabolism, and regulation of immune function and have significant effects outside the gastrointestinal tract. Changes in the diversity and function of these communities are associated with profound effects on host health and have been associated with many disorders, including functional bowel diseases, inflammatory bowel diseases and other immune-mediated diseases (celiac disease, allergies) and metabolic disorders (type 2 diabetes, NASH).

[0007] Dysbiosis (also known as microbiota dysbiosis) is a term for a microbial imbalance or maladaptation (such as impaired microbiota) in or on the body. For example, the bacterial communities that colonize certain surface areas of the host, such as the gut microbiota, skin microbiota or vaginal microbiota, can be modified / altered, with the normally dominant species being underrepresented and the normally outcompeted or included species increasing to undesired levels. The collection of bacteria in such communities is collectively referred to as the microbiota. The local microbiota together with other microorganisms (including yeasts, fungi, viruses and parasites present in such niches) are collectively referred to as the microbiome. Dysbiosis is not limited to imbalances in the microbiota but can also involve other microorganisms in the microbiome (such as viruses, archaea and fungi).

[0008] When the microbiome is in good balance (referred to as normobiosis), the microorganisms occupying a particular niche form more or less stable communities that are well adapted to local conditions, have the metabolic capacity to survive on available substrates, effectively handle stressors and regulation of substrate availability, and have regulatory mechanisms in place that contribute to the metastability of the community and to the long-term health of its host.

[0009] Dysbiosis is most often studied in gastrointestinal conditions, but it can affect any body cavity, mucosal and skin surface colonized by microbial communities.

[0010] Dysbiosis is associated with host diseases; for example, gut dysbiosis is associated with inflammatory bowel disease, chronic fatigue syndrome, obesity, cancer, cardiometabolic conditions, insulin insensitivity, diabetes (prediabetes), bacterial vaginosis, and colitis.

[0011] The composition and stability of the microbiota are influenced by the host's genetic background, environmental conditions, or stressors, which include, for example, diet, lifestyle, use of medications (such as antibiotics), and the host's developmental stage (age). This translates into a permanent and complex interaction between the host and the major components of the local microbial ecosystem. These components include the microbiota, the host immune system, the local epithelial barrier, and the enteric nervous system (in the case of the gut).

[0012] The neonatal microbiota establishes itself from birth and fluctuates significantly in the first weeks and months of life to approximate a core mature microbiota at around 3 - 4 years of age. This progressive microbial colonization of the gut is crucial for the cultivation and maturation of the host immune and enteric nervous systems, gut barrier and function, and the host's metabolic programming, which has implications for short - and later - life health status and disease risk. The neonatal microbiota is influenced by maternal diet and microbiota, mode of delivery, infant nutrition (breastfeeding or infant formula), and the surrounding environmental conditions.

[0013] In contrast, the healthy adult core microbiota is more stable in the sense that when disrupted by various stressors (such as the use of antibiotics or medications), it recovers to near its original composition in healthy subjects, which is referred to as the resilience of the microbiota. Loss of variability, lack or low abundance of beneficial microorganisms, and loss of resilience are all associated with disease.

[0014] The microbiota of aged or elderly subjects differs from that of healthy adult populations in terms of compositional changes, resulting in less bacterial diversity, fewer beneficial microorganisms, and reduced resilience. All of these changes are associated with changes in the health status.

[0015] The microbiota is composed of a wide variety of species that compete for space and resources / nutrients, but can also feed on each other's fermentation products, leading to cooperation among the microorganisms that are symbiotic with the mammalian host in a healthy state. High microbial diversity within the gut microbiota is thought to be beneficial for the host's health because diversity makes the microbiota more resilient to factors that disrupt the gut microbiota (such as antibiotics, diet changes, invasion by new species). Also important is the cooperation of microorganisms, that is, a metastable combination of different microorganisms that feed on each other's fermentation products and use available substrates (such as from mucus, cell shedding, and diet) to jointly form an approximately stable ecosystem that thrives in a specific ecological niche.

[0016] Most of the typical microbial species found on or in the body are beneficial or harmless. As long as the pathobionts or even pathogens remain below the critical level, they are also part of the "normal" microbiota. The mammalian gut microbiota performs a series of helpful and necessary functions, such as aiding digestion, providing energy from food, supplying specific (trace) nutrients to the host, producing key metabolites such as short-chain fatty acids, and cultivating (newborns and infants) or maintaining (adults) the host's immune system. They also help protect the body from incoming pathogenic microorganisms or toxic compounds.

[0017] Microbial species also secrete many different types of waste by-products. Using different waste removal mechanisms, under normal circumstances, the human body effectively manages these by-products with little trouble. Unfortunately, due to increased numbers, an overabundant microbial population and the dominance of inappropriate microbial species secrete increased amounts of these by-products. As the amount of microbial by-products increases, higher levels of waste by-products can overload the body's waste removal mechanisms. An example of this situation is the formation of ammonia from protein fermentation, which can be further fermented into compounds harmful to the host.

[0018] It is the relative dominance or underrepresentation of specific microbial species, the low diversity of microbial metabolites, and / or the production of disturbances that result in many of the negative health symptoms observed in subjects with dysbiosis.

[0019] Consuming probiotics and / or prebiotics can have a beneficial effect on the gut microbiota.

[0020] Probiotics are "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host" (definition by the World Health Organization).

[0021] Prebiotics are indigestible food ingredients that beneficially affect the host by selectively stimulating the growth and / or activity of one or a limited number of microbial species in the community.

[0022] Most known prebiotics are simple oligomers of the same sugar (such as fructose, galactose or arabinose) linked by glycosidic bonds. These stimulate the selective growth of microbial species with the metabolic ability to (rapidly) ferment these relatively simple substrates to produce beneficial metabolites such as short-chain fatty acids. Typical side effects of using such readily fermentable substrates include intestinal discomfort, flatulence and reflux. These side effects are caused by the rapid fermentation production of gas.

[0023] Pectin is a structural heteropolysaccharide present in the primary cell walls of terrestrial plants.

[0024] Pectin polysaccharides are a heterogeneous group of polysaccharides that contain varying amounts of the following polysaccharide components:

[0025] (i) Homogalacturonan (HG),

[0026] (ii) Xylogalacturonan (XG)

[0027] (iii) Arabinogalacturonan (AG)

[0028] (iv) Rhamnogalacturonan-1 (RG-I) and

[0029] (v) Rhamnogalacturonan-II (RG-II).

[0030] Figure 1 A schematic diagram of the structure of pectin polysaccharides (including the above 4 polysaccharide components) is provided. Note that the polysaccharide components HG, XG, and RG-II usually represent only a small fraction of the pectin polysaccharides.

[0031] The polysaccharide components HG, XG, and RG-II each contain a main chain composed of a linear chain of α-(1-4)-linked D-galacturonic acid monosaccharide units.

[0032] Only RG-I contains a main chain composed of a linear chain of repeating disaccharide units: 4)-α-D-galacturonic acid-(1,2)-α-L-rhamnose-(1. A schematic diagram of the structure of RG-I is shown in Figure 2 in.

[0033] The composition and fine structure of pectin polysaccharides vary greatly depending on the plant source and the extraction conditions of the application. The homogalacturonan domain can have a length of up to about 100 consecutive D-GalA residues. The RG-I domain containing side chains is usually referred to as the 'branching region' or 'hairy region', while the homogalacturonan domain (between two RG-I domains) is usually not substituted by oligosaccharides.

[0034] The GalA residues in RG-I are linked to Rha residues via positions 1 and 4, while the Rha residues are linked to GalA residues via the anomeric and 2-OH positions. Typically, about 20 - 80% of the Rha residues are branched at the 4-OH position (depending on the plant source and isolation method), with neutral and acidic side chains. These side chains are mainly composed of Ara and Gal residues linked in various ways, forming polymers called arabinogalactan I (AG-I) and / or AG-II. AG I consists of a β-(1,4)-linked D-Gal backbone with substitutions at the 3-OH of α-L-arabinose; the Gal backbone can have intervening α(1,5)-L-Ara units. AG-II consists of highly branched galactans, where predominantly internal β(1,3)-linked D-Gal has substitutions with short (1,6)-linked chains on the outside. The latter also has linkages of (1,3)- and / or α(1,5)-linked L-Ara. The oligosaccharide side chains can be linear or branched, and some of these side chains can be terminated with α-L-fucosyl, β-D-glucuronide, and 4-O-methyl-β-D-glucuronide residues.

[0035] Gómez 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) described the results of a study using two mixtures of pectic oligosaccharides (designated as SBPOS and LPOS, respectively) obtained from sugar beet pulp (SBP) and lemon peel wastes (LPW). By in vitro fermentation and fluorescence in situ hybridization, using human fecal inoculum and 8 different bacterial probes, the applicability of pectic oligosaccharides, pectins from SBP and LPW, and commercial FOS to elicit a prebiotic effect was compared. In media containing LPOS, SBPOS, and FOS, the combined populations of Bifidobacterium and Lactobacillus increased from 19% to 29%, 34%, and 32%, respectively. All substrates (especially in the case of LPOS) also increased the counts of Faecalibacterium and Roseburia. The highest concentrations of organic acids were observed in media containing oligosaccharides. According to the authors, the work confirmed that pectic oligosaccharides exhibit better prebiotic properties than pectins and exhibit prebiotic properties similar to or better than FOS.

[0036] Chatterjee et al. (Effect of Fruit Pectin on Growth of Lactic Acid Bacteria, J Prob Health 2016, 4:2) reported a study in which the effects of pectins extracted from different types of fruit wastes (banana (Musa sp.), sweet lime (Citrus limetta), watermelon rind (rind of Citrullus lanatus), tomato (Solanum lycopersicum), and guava (Psidium guajava) rotten fruits) on the growth of lactic acid bacteria (LAB) and bifidobacteria (Lactobacillus casei, L. acidophilus, and Bifidobacterium bifidum) were tested. It was observed that pectin could significantly enhance bacterial growth and titratable acidity. The authors concluded that pectin extracted from fruit wastes could be used to promote the growth of lactobacilli and bifidobacteria.

[0037] Babbar et al. (Pectic oligosaccharides from agricultural by-products: production, characterization and health benefits, Crit. Rev. Biotech. 2016; 36(4) 594 - 606) mentioned that agricultural by-products containing pectin are potential sources of a new class of prebiotics called pectic oligosaccharides (POS). Controlled hydrolysis of pectin-containing agricultural by-products such as sugar beet, apple, olive, and citrus by chemical, enzymatic, and hydrothermal treatments can be used to produce oligo-galacturonides, galacto-oligosaccharides, rhamnogalacturonan-oligosaccharides, etc.

[0038] 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) prepared four polysaccharide fractions from barley leaves and compared their intestinal immunostimulatory activities in vitro. Among these fractions, the high-molecular-weight fraction (BLE-P) prepared by enzymatic extraction showed potency in activating bone marrow cell proliferation and stimulating cytokine production in vitro through Peyer's patches (PP). BLE-P was identified as a mixture of hemicellulose glucuronoarabinoxylan and pectin rhamnogalacturonan I, accounting for more than 80%. Subsequently, BLE-P was orally administered to mice for 20 days to study its effect on intestinal immunostimulatory activity in vivo. BLE-P administration not only increased the production of immunoglobulin A (IgA), but also increased the levels of IgA-related cytokines such as transforming growth factor-β and interleukin-10.

[0039] US2014 / 275233 describes a method for treating gastrointestinal disorders in a subject, which comprises orally administering to the subject an effective amount of a composition comprising isolated plant tissue having at least 0.25 mg glyceollin content / gram of plant tissue. When plants are exposed to soil microorganisms, ultraviolet (UV) light, or heavy metals, soybeans produce three very similar phytoalexins called glyceollin I, glyceollin II, and glyceollin III.

[0040] WO2011 / 069781 describes a polysaccharide capable of modulating an immune response, which is obtained from a plant of the species Camellia sinensis, wherein the backbone of the polysaccharide comprises an alternating rhamnogalacturonan-I domain and an α(1,4)-linked polygalacturonate or α(1,4)-linked oligogalacturonate domain, 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.

[0041] WO 2012 / 148277 describes a preparation having a dry matter content of at least 20% by weight, said preparation comprising at least 50% by weight of the dry matter of a mixture of pectic polysaccharides, including at least 20% (calculated on the weight of the pectic polysaccharides) of rhamnogalacturonan-I pectin having a molecular weight greater than 40 kDa, the mixture of pectic polysaccharides being characterized in that:

[0042] · the degree of methylation of galacturonic acid residues does not exceed 20%;

[0043] ● the degree of acetylation of galacturonic acid residues does not exceed 20%;

[0044] wherein when the preparation is diluted with an aqueous solution of 50 mM ammonium bicarbonate to a solids content of 2.5% by weight, the preparation does not form a gel. The use of the preparation as a medicament for modulating the immune response is also described. Summary of the Invention

[0046] The inventors of the present application have unexpectedly found that conditions associated with a disrupted composition or function of the gut microbiome, in particular 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 beets, said RG-I polysaccharides having a molecular weight of more than 15 kDa and having a backbone comprising a rhamnogalacturonan I domain and an optional α(1,4)-linked homogalacturonan domain, wherein the molar ratio of galacturonic acid residues to rhamnose residues in the backbone is in the range of 20:1 to 1:1.

[0047] Although the inventors do not wish to be bound by theory, it is believed that RG-I polysaccharides act as prebiotics by promoting high microbial diversity within the gut microbiota, by stimulating the growth or activity of beneficial bacteria (such as Akkermansia muciniphila and Bifidobacterium) and / or by increasing the resilience of the gut microbiota against perturbations.

[0048] It has further been found that intestinal fermentation of RG-I polysaccharides results in the formation of beneficial short-chain fatty acids. Surprisingly, the fermentation conversion of RG-I polysaccharides into short-chain fatty acids is accompanied by significantly less gas production than that observed for classical prebiotics such as inulin.

[0049] Accordingly, one aspect of the present invention relates to a prebiotic composition for modulating the gut microbiota of a subject, said use comprising orally administering said prebiotic composition to said subject, wherein said composition comprises at least 0.1% by weight of the dry matter of RG-I polysaccharide derived from fruits, carrots, peas, chicory or beets, said RG-I polysaccharide having a molecular weight of more than 15 kDa and having a backbone composed of galacturonic acid residues and rhamnose residues, said rhamnose residues being contained in α(1→4)-galacturonic acid-α(1→2)-rhamnose residues, wherein the molar ratio of galacturonic acid residues to rhamnose residues in said RG-I polysaccharide is in the range of 20:1 to 1:1.

[0050] The RG-I polysaccharide used according to the present invention can be isolated from fruits, carrots, peas, chicory or beets by water extraction (optionally in combination with enzymatic treatment (using, for example, polygalacturonase)).

[0051] Another aspect of the present invention relates to a prebiotic composition comprising:

[0052] · at least 0.1% by weight of the dry matter of the aforementioned RG-I polysaccharide; and

[0053] ● at least 1% by weight of the dry matter of one or more prebiotics selected from lactulose, inulin, fructooligosaccharides, galactooligosaccharides, lactooligosaccharides, guar gum, arabic gum or any combination thereof.

[0054] Yet another aspect of the present invention relates to a synbiotic composition comprising:

[0055] ● at least 0.1% by weight of the dry matter of RG-I polysaccharide; and

[0056] ● one or more probiotic microbial strains in the form of live microorganisms, inactivated microorganisms, microbial fragments and combinations thereof. DETAILED DESCRIPTION OF THE INVENTION

[0058] One aspect of the present invention relates to a prebiotic composition for modulating the gut microbiota of a subject, said use comprising orally administering said prebiotic composition to said subject, wherein said composition comprises at least 0.1% by weight of the dry matter of rhamnogalacturonan I (RG-I) polysaccharide derived from fruits, carrots, peas, chicory or beets, said RG-I polysaccharide having a molecular weight of more than 15 kDa and having a backbone composed of galacturonic acid residues and rhamnose residues, said rhamnose residues being contained in α(1→4)-galacturonic acid-α(1→2)-rhamnose residues, wherein the molar ratio of galacturonic acid residues to rhamnose residues in said RG-I polysaccharide is in the range of 20:1 to 1:1.

[0059] As used herein, the term "condition related to a disrupted composition or function of the gut microbiome" encompasses dysbiotic conditions (disrupted composition) and conditions related to an insufficient fermentation of the gut microbiome to produce essential metabolites (disrupted function). Short-chain fatty acids (acetates, propionates, and butyrates) are examples of such essential metabolites.

[0060] As used herein, the term "dysbiotic condition" refers to a condition that adversely affects the health of a subject and is caused by a significant deviation from a balanced gut microbiome (eubiotic).

[0061] As used herein, the term "branched polysaccharide" refers to a polysaccharide having a linear backbone of monosaccharide units linked together by glycosidic bonds, wherein at least one monosaccharide unit within the backbone carries a side chain of one or more glycosidically linked monosaccharide units.

[0062] The terms "backbone chain" and "backbone" are synonyms.

[0063] As used herein, the term "pectic polysaccharide" refers to an optionally branched polysaccharide having a molecular weight of more than 15 kDa and having a backbone composed of galacturonic acid residues and rhamnose residues, the rhamnose residues being included in α(1→4)-galacturonic acid-α(1→2)-rhamnose residues.

[0064] As used herein, the term "segment" refers to a sequence of two or more glycosidically linked monosaccharides within a polysaccharide backbone, excluding any side chains attached thereto.

[0065] As used herein, the term "domain" refers to a segment plus any side chains attached to the segment.

[0066] The term "rhamnogalacturonan-I segment" or "RG-I segment" refers to a segment composed of galacturonic acid (GalA) and rhamnose (Rha) pairs, wherein the GalA residue is linked to the Rha residue via positions 1 and 4, and the Rha residue is linked to the GalA residue via the anomeric and 2-OH positions, i.e., alternating α(1→4)-galacturonic acid-α(1→2)-rhamnose residues. The carboxyl groups of the galacturonic acid residues within RG-I can be esterified. The esterified galacturonic acid can be present in the form of a methyl ester or an acetyl ester.

[0067] The RG-I domain can contain side chains such as, for example, galactan, arabinan, and arabinogalactan side chains.

[0068] The term "rhamnogalacturonan-I polysaccharide" or "RG-I polysaccharide" refers to an optionally branched pectin polysaccharide that contains a backbone comprising one or more rhamnogalacturonan-I segments.

[0069] The term "α(1,4)-linked galacturonic acid segment" refers to a segment composed of α(1→4)-galacturonic acid residues.

[0070] In addition to the RG-I domain, the RG-I polysaccharides of the present invention may further comprise one or more of the following domains:

[0071] · Homogalacturonan (HG),

[0072] · Xylogalacturonan (XG),

[0073] · Apiogalacturonan (AG),

[0074] · Rhamnogalacturonan-II (RG-II).

[0075] The domains XG, AG, and RG-II typically represent only a small fraction of the RG-I polysaccharide.

[0076] The HG domain, XG domain, AG, and RG-II domain optionally present in the RG-I polysaccharides of the present invention comprise a backbone consisting of a linear chain of two or more α-(1-4)-linked D-galacturonic acids. The carboxyl groups of the galacturonic acid residues within the backbone of these domains may be esterified. The esterified galacturonic acid may be present in the form of a methyl ester or an acetyl ester.

[0077] The HG domain does not contain any side chains.

[0078] The backbone of the XG domain contains one or more side chains in the form of D-xylose.

[0079] The backbone of the AG domain contains one or more side chains composed of one or more D-apiose residues.

[0080] The backbone of RG-II contains one or more side chains that are not composed solely of D-xylose or D-apiose.

[0081] As used herein, the term "fruit" refers to the seed-bearing structure in flowering plants.

[0082] As used herein, the term "prebiotic" refers to a substance that selectively induces the growth or activity of microorganisms that contribute to the well-being of its host.

[0083] As used herein, the term "probiotic" refers to microorganisms that provide health benefits when administered orally in sufficient amounts. These microorganisms are selected from live microorganisms, inactivated microorganisms, microbial fragments, and combinations thereof.

[0084] The term "synbiotic" refers to a composition comprising a combination of (a) one or more prebiotics and (b) one or more probiotics.

[0085] The concentration of different polysaccharides and their monosaccharide composition can be determined by analytical techniques known to those skilled in the art. After acid hydrolysis, the monosaccharide composition can be appropriately determined by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

[0086] The molecular size distribution can be determined by high performance size exclusion chromatography using refractive index (RI) detection (for concentration), light scattering detection (for molecular weight detection), UV detection (to indicate the presence of proteins), and differential pressure detection (for intrinsic viscosity detection).

[0087] The above-mentioned analytical methods are described in: Analytical Biochemistry, Vol. 207, No. 1, 1992, pg 176 (for neutral sugar analysis) and Mol. Nutr. Food Res., Vol. 61, No. 1, 2017, 1600243 (for galacturonic acid analysis and molecular size distribution).

[0088] Unless otherwise indicated, all percentages mentioned herein refer to percentages by weight.

[0089] The subject to which the composition containing RG-I polysaccharide of the present invention is administered orally is preferably a mammal, more preferably a human subject. According to a preferred embodiment, the human subject is an infant (<4 years old) who is still developing its core mature microbiota or an elderly person (>50 years old) at risk of losing the diversity and resilience of its core mature microbiota.

[0090] Oral administration in the context of the methods of the present invention for treatment encompasses self-administration.

[0091] According to a preferred embodiment, the subject receiving the composition containing RG-I polysaccharide administered orally has a metabolic disorder or is at risk of having a metabolic disorder. Most preferably, the subject has a metabolic disorder.

[0092] Metabolic disorders that can be successfully treated (therapeutically or prophylactically) by the treatment of the present invention include overweight, obesity, metabolic syndrome, insulin deficiency or insulin resistance-related disorders, type 2 diabetes, glucose intolerance, dyslipidemia, hyperglycemia, hepatic steatosis, abnormal blood lipid levels, hypercholesterolemia, and elevated triglycerides. The treatment of the present invention is particularly suitable for the therapeutic or prophylactic treatment of overweight or obesity and insulin resistance.

[0093] According to another preferred embodiment, the subject has or is at risk of having intestinal barrier dysfunction. More preferably, the subject has intestinal barrier dysfunction.

[0094] The intestinal barrier or intestinal mucosal barrier refers to the properties of the intestinal mucosa that ensure adequate containment of unwanted luminal contents within the intestine while maintaining the ability to absorb nutrients. The separation it provides between the body and the luminal contents of the intestine prevents the uncontrolled transfer of luminal contents into the body. Its role in protecting mucosal tissues and the circulatory system from exposure to pro-inflammatory pathogens, toxins, and antigens is crucial for maintaining health and well-being. Intestinal barrier dysfunction is associated with many 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.

[0095] The subject receiving treatment for an intestinal disorder condition according to the present invention is preferably a subject having or at risk of having a pathogenic intestinal disorder condition, and most preferably a subject having such a pathogenic intestinal disorder condition. As used herein, "pathogenic" means that the condition is capable of causing or exacerbating a disease.

[0096] According to another preferred embodiment, the prebiotic composition is used to increase the intestinal fermentation production of short-chain fatty acids.

[0097] The RG-I polysaccharide used according to the present invention is preferably derived from plant sources selected from apples, bell peppers, blueberries, carrots, citrus fruits, grapes, peas, chicory, beets, and olives, okra, and combinations thereof. Even more preferably, the RG-I polysaccharide is derived from plant sources selected from apples (such as apple pomace), bell peppers, carrots, citrus peels, grapes, chicory, beets (such as beet pulp), olives (such as olive pulp), okra, and combinations thereof. Most preferably, the RG-I polysaccharide is derived from carrots or apples.

[0098] The RG-I polysaccharide is preferably incorporated into the prebiotic composition in the form of a pectin polysaccharide isolate rich in RG-I polysaccharide. Thus, in a particularly preferred embodiment, the RG-I polysaccharide accounts for at least 20% by weight, more preferably at least 40% by weight, even more preferably at least 50% by weight, and most preferably at least 60% by weight of the pectin polysaccharide present in the prebiotic composition.

[0099] The RG-I polysaccharide has a backbone comprising a rhamnogalacturonan-I segment and optionally an α(1,4)-linked homogalacturonan segment. The molar ratio of galacturonic acid residues to rhamnose residues in the RG-I polysaccharide ranges from 20:1 to 1:1. Preferably, the molar ratio of galacturonic acid residues to rhamnose residues in the RG-I polysaccharide ranges from 15:1 to 1:1, more preferably from 12:1 to 1:1, even more preferably from 10:1 to 1:1, and most preferably from 9:1 to 1:1.

[0100] Preferably, the rhamnose residues account for 3 - 50% of the monosaccharide residues in the backbone of the RG-I polysaccharide, more preferably 5 - 50%, and most preferably 10 - 50%.

[0101] The rhamnose residues generally account for 3 - 50% of all the monosaccharide residues contained in the RG-I polysaccharide (i.e., including the monosaccharide residues contained in the side chains), more preferably 3.5 - 40%, and most preferably 4 - 35%.

[0102] The galacturonic acid residues generally account for 50 - 97% of the monosaccharide residues in the backbone of the RG-I polysaccharide, more preferably 50 - 95%, and most preferably 50 - 90%.

[0103] The galacturonic acid residues generally account for 10 - 80% of all the monosaccharide residues contained in the RG-I polysaccharide (i.e., including the monosaccharide residues contained in the side chains), more preferably 15 - 70%, and most preferably 20 - 65%.

[0104] The RG-I polysaccharide generally has a molecular weight of at least 20 kDa. Preferably, the RG-I polysaccharide has a molecular weight of 25 kDa to 2,000 kDa, more preferably 30 kDa to 1,500 kDa, even more preferably 35 kDa to 1,200 kDa, and most preferably 40 kDa to 1,000 kDa.

[0105] The average molecular weight of the RG-I polysaccharide contained in the composition of the present invention preferably exceeds 30 kDa, more preferably exceeds 40 kDa, and most preferably exceeds 60 kDa.

[0106] Preferably, less than 85% of the galacturonic acid residues in the RG-I polysaccharide are esterified in the form of methyl esters. More preferably, the RG-I polysaccharide has an esterification degree of 0% to 70%, more preferably 0% to 60%, even more preferably 0% to 55%, and most preferably 0% to 50%.

[0107] Preferably, 0 - 95% of the galacturonic acid residues in the RG-I polysaccharide are esterified in the form of acetate esters. More preferably, the RG-I polysaccharide has an esterification degree of 5% to 90%, more preferably 7% to 50%, and most preferably 8% to 30%.

[0108] The main chain of RG-I polysaccharide is composed of galacturonic acid residues and rhamnose residues. If the RG-I polysaccharide contains one or more side chains, the polysaccharide may additionally contain residues of arabinose and / or galactose. In addition, the side chains of RG-I polysaccharide may provide a small amount of monomeric fucose, glucose, glucuronic acid, xylose and / or glucuronic acid residues. One or more side chains are preferably selected from galactan side chains, arabinan side chains and arabinogalactan side chains.

[0109] The arabinan side chain contains at least one or more α(1,5)-linked arabinose residues and is substituted at the 4-OH position of the rhamnose residue in the RG-I domain. The arabinan side chain can be linear or branched. If the side chain is linear, the side chain is composed of α(1,5)-linked arabinose residues. If the arabinan side chain is a branched side chain, one or more α-arabinose residues are linked to the 2-OH and / or 3-OH of α(1,5)-linked arabinose. 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.

[0110] The galactan side chain contains at least one or more β(1,4)-linked galactose residues and is substituted at the 4-OH position of the rhamnose residue in the RG-I domain.

[0111] The galactan side chain is preferably substantially linear (unbranched), i.e., less than 10 mol% of the galactose residues in the chain are β(1,3)-linked or β(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.

[0112] The arabinogalactan side chain is substituted at the 4-OH position of the 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 main chain, on which monomeric Galp units can be substituted at the O-6 or at the O-3 position. AGI is further substituted by 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 main chain modified with a (1→6)-β-D-Galp secondary chain, which is arabinosylated.

[0113] 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.

[0114] The molar ratio of galactose residues 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.

[0115] According to a preferred embodiment, at least 20% of the rhamnose residues in the RG-I segment are substituted at the 4-OH position. More preferably at least 30%, even more preferably at least 40%, and most preferably at least 45% of these rhamnose residues are substituted at the 4-OH position. Preferably, at most 90%, more preferably at most 80% of these rhamnose residues are substituted at the 4-OH position.

[0116] The prebiotic composition for use in the method of the present invention preferably comprises at least 0.2% by weight, more preferably 0.3 - 10% by weight, and most preferably 0.4 - 5% by weight of the dry matter of the RG-I polysaccharide as defined herein.

[0117] In addition to the RG-I polysaccharide, the prebiotic composition for use in the method of the present invention advantageously comprises one or more other prebiotics. Preferably, the composition comprises at least 1% by weight of the dry matter of one or more prebiotics, more preferably at least 3% by weight of the dry matter of one or more prebiotics, and the prebiotics are selected from lactulose, inulin, fructooligosaccharides, galactooligosaccharides, lactooligosaccharides, guar gum, and arabic gum.

[0118] If the RG-I polysaccharide is no longer entangled in the matrix of the cell wall material, it is considered to be particularly effective in the treatment method of the present invention. Thus, in a particularly preferred embodiment, the composition containing the RG-I polysaccharide of the present invention comprises at least 0.05% by weight of dry matter, more preferably at least 0.1% by weight of dry matter, even more preferably at least 0.2% by weight of dry matter, and most preferably at least 0.3% by weight of the dry matter of the water-soluble RG-I polysaccharide. The concentration of the water-soluble RG-I polysaccharide in the composition containing the RG-I polysaccharide can be determined by combining 100 ml of deionized water (20 °C) with a sufficient amount of the composition containing the RG-I polysaccharide to provide 2.5 g of dry matter, then stirring for 5 minutes and filtering through a 100 μm filter. The RG polysaccharide in the filtrate is the water-soluble RG-I polysaccharide.

[0119] According to a particularly preferred embodiment of the present invention, the composition of the present invention is orally administered to a subject in an amount providing at least 1 mg of RG-I polysaccharide / kg body weight / day over a period of at least 2 days. More preferably, over a period of at least 7 days, most preferably over a period of at least 14 days, an amount of at least 4 mg of RG-I polysaccharide / kg body weight / day, even more preferably at least 15 mg / kg body weight / day, and most preferably at least 20 - 100 mg of RG-I polysaccharide / kg body weight / day is provided by administering a composition containing RG-I polysaccharide.

[0120] According to another preferred embodiment, a composition containing RG-I polysaccharide is orally administered to a subject over a period of at least 21 days to provide an amount of RG-I polysaccharide of at least 4 mg of RG-I polysaccharide / kg body weight / day, more preferably 15 - 300 mg of RG-I polysaccharide / kg body weight / day.

[0121] It has been found that the prebiotic composition of the present invention is capable of inducing the growth of gut microbiota, which are believed to provide health benefits, in particular Akkermansia muciniphila and Bifidobacterium. Thus, in another preferred embodiment, a composition containing RG-I polysaccharide is capable of inducing the growth or activity of Akkermansia muciniphila and / or Bifidobacterium in the gut microbiota of a subject. Most preferably, the composition is capable of inducing the growth or activity of Akkermansia muciniphila in the gut microbiota of a subject.

[0122] It has been found that the prebiotic composition of the present invention is capable of inducing the production of short-chain fatty acids by the gut microbiota, with significantly less gas production compared to classical prebiotics such as inulin. Thus, in another preferred embodiment, a composition containing RG-I polysaccharide is capable of enhancing the gut fermentation production of short-chain fatty acids and reducing side effects associated with rapid gas production, such as gut discomfort, flatulence, and reflux.

[0123] According to a particularly preferred embodiment, the composition is selected from oral dosage units, powders, and spreads.

[0124] The oral dosage unit is preferably a capsule or a tablet. The oral dosage unit preferably has a weight of 50 to 1500 mg, more preferably 100 to 800 mg. The oral dosage unit typically contains at least 1 wt%, more preferably at least 20 wt%, and most preferably 40 - 90 wt% of RG-I polysaccharide.

[0125] Typically, the powder contains at least 0.5 wt%, more preferably at least 5 wt%, and most preferably 10 - 75 wt% of RG-I polysaccharide.

[0126] The composition in the form of a spread is preferably a water-in-oil emulsion, preferably a water-in-oil emulsion comprising 20 - 90% by weight of a fatty phase and 10 - 80% by weight of an aqueous phase. The spread preferably comprises at least 0.3% by weight, more preferably at least 1 - 10% by weight, and most preferably 1.5 - 16% by weight of RG-I polysaccharide.

[0127] Another aspect of the invention relates to a prebiotic composition, which comprises:

[0128] · at least 0.1% by weight of the dry matter of RG-I polysaccharide as defined above; and

[0129] · at least 1% by weight, preferably at least 3% by weight of the dry matter of one or more prebiotics selected from lactulose, inulin, fructooligosaccharides, galactooligosaccharides, lactooligosaccharides, guar gum and arabic gum.

[0130] Even more preferably, the product comprises at least 1% by weight of dry matter, more preferably at least 3% by weight of the dry matter of one or more prebiotics selected from lactulose, inulin, fructooligosaccharides, galactooligosaccharides and lactooligosaccharides.

[0131] The preferred embodiment of the prebiotic composition is the same as that described above for the prebiotic composition used in the method of the invention.

[0132] Yet another aspect of the invention relates to a synbiotic composition, which comprises:

[0133] · at least 0.1% by weight of the dry matter of RG-I polysaccharide as defined above; and

[0134] · one or more probiotic microbial strains in the form of live microorganisms, inactivated microorganisms, microbial fragments and combinations thereof.

[0135] The preferred embodiment of the synbiotic composition is the same as that described above for the prebiotic composition used in the method of the invention.

[0136] One or more probiotic microbial strains in the synbiotic composition are preferably live microbial strains, more preferably live bacterial strains.

[0137] One or more probiotic microbial strains may be selected from yeast strains, mold 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 mold strains include strains belonging to Aspergillus, Rhizopus, Mucor, Penicillium, and combinations thereof. Examples of suitable bacterial strains include strains belonging to Bifidobacterium, Bacteroides, Fusobacterium, Melissococcus, Propionibacterium, Enterococcus, Lactococcus, Staphylococcus, Peptostreptococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Faecalibacterium, Akkermansia, Oenococcus, Lactobacillus, Allobaculum, Eubacterium, and combinations thereof.

[0138] According to a particularly preferred embodiment, 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, Lactobacillus rhamnosus, Lactobacillus sake, Lactococcus lactis, Micrococcus varians, Pediococcus acidilactici, Pediococcus pentosaceus, Pediococcus halophilus, Streptococcus salivarius, Streptococcus thermophilus, Staphylococcus carnosus, Staphylococcus xylosus, Staphylococcus epidermidis, Akkermansia muciniphila, Faecalibacterium prausnitzii, Roseburia hominis, and Eubacterium hallii.

[0139] In the case where the probiotic microbial strain is applied in an inactive form, the synbiotic composition preferably contains one or more probiotic microbial strains or their equivalents at a concentration of 10 4 -10 10 cfu. More preferably, the synbiotic composition contains one or more probiotic microbial strains or their equivalents at a concentration of 10 5 -10 9 cfu.

[0140] Akkermansia muciniphila is preferably contained in the probiotic composition at a concentration of 10 5 -10 10 cfu / g, more preferably 10 6 -10 9 cfu / g.

[0141] Bifidobacterium is preferably at 10 6 -10 10cfu / g, more preferably 10 7 -10 9 cfu / g is included in the probiotic composition.

[0142] The present invention is further illustrated by the following non-limiting examples. Examples

[0143] Example 1

[0144] Using the procedure described below, the RG-I polysaccharide fraction was isolated from pilot-scale dried paprika powder (Paprika Mild 80 - 100 AtsaSteamtr - Felix Reverte S.A.).

[0145] The paprika material (100 kg) was washed three times with an 80% aqueous ethanol solution under gentle agitation, i.e., washed twice at 80 °C for 2 hours and then washed overnight at room temperature; 12.5% (w / v) was used each time to remove ethanol-soluble materials. The ethanol-insoluble residue was recovered each time by centrifugation (1000 G for 10 min). The ethanol-insoluble residue obtained after 3 washing cycles was dried, and 90 kg was extracted twice with 1000 L of hot water at a temperature of 95 °C for 90 minutes. Each time, the supernatant was retained after centrifugation at 1000 G for 10 minutes. Subsequently, the collected supernatant was filtered through a cloth and ultrafiltered using a 2 KDa molecular weight cut-off membrane to remove low molecular weight materials. The dried RG-I-rich extract was obtained by freeze-drying the retentate, yielding approximately 5 kg of dried RG-I-rich polysaccharide extract.

[0146] Characterization of the RG-I polysaccharide-rich extract

[0147] Molecular weight distribution:

[0148] The molecular weight distribution of the polysaccharide sample was determined by high performance size exclusion chromatography using refractive index (RI) detection (for concentration), light scattering detection (for molecular weight detection), UV detection (to indicate the presence of proteins) and differential pressure detection (for intrinsic viscosity detection). Pullulan molecular weight standards were used for calibration.

[0149] Monosaccharide composition:

[0150] The polysaccharide sample was dissolved in 0.5 M aqueous trifluoroacetic acid and hydrolyzed at 120 °C for 2 h. The sample was then neutralized with NaOH and kept frozen until analyzed by high-pH anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). The uronic acid in the sample was determined by using a colorimetric metahydroxybiphenyl assay (Achmed & Labavitch, J. Food Biochem, 1978, 361)) automated on an autoanalyzer (Skalar).

[0151] Degree of esterification

[0152] The polysaccharide sample was treated with sodium hydroxide (0.25 M, 5 h, 20 °C) and then neutralized. After incubation with the developing reagent (acetylacetone and acetic acid in 2 M ammonium acetate), the absorbance of the released methanol was measured at 420 nm. The released acetic acid was determined using a K-ACETAF acetic acid assay kit (Megazyme). Beet pectin with known degrees of methylation and acetylation was used as a standard. The degree of esterification was expressed as the percentage of the molar amounts of released methanol and acetic acid relative to the amount of uronic acid.

[0153] The molecular characteristics of the RG-I polysaccharide fraction are shown in Tables 1a and 1b.

[0154] Table 1a

[0155]

[0156]

[0157] Table 1b

[0158] Molecular ratio Red bell pepper GalA / Rha 14 Ara / Rha 1.8 Gal / Rha 1.8 Degree of methylation % 39 Degree of acetylation % 9.0

[0159] Example 2

[0160] Six-week-old specific pathogen-free female C57BL / 6 mice were allowed free access to sterile drinking water and a semi-synthetic irradiated AIN-93G diet (Research Diet Services, Wijk bij Duurstede, The Netherlands) containing 30 mmol / kg calcium (non-infected and infected groups) or the same diet supplemented with 1% (w / w) of an RG-I-rich extract from bell peppers.

[0161] Mice were randomly assigned to treatment groups and housed three per cage for 2 weeks, then singly housed for an additional week. Reeves et al. described the composition of the AIN-93G diet (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).

[0162] Feces were collected at baseline before dietary intervention and 3 weeks after dietary intervention. Genomic DNA was extracted from fecal samples according to the manufacturer's protocol (Zymoresearch). Microbial identification of the isolated gDNA was performed by 16S rRNA gene sequencing. Specific primers were used for PCR amplification of the target genomic region, such as the V3-V4 or V4 hypervariable region of the 16S rRNA gene. Paired-end sequence reads were generated using the Illumina MiSeq system. FASTQ sequence files were generated using the Illumina Casava pipeline version 1.8.3. Initial quality assessment was based on Illumina Chastity filtering. Subsequently, reads containing PhiX control signals were removed using an in-house filtering protocol (Baseclear). Additionally, reads containing (partial) adapters were trimmed (down to a minimum read length of 50 bp). A second quality assessment was based on the remaining reads using the FASTQC quality control tool version 0.10.0.

[0163] Bacterial DNA sequencing

[0164] Illumina Miseq data was analyzed using a workflow that employed the Quantitative Insights Into Microbial Ecology (QIIME, v8) pipeline (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). The data was demultiplexed and filtered for mismatched barcodes and short reads (>50 nt). Open-reference OTU picking was performed in QIIME using the Silva 111 database, chimeras were detected via USEARCH and filtered out from the OTUs. The Silva 111 database was used again to generate biom files and phylogenetic tree files from the filtered OUTs. Further outputs were generated via QIIME, such as filtered reads per sample, PD whole-tree diversity measurements, and taxonomic distributions at levels 1 - 6 with relative abundances.

[0165] Effect of RG-I polysaccharide-rich extract on the microbial composition

[0166] Illumina 16S rRNA sequencing was performed on individual fecal samples from each group of 12 mice to further understand the microbial composition. 26x10 3 to 79x10 4 reads were obtained per sample. The results are shown in Tables 2, 3, and 4.

[0167] Table 2

[0168]

[0169] * indicates significance relative to the RG-I baseline

[0170] Table 3

[0171]

[0172] * indicates significance relative to the RG-I baseline

[0173] Table 4

[0174]

[0175] *Indicates significance relative to the RG-I baseline

[0176] At the phylum level, the addition of RG-I-rich polysaccharide extract to the diet significantly increased the abundances of Actinobacteria and Verrucomicrobia, while the abundances of Bacteroidetes and Proteobacteria decreased (>0.5% abundance; Wilcoxon, P < 0.05), resulting in an increased Firmicutes / Bacteroidetes ratio.

[0177] At the genus level, redundancy analysis indicated that the addition of RG-I-rich polysaccharide extract to the diet had a significant effect on the microbial composition (P < 0.05; Monte Carlo permutation). Inclusion of RG-I-rich polysaccharide extract in the diet led to higher abundances of the genus Bifidobacterium (Actinobacteria), Allobaculum (Firmicutes), Akkermansia (Verrucomicrobia), and unassigned bacterial groups.

[0178] Example 3

[0179] Indigestible polysaccharides are mainly digested by the gut microbiota in the human large intestine. This may lead to the growth of health-beneficial bacteria, a decrease in pH, an increase in resistance to gut pathogens, and the regulation of the metabolic activity of the microbiota. Beneficial (prebiotic) functional carbohydrates will promote the production of health-beneficial metabolites such as short-chain fatty acids (SCFAs, i.e., acetate, propionate, and butyrate), while reducing the production of unwanted metabolites from protein metabolism (such as branched-chain SCFAs and ammonia).

[0180] The effect of plant-derived polysaccharide extracts on the metabolic activity of the microbiota was tested using an established short-term colon incubation model.

[0181] At the start of the incubation, a sugar-depleted basal colon medium containing nutrients present in the colon, such as host-derived glycans like mucins, was introduced into 70 mL penicillin bottles that already contained the test extract (5 g / L final concentration). The bottles were sealed with rubber stoppers and an anaerobic habitat was obtained by flushing with N2. Subsequently, a human faecal inoculum was prepared by mixing freshly collected faecal samples with anaerobic phosphate buffer. After homogenization and removal of particles by centrifugation (2 min, 500 g), the faecal inoculum was added to the different bottles. At this point, incubation was started for 48 h, during which the temperature was controlled at 37 °C and continuous mixing was ensured by an oscillator (90 rpm). Samples were taken after 6 h, 24 h and 48 h of incubation for pH (Senseline F410; ProSense, Oosterhout, The Netherlands), barometric pressure (handheld pressure indicator CPH6200; Wika, Echt, The Netherlands) and SCFA analysis. SCFAs (which are acetate, propionate, butyrate) and branched SCFAs (isobutyrate, isovalerate and isohexanoate) 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).

[0182] The sugar-depleted colon medium and inulin (which is a well-known prebiotic (Beneo, DP ≥ 23, ∼100% inulin)) were used as negative and positive references, respectively.

[0183] Sample A was produced from paprika powder (Paprika poeder, Natural Spices Mijdrecht, the Netherlands) by water extraction (10% w / w, 2 h at 90 °C), centrifugation to remove insoluble residues, filtration (cut-off 40 kDa) to remove small molecules and drying to obtain a powder.

[0184] Sample B was produced from dried carrot pomace (residue from carrot juice production (carrot fibre powder, GreendFields, Poland)). Sample B was produced by using pectinase ( It is produced by subjecting Ultra Mash, Novozymes) to aqueous extraction (10% w / w, 2 hours at 45 °C), heat inactivation (90 °C, 10 minutes), removal of insoluble residues by decantation, ultrafiltration (40 kDa cut-off), and finally drying.

[0185] Sample C was extracted from apple pomace powder (apple pomace, GreendFields, Poland) in the same manner as sample B.

[0186] Sample D was produced from Ground Okra, My Foods, Blue mountain peak, UK) by using hot water extraction (10% w / w, 2 hours at 90 °C) and dialysis against water for several hours to remove low molecular weight materials. The dialyzed extract was then lyophilized.

[0187] The determination of the monosaccharide composition of the above samples was carried out as described in Example 1. The results are shown in Table 5.

[0188] Table 5

[0189]

[0190]

[0191] The results of the incubation experiments are shown in Table 6.

[0192] Table 6

[0193]

[0194] The results showed that all four plant-derived RG-I polysaccharide extracts were readily fermented by the gut microbiota.

[0195] All RG-I polysaccharide extracts increased the production of SCFAs. In fact, all extracts were found to increase SCFA production to levels similar to or exceeding those observed for inulin.

[0196] All RG-I polysaccharide extracts decreased the production of branched SCFAs and ammonia.

[0197] Surprisingly, the gas production observed for all RG-I polysaccharide extracts was substantially lower than that observed for inulin. Excessive gas production can lead to intestinal discomfort and flatulence, which are well-described undesirable side effects of most classical prebiotics, including inulin.

[0198] Example 4

[0199] RG-I polysaccharide fractions were extracted from different source materials for testing in the short-term colon incubation model described in Example 3. Sugar-depleted colon medium and inulin (which is a well-known prebiotic (Beneo, DP≥23, ∼100% inulin)) were used as negative and positive references, respectively.

[0200] Sample A was produced from dried and ground pea hulls (from (ex) Cosucra, Warcoing, Belgium). The powder was dispersed in demineralized water (100 g / L), enzymatically pre-hydrolyzed with heat-stable α-amylase (Megazyme) at 90 °C for 30 min, and further hydrolyzed with pectinase (2 h at 45 °C, 0.2 v / v% Ultra Mash, Novozymes). Enzymolysis was terminated by heating at 100 °C for 10 min, then centrifuged (18,000 g, 10 min), and the supernatant was extensively dialyzed using a 12 - 14 kDa cut-off membrane (Visking, London, UK). The material was then lyophilized.

[0201] Sample B was produced from dried and ground sugar beet pulp (from Suiker Unie, Dinteloord, NL) using the same method, but omitting the α-amylase pre-incubation step.

[0202] Sample C was produced from dried and ground chicory pulp (from Cosucra, Warcoing, Belgium) using the same method, but omitting the α-amylase pre-incubation step.

[0203] The monosaccharide composition of the above samples was determined using the method described in Example 1. The results are shown in Table 10.

[0204] Table 10

[0205]

[0206] The results of the incubation experiments are shown in Table 11.

[0207] Table 11

[0208]

[0209]

[0210] These results show that these RG-I polysaccharide extracts from different plant sources are readily fermented by the gut microbiota.

[0211] All RG-I polysaccharide extracts increased SCFA production. In fact, all extracts were found to increase SCFA production to levels similar to or exceeding those observed for inulin.

[0212] All RG-I polysaccharide extracts decreased the production of branched-chain SCFAs and ammonia compared to the media control.

Claims

1. Use of a pectic polysaccharide of rhamnogalacturonan I (RG-I) polysaccharide in the preparation of a prebiotic composition for modulating the gut microbiota of a subject, said use comprising orally administering said prebiotic composition to said subject, wherein said composition comprises 0.1 - 10% by weight of the dry matter of RG-I polysaccharide derived from apple, carrot, chicory or beet, said RG-I polysaccharide having a molecular weight of more than 15 kDa and having a backbone composed of galacturonic acid residues and rhamnose residues, said rhamnose residues being contained in α(1→4)-galacturonic acid-α(1→2)-rhamnose residues, wherein the molar ratio of galacturonic acid residues to rhamnose residues in said RG-I polysaccharide is in the range of 20:1 to 1:1, wherein the molar ratio of arabinose residues to rhamnose residues in said RG-I polysaccharide does not exceed 8:1, wherein the molar ratio of galactose residues to rhamnose residues in said RG-I polysaccharide does not exceed 5:1, wherein said galacturonic acid accounts for 20 - 65 wt.% of the monosaccharide residues in said RG-I polysaccharide, and rhamnose accounts for 4 - 35 wt.% of the monosaccharide residues in said RG-I polysaccharide, and wherein said RG-I polysaccharide is isolated from apple, carrot, chicory or beet by aqueous extraction.

2. The use according to claim 1, wherein said composition is ingested by said subject in an amount providing at least 1 mg of RG-I polysaccharide / kg body weight / day over a period of at least 3 days.

3. The use according to claim 1 or 2, wherein said RG-I polysaccharide accounts for at least 20% by weight of the pectic polysaccharides present in said prebiotic composition.

4. The use according to claim 1 or 2, wherein the molar ratio of galacturonic acid residues to rhamnose residues in said RG-I polysaccharide does not exceed 15:

1.

5. The use according to claim 1 or 2, wherein the molar ratio of galacturonic acid residues to rhamnose residues in said RG-I polysaccharide does not exceed 12:

1.

6. The use according to claim 1 or 2, wherein the molar ratio of galacturonic acid residues to rhamnose residues in said RG-I polysaccharide does not exceed 10:

1.

7. The use according to claim 1 or 2, wherein less than 85% of the galacturonic acid residues in said RG-I polysaccharide are esterified in the form of methyl esters.

8. A synbiotic composition, comprising: ● 0.1 - 10% by weight of the dry matter of rhamnogalacturonan I (RG-I) polysaccharide derived from carrot or apple, said RG-I polysaccharide having a molecular weight of more than 15 kDa and having a main chain composed of galacturonic acid residues and rhamnose residues, said rhamnose residues being contained in α(1→4)-galacturonic acid-α(1→2)-rhamnose residues, wherein the molar ratio of galacturonic acid residues to rhamnose residues in said RG-I polysaccharide is in the range of 20:1 to 1:1, wherein the molar ratio of arabinose residues to rhamnose residues in said RG-I polysaccharide does not exceed 8:1, wherein the molar ratio of galactose residues to rhamnose residues in said RG-I polysaccharide does not exceed 5:1, wherein the galacturonic acid accounts for 20 - 65 wt.% of the monosaccharide residues in said RG-I polysaccharide, and rhamnose accounts for 4 - 35 wt.% of the monosaccharide residues in said RG-I polysaccharide, and wherein said RG-I polysaccharide is isolated from apple or carrot by aqueous extraction; and ● 10 4 -10 10 cfu / g of Akkermansia muciniphila and / or 10 6 -10 10 cfu / g of Bifidobacterium.

9. The composition according to claim 8, wherein the composition is selected from capsules, tablets, powders and spreads.

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