Prebiotic compositions to stabilize healthy gut flora

Abstract

The present inventors have discovered that a healthy gut microbiota can be stabilized in a subject by orally administering a prebiotic composition comprising at least 0.1% by weight of a dry matter of rhamnogalacturonan I (RG-I) polysaccharide having a molecular weight greater than 10 kDa and having a backbone composed of galacturonic acid and rhamnose residues, wherein the rhamnose residues are contained in alpha(1→4)-galacturonic-alpha(1→2)-rhamnose residues, and wherein the molar ratio of galacturonic acid to rhamnose residues in the RG-I polysaccharide is within the range of 20:1 to 1:1.

Description

[Technical Field]

[0001] The present invention relates to the use of a prebiotic composition for stabilizing a healthy gut microbiota, comprising a rhamnogalacturonan I (RG-I) polysaccharide having a molecular weight greater than 10 kDa and a backbone composed of galacturonic acid and rhamnose residues, the rhamnose residues being contained in alpha(1→4)-galacturonic-alpha(1→2)-rhamnose residues, and the molar ratio of galacturonic acid to rhamnose residues in the RG-I polysaccharide being in the range of 20:1 to 1:1. [Background technology]

[0002] The human gastrointestinal tract provides habitat and nutrients for a large and diverse ecosystem of microorganisms that play a key role in regulating digestion, metabolism, and immune function, and have a profound impact on the gastrointestinal tract. It is now well established that a healthy gut microbiota contributes to the overall health of the host.

[0003] The composition and stability of the microbiota are influenced by the host's genetic background, environmental conditions or stressors, including diet, lifestyle, and the use of medications (e.g., antibiotics), as well as the host's developmental stage (age). This results in a constant and complex interaction between the host and key components of the local microbial ecosystem: the microorganisms, the host immune system, the local epithelial barrier, and, in the case of the gut, the enteric nervous system.

[0004] Consumption of probiotics and / or prebiotics can have a favorable effect on the intestinal microflora. Probiotics are "live microorganisms that, when ingested in adequate amounts, confer a beneficial effect on the health of the host" (as defined by the World Health Organization). 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 microorganisms in the gastrointestinal tract.

[0005] Most known prebiotics are simple oligomers of identical sugars (such as fructose, galactose, or arabinose) linked by glycosidic bonds. They stimulate the selective growth of microbial species that have 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 easily fermentable substrates include intestinal discomfort, bloating, and vomiting. These side effects are caused by the rapid production of fermentable gases.

[0006] WO2011 / 069781 describes a polysaccharide capable of modulating immune responses, the polysaccharide being obtained from a plant of the species Camellia sinensis, the polysaccharide backbone comprising alternating rhamnogalacturonan I domains and alpha(1,4)-linked polygalacturonic acid or alpha(1,4)-linked oligogalacturonic acid domains, the molar ratio of galacturonic acid residues to rhamnosyl residues in the polysaccharide backbone being in the range of 2.5:1 to 1:1, and the polysaccharide having a molecular weight of at least 70 kDa.

[0007] WO2012 / 148277 describes a preparation having a dry matter content of at least 20% by weight, which comprises at least 50% by weight of a dry matter mixture of pectic polysaccharides, including at least 20% by weight, calculated on the weight of pectic polysaccharides, of rhamnogalacturonan I pectin having a molecular weight of more than 40 kDa, said mixture of pectic polysaccharides comprising: a degree of methylation of galacturonic acid residues of 20% or less; degree of acetylation of galacturonic acid residues of 20% or less; and the preparation does not form a gel when diluted to 2.5% solids by weight with an aqueous solution of 50 mM ammonium bicarbonate. The use of the preparation as a pharmaceutical for modulating immune responses is also described.

[0008] WO2020 / 0048609 describes a prebiotic composition for use in a method for the therapeutic or preventive treatment of a disorder associated with a disruption in the composition or functionality of the intestinal microbiome in a subject, the use comprising oral administration of the prebiotic composition to the subject, the composition comprising at least 0.1% by weight of a dry matter of RG-I polysaccharide derived from fruit, carrot, pea, chicory, or sugar beet, the RG-I polysaccharide having a molecular weight of greater than 15 kDa and a backbone consisting of galacturonic acid residues and rhamnose residues, the rhamnose residues being contained in alpha(1→4)-galacturonic-alpha(1→2)-rhamnose residues, and the molar ratio of galacturonic acid to rhamnose residues in the RG-I polysaccharide being within the range of 20:1 to 1:1.

[0009] Van den Abbeele et al., A Novel Non-Digestible, Carrot-Derived Polysaccharide (cRG-I) Selectively Modulates Human Gut Microbiota while Promoting Gut Integrity: An Integrated In Vitro Approach, Nutrients 2020, 12, 1917; doi:10.3390 / nu12071917, describes three different in vitro models in which the prebiotic properties of carrot RG-I (cRG-I) were evaluated.

[0010] McKay et al., Development of an affordable, sustainable, and efficacious plant-based immunomodulatory food ingredient based on bell pepper or carrot RG-I pectic polysaccharides, Nutrients 2021, 13, 963, https: / / doi.org / 10.3390 / nu13030963, describes an in vitro fermentation assay in which the prebiotic properties of cRG-I and bell pepper RG-I were evaluated by measuring the short-chain fatty acids produced and changes in microbial species at the phylum level.

[0011] Van den Abbeele et al., Consistent Prebiotic Effects of Carrot RG-I on the Gut Microbiota of Four Human Adult Donors in the SHIME® Model Despite Baseline Individual Variability, Microorganisms 2021, 9, 2142, https: / / doi.org / 10.3390 / microorganisms9102142, describes the evaluation of the impact of cRG-I on gut microbiota composition and function upon repeated administration using M-SHINE technology (three doses per day for three weeks resulting in ≤5 g / day of cRG-I). Consistent effects across the four stimulated adult donors included enhanced levels of acetate (+21.1 mM), propionate (+17.6 mM), and to a lesser extent, butyrate (+4.1 mM), as well as consistent microbial changes across different donors by the end of the experiment.

[0012] Cantu-Jungles et al. (New View on Dietary Fiber Selection for Predictable Shifts in Gut Microbiota, ASM Journals mBio(2020),11(1)e02179019) propose that dietary fibers can be hierarchically classified according to their specificity for gut microbiota. Highly specific fibers possess chemical and physical properties that reduce competition for their substrates and allow their utilization by only a narrow group of bacteria in the gut. The use of such fibers as prebiotics targeting specific microorganisms results in predictable changes that are independent of the background microbial composition. Summary of the Invention

[0013] The gut microbiota of healthy subjects is constantly adapting and responding to external stress factors / stimuli, and is greatly influenced by diet.The present inventors have surprisingly discovered that oral administration of RG-I polysaccharide can improve the stability of the gut microbiota of healthy subjects.More specifically, the present inventors have discovered that administration of RG-I polysaccharide helps to form a gut microbiota that exhibits a wide range of species diversity while at the same time being less susceptible to strain factors such as antimicrobial diet, medication, or stress, thereby becoming more robust.

[0014] The present invention relates to a prebiotic composition for use in stabilizing a healthy gut microbiota in a subject by selective prebiotic stimulation of at least one taxonomic group of commensal bacteria, the prebiotic composition comprising at least 0.1% by weight of a dry matter of rhamnogalacturonan I (RG-I) polysaccharide having a molecular weight greater than 10 kDa and having a backbone composed of galacturonic acid and rhamnose residues, the rhamnose residues being contained in alpha(1→4)-galacturonic-alpha(1→2)-rhamnose residues, the molar ratio of galacturonic acid to rhamnose residues in the RG-I polysaccharide being in the range of 20:1 to 1:1, and the use comprising oral administration of 0.1 to 5 grams of RG-I polysaccharide per day to the subject for at least three days.

[0015] The stabilization of healthy gut flora achieved by the present invention entails a reduction in the intra-individual variability of gut microbiota and a reduction in the inter-individual variability of gut microbiota within a group of subjects.

[0016] Although the inventors do not wish to be bound by theory, the stabilizing effect on the intestinal microbiota achieved by oral administration of RG-I polysaccharide is thought to be related to the moderate to high specificity of RG-I polysaccharide as a dietary fiber. The inventors have discovered that fibers with moderate to high specificity are particularly suitable for stabilizing a healthy intestinal microbiota. Low-specificity fibers, such as inulin, are utilized by most microbial species in the intestinal microbiota, thereby maintaining the same microbiota diversity as before exposure to inulin, leading to large differences between subjects. High-specificity fibers, such as xanthan, are utilized by only a narrow range of microbial species but are not consistently present in the microbiota of each subject. Xanthan is fermented only when specific species capable of utilizing xanthan are present in the intestinal microbiota, resulting in responders and non-responders.

[0017] RG-I polysaccharide, with its moderate to high specificity, increases the abundance of bacterial subsets commonly present in healthy adult humans, thereby achieving a significant homogenization effect on the intestinal microbiota and reducing interindividual variability, providing the advantage of consistent and predictable effects on the intestinal microbiota when orally administered. Because prebiotics such as inulin and xanthan do not have this stabilizing effect, the effect on stabilizing healthy intestinal microbiota is a unique effect secondary to the prebiotic properties of RG-I polysaccharide.

[0018] Reducing intra-individual variation in the microbiota over time is advantageous, as variations in the composition of the microbiota in response to various stresses can lead to intestinal discomfort or variations in metabolism and / or uptake of nutrients, dietary supplements and / or drugs.

[0019] The reduction in inter-individual variation in the microbiota provides the benefit of, for example, making it easier to design dietary supplements or drugs that work well in most individuals. Since tested compounds "land" on a heterogeneous group of subjects, all characterized by their individual microbiota, the substantial inter-individual variation in a group of healthy subjects can lead to variable efficacy or inaccurate recommended dosages for dietary supplements / drugs, or inconclusive results in human intervention studies.

[0020] The beneficial effects of orally administered RG-I polysaccharide on the gut microbiota can be enhanced by combining RG-I polysaccharide with one or more other medium to high specificity or high specificity fibers. at least 0.1% by weight of the dry matter of rhamnogalacturonan I (RG-I) polysaccharide as defined above; and At least 0.1% by weight of a dry matter of prebiotic fiber selected from beta-glucan, arabinoxylan, type 2 resistant starch, and combinations thereof. The present invention relates to a prebiotic composition comprising: [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 shows a schematic diagram of the structure of a pectin polysaccharide, comprising the five polysaccharide domains described above. [Figure 2] FIG. 1 shows a schematic representation of the structure of RG-I. [Figure 3] FIG. 1 shows the results of intra-individual Bray-Curtis dissimilarity for the three treatment groups in the Example. [Figure 4] FIG. 1 shows that the mean generalized UniFrac distance from subjects in the Examples to other subjects in the same group is lower at the end of the RG-I supplementation period than before the start of cRG-I supplementation. [Figure 5] FIG. 1 shows the results of inter-individual weighted UniFrac distances for various experimental groups in the Examples. DETAILED DESCRIPTION OF THE INVENTION

[0022] A first aspect of the present invention relates to a prebiotic composition for use in stabilizing a healthy gut microbiota in a subject by selective prebiotic stimulation of at least one taxonomic group of commensal bacteria, the prebiotic composition comprising at least 0.1% by weight of a dry matter of rhamnogalacturonan I (RG-I) polysaccharide having a molecular weight greater than 10 kDa and having a backbone composed of galacturonic acid and rhamnose residues, the rhamnose residues being contained in alpha(1→4)-galacturonic-alpha(1→2)-rhamnose residues, the molar ratio of galacturonic acid to rhamnose residues in the RG-I polysaccharide being in the range of 20:1 to 1:1, and the use comprising oral administration of 0.1 to 5 grams of RG-I polysaccharide per day to the subject for at least three days.

[0023] In some aspects, the invention also relates to a method for stabilizing a healthy gut microbiota in a subject by selective prebiotic stimulation of at least one taxonomic group of commensal bacteria, comprising administering a prebiotic composition comprising at least 0.1% by weight of a dry matter of rhamnogalacturonan I (RG-I) polysaccharide having a molecular weight greater than 10 kDa and having a backbone composed of galacturonic acid and rhamnose residues, wherein the rhamnose residues are contained in alpha(1→4)-galacturonic-alpha(1→2)-rhamnose residues, and wherein the molar ratio of galacturonic acid to rhamnose residues in the RG-I polysaccharide is in the range of 20:1 to 1:1, and the method comprises orally administering to the subject 0.1 to 5 grams of RG-I polysaccharide per day for at least three days.

[0024] In some aspects, the invention also relates to the use of RG-I polysaccharide in the manufacture of a prebiotic composition for stabilizing a healthy gut microbiota in a subject through selective prebiotic stimulation of at least one taxonomic group of commensal bacteria, the prebiotic composition comprising at least 0.1% by weight of a dry matter of rhamnogalacturonan I (RG-I) polysaccharide having a molecular weight greater than 10 kDa and having a backbone composed of galacturonic acid and rhamnose residues, the rhamnose residues being contained in alpha(1→4)-galacturonic-alpha(1→2)-rhamnose residues, wherein the molar ratio of galacturonic acid to rhamnose residues in the RG-I polysaccharide is within the range of 20:1 to 1:1, and the use comprises oral administration of 0.1 to 5 grams of RG-I polysaccharide per day to the subject for at least three days.

[0025] In some aspects, the invention also relates to use of a prebiotic composition for stabilizing a healthy gut microbiota in a subject through selective prebiotic stimulation of at least one taxonomic group of commensal bacteria, the prebiotic composition comprising at least 0.1% by weight of a dry matter of rhamnogalacturonan I (RG-I) polysaccharide having a molecular weight greater than 10 kDa and having a backbone composed of galacturonic acid and rhamnose residues, the rhamnose residues being contained in alpha(1→4)-galacturonic-alpha(1→2)-rhamnose residues, wherein the molar ratio of galacturonic acid to rhamnose residues in the RG-I polysaccharide is within the range of 20:1 to 1:1, and the use comprises oral administration of 0.1 to 5 grams of RG-I polysaccharide per day to the subject for at least three days.

[0026] In some jurisdictions, administration of a prebiotic composition to a subject is considered non-therapeutic. In these instances, the invention may have the language defined above by a method comprising administering a prebiotic composition. For clarity, the method may also be defined as a non-therapeutic method. By definition, the term "non-therapeutic" excludes any therapeutic effect.

[0027] The term "microbiota" as used herein refers to the collection of microorganisms found in a particular environment. The gut microbiota refers to the collection of microorganisms that inhabit the intestine.

[0028] The term "intra-individual variation" as used herein refers to the variation of the gut microbiota over time within the same subject.

[0029] The term "inter-individual variation" as used herein refers to the variation in gut microbiota when comparing gut microbiota within a group of subjects.

[0030] The term "diversity index" as used herein refers to a quantitative measure that reflects how many different species are present in a given gut microbiota. Suitable diversity indices include, for example, the Bray-Curtis dissimilarity index; weighted, unweighted, or generalized UniFrac distance; Jaccard distance; the Shannon diversity index; and the Simpson diversity index, which represents interrelationships.

[0031] The term "low specificity fiber" as used herein refers to indigestible fibers, such as inulin, that possess chemical and physical properties that allow them to be utilized as substrates by a wide variety of commensal gut bacteria.

[0032] The term "moderate to high specificity fiber" as used herein refers to indigestible fibers, such as RG-I polysaccharide or beta-glucan, that possess chemical and physical properties that allow them to be utilized as substrates by a small group of gut commensal organisms that commonly inhabit healthy subjects.

[0033] The term "highly specific fiber" as used herein refers to indigestible fibers, such as xanthan, that possess chemical and physical properties that allow them to be utilized as substrates by only a small subset of gut commensal bacteria that do not reside in all healthy individuals.

[0034] The term "or" also includes "and," unless otherwise indicated. In other words, the phrase "a composition comprising A or B" includes compositions comprising A and B.

[0035] The subject is preferably a mammalian subject, more preferably a human subject.

[0036] The use of the prebiotic composition according to the present invention preferably stimulates the growth of probiotic bacteria of at least one taxonomic group selected from the phyla Bacteroidetes, Actinobacteria, Firmicutes and combinations thereof.

[0037] In a preferred embodiment, the use reduces the intraindividual variability of the gut microbiota in a subject. The change in intraindividual variability is i. collecting fecal samples from the same subject at different time points, wherein a first fecal sample is collected before the start of oral administration of the prebiotic composition and a second fecal sample is collected at least 3 days after the start of oral administration of the prebiotic composition; ii. To determine the microbiota composition in these fecal samples; and iii. Calculating diversity indices to measure the (dis)similarity between the microbiota compositions of these fecal samples can be appropriately determined by

[0038] In another preferred embodiment, the use reduces the inter-individual variability of the gut microbiota within a group of subjects. i. collecting a fecal sample from each of the subjects in the group; ii. To determine the microbiota composition in these fecal samples; and iii. Calculating diversity indices to measure the (dis)similarity between the microbiota compositions in these fecal samples can be appropriately determined by

[0039] The change in inter-individual variability within a group of subjects is preferably determined by comparing the inter-individual variability of the group before the group began oral administration of the prebiotic composition with the inter-individual variability of the group at least 3 days after the group began oral administration of the prebiotic composition.

[0040] The diversity index is preferably selected from Bray-Curtis dissimilarity, unweighted UniFrac distance, weighted UniFrac distance, and generalized UniFrac distance. More preferably, for determining intra-individual variation, Bray-Curtis dissimilarity is used as the diversity index. Alternatively, more preferably, for determining inter-individual variation, generalized UniFrac distance is used as the diversity index.

[0041] The use preferably comprises oral administration of a dietary supplement or drug before, simultaneously with, or after oral administration of a prebiotic composition, where the dietary supplement or drug affects the intestinal flora or requires metabolism by the intestinal flora for its efficacy. Expressions such as "oral administration of a dietary supplement or drug before, simultaneously with, or / and after oral administration of a prebiotic composition" refer herein to administration of the dietary supplement or drug occurring within a time interval of -8 hours to +8 hours relative to the time of oral administration of the prebiotic composition (0 hours).

[0042] In a preferred embodiment, the efficacy of a dietary supplement or drug in a subject is improved by reducing the intra-individual variability of the gut microbiota. In another preferred embodiment, reducing the inter-individual variability of the gut microbiota within a group of subjects is applied to a method for evaluating the efficacy of a dietary supplement or drug in a group of subjects.

[0043] The RG-I polysaccharide of the present invention is a type of pectic polysaccharide (or pectin), which is a structural heteropolysaccharide present in the primary cell walls of land plants.

[0044] Pectic polysaccharides contain variable amounts of the following polysaccharide domains: (i) Homogalacturonan (HG) (ii) Xylogalacturonan (XG) (iii) Apiogalacturonan (AG) (iv) rhamnogalacturonan I (RG-I) and (v) Rhamnogalacturonan II (RG-II) It is a heterogeneous group of polysaccharides, including:

[0045] A schematic diagram of the structure of a pectin polysaccharide, including the five polysaccharide domains mentioned above, is shown in Figure 1. Note that polysaccharide domains AG, XG, and RG-II typically represent only a small portion of the pectin polysaccharide.

[0046] The polysaccharide domains HG, AG, XG and RG-II each contain a backbone consisting of a linear chain of alpha(1→4)-linked D-galacturonic acid monosaccharide units (GalA).

[0047] Only RG-I contains a backbone consisting of a linear chain of repeating disaccharide units (4)-alpha-D-galacturonic acid-(1,2)-alpha-L-rhamnose-(1). Figure 2 shows a schematic representation of the structure of RG-I.

[0048] Pectin polysaccharide composition and microstructure vary depending on the plant source and the extraction conditions applied. Homogalacturonan domains can have lengths of up to approximately 100 consecutive GalA residues. The RG-I domains containing the side chains are usually referred to as the "branch region" or "hairy region," but the homogalacturonan domains (attached to the RG-I domains) are typically not substituted with glycosides or glycosidic side chains.

[0049] GalA residues in RG-I are linked to rhamnose (Rha) residues through the 1- and 4-OH positions, while Rha residues are linked to GalA residues through the anomeric and 2-OH positions. Generally, approximately 20–80% of Rha residues are branched at the 4-OH position (depending on the plant source and isolation method) with neutral and acidic chains. These side chains consist of variously linked Ara and Gal residues, forming polymers known as arabinans, arabinogalactan I (AG-I), or arabinogalactan II (AG-II). AG-I consists of a beta-(1,4)-linked D-Gal backbone substituted at the 3-OH position with an alpha-L-arabinosyl group, and the Gal backbone may contain spaced alpha(1,5)-L-Ara units. AG-II consists of highly branched galactans with predominantly internal beta(1,3)-linked D-Gal substituted with short external (1,6)-linked chains. The latter have additional attachments of (1,3)- and / or alpha(1,5)-linked L-Ara. The polysaccharide side chains can be linear or branched.

[0050] The term "branched polysaccharide" as used herein refers to a polysaccharide comprising a linear backbone chain of monosaccharide units linked together by glycosidic bonds, at least one of the monosaccharide units in the backbone chain bearing one or more glycosidically linked monosaccharide unit side chains.

[0051] The terms "backbone chain" and "backbone" are synonymous.

[0052] The term "pectic polysaccharide" as used herein refers to an optionally branched polysaccharide having a molecular weight of more than 10 kDa and comprising a backbone consisting of galacturonic acid residues or a combination of galacturonic acid and rhamnose residues, the rhamnose residues being contained in alpha(1→4)-galacturonic-alpha(1→2)-rhamnose residues.

[0053] The term "stretch" as used herein refers to a sequence of two or more glycosidically linked monosaccharide units within the polysaccharide backbone, excluding any side chains attached thereto.

[0054] The term "domain" as used herein refers to a stretch and any side chains attached to the stretch.

[0055] The term "rhamnogalacturonan I stretch" or "RG-I stretch" refers to a stretch of galacturonic acid (GalA) and rhamnose (Rha) pairs, i.e., alternating alpha(1→4)-galacturonic-alpha(1→2)-rhamnose residues, where the GalA residues in RG-I are linked to Rha residues through positions 1 and 4, while the Rha residues are linked to GalA residues through the anomeric and 2-OH positions. The RG-I domain can include side chains such as, for example, galactan, arabinan, and arabinogalactan side chains.

[0056] The term "rhamnogalacturonan I polysaccharide" or "RG-I polysaccharide" refers to an optionally branched pectin polysaccharide that includes a backbone that includes one or more stretches of rhamnogalacturonan I. The backbone of the RG-I polysaccharide may optionally have one or more side chains. These side chains consist of arabinose and / or galactose residues.

[0057] The term "strand of alpha(1,4)-linked galacturonic acid" refers to a stretch consisting of alpha(1→4)-galacturonic residues.

[0058] The HG domain, XG domain, AG and RG-II domains optionally present in the RG-I polysaccharide of the present invention comprise a backbone consisting of two or more linear chains of alpha-(1-4) linked D-galacturonic acid.

[0059] The HG domain does not contain any side chains. The carboxyl groups of the galacturonic acid residues in the backbone of the HG domain can be esterified. The esterified galacturonic acid can occur in the form of a methyl ester or an acetyl ester.

[0060] The backbone of the XG domain contains one or more side chains in the form of D-xylose.

[0061] The backbone of the AG domain contains one or more side chains composed of one or more D-apiose residues.

[0062] The backbone of RG-II contains one or more side chains composed exclusively of D-xylose or D-apiose. The carboxyl groups of galacturonic acid residues within the backbone of the RG-II domain can be esterified. Galacturonic acid can be esterified with either a methyl or acetyl group to form a methyl or acetyl ester, respectively.

[0063] The term "degree of acetylation" is expressed as a percentage and refers to the number of acetyl residues per galacturonic acid residue.

[0064] The term "degree of methylation" is expressed as a percentage and refers to the number of methyl residues per galacturonic acid residue.

[0065] The concentrations of the various polysaccharides and their monosaccharide composition can be determined by analytical techniques known to those skilled in the art. After acid hydrolysis (methanolysis), the monosaccharide composition of neutral sugars can be suitably determined by high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

[0066] Uronic acids (galacturonic acid is the major form of uronic acid) can be determined using the colorimetric m-hydroxydiphenyl method.

[0067] Molecular size distribution can be determined by high performance size exclusion chromatography (HPSEC) with refractive index (RI) detection (concentration).

[0068] The above analytical methods are described in Analytical Biochemistry Vol. 207, Issue 1, 1992, pg. 176 (for neutral sugar analysis) and Mol. Nutr. Food Res., Vol. 61, Issue 1, 2017, 1600243 (for uronic acid analysis and molecular size distribution).

[0069] All percentages stated herein refer to weight percent unless otherwise stated.

[0070] Oral administration within the context of this use includes self-administration.

[0071] The use comprises oral administration of 0.1 to 5 grams of RG-I polysaccharide per day for at least 3 days, preferably at least 6 days, more preferably at least 8 days, even more preferably at least 10 days, even more preferably at least 20 days, and most preferably at least 30 days. In a particularly preferred embodiment, the use comprises oral administration of RG-I polysaccharide in a daily dose of 0.1 to 3 grams, more preferably 0.15 to 2.5 grams, and most preferably 0.2 to 2 grams for the aforementioned periods.

[0072] The use preferably involves at least daily oral administration, most preferably once daily, of a prebiotic composition comprising RG-I polysaccharide.

[0073] The use preferably involves oral administration of the prebiotic composition at least once a day for at least 10 days, more preferably at least 20 days, most preferably at least 30 days.

[0074] The prebiotic composition preferably contains 0.2 to 75% by weight of dry matter of RG-I polysaccharide, more preferably 0.3 to 50% by weight of dry matter, even more preferably 0.4 to 25% by weight of dry matter, and most preferably 0.5 to 10% by weight of dry matter.

[0075] The RG-I polysaccharide for this use can be obtained from various crops. In a preferred embodiment, the RG-I polysaccharide is obtained from one or more crops selected from fruits (including tomatoes), carrots, olives, peas, sugar beets, red beets, chicory, okra, soybeans, sunflowers, rapeseed, and corn. More preferably, the RG-I polysaccharide is obtained from one or more crops selected from apples, pears, citrus fruits, carrots, sugar beets, and chicory. Even more preferably, the RG-I polysaccharide is obtained from one or more crops selected from apples, pears, carrots, and chicory. Most preferably, the RG-I polysaccharide is obtained from carrots, apples, and / or chicory.

[0076] RG-I polysaccharide is preferably incorporated into the prebiotic composition in the form of a pectin polysaccharide isolate enriched in RG-I polysaccharide. Thus, in a particularly preferred embodiment, RG-I polysaccharide represents at least 10% by weight, more preferably at least 20% by weight, even more preferably at least 30% by weight, even more preferably at least 60% by weight, and most preferably at least 80% by weight of the pectin polysaccharides present in the prebiotic composition.

[0077] The RG-I polysaccharide of the present invention has a backbone comprising stretches of rhamnogalacturonan I and, optionally, stretches of alpha(1,4)-linked homogalacturonic acid. Preferably, the molar ratio of galacturonic acid residues to rhamnose residues in the RG-I polysaccharide does not exceed 15:1, more preferably does not exceed 12:1, even more preferably does not exceed 10:1, even more preferably does not exceed 8:1, and most preferably does not exceed 5:1.

[0078] The RG-I polysaccharide preferably has a monosaccharide composition having one or more of the following characteristics: 20 to 60 mol % galacturonic acid residues, where individual galacturonic acids may be methylated and / or acetyl esterified; 8–50 mol % rhamnose residues; 0–40 mol % arabinose residues; 0-40 mol% galactose residues; a molar ratio of galacturonic acid residues to rhamnose residues ranging from 5:1 to 1:1; galacturonic acid residues, rhamnose residues, arabinose residues and galactose residues together constitute at least 85 mole % of the monosaccharide residues in the RG-I polysaccharide; It has.

[0079] Galacturonic acid residues typically represent 21 to 55%, more preferably 22 to 50%, and most preferably 23 to 45% of all monosaccharide residues contained in the RG-I polysaccharide, i.e., monosaccharide residues including those contained in side chains.

[0080] Rhamnose residues typically represent 9 to 45%, more preferably 10 to 40%, and most preferably 11 to 35% of all monosaccharide residues contained in the RG-I polysaccharide, i.e., monosaccharide residues including those contained in side chains.

[0081] Arabinose residues typically represent 4 to 38%, more preferably 6 to 36%, and most preferably 8 to 34% of all monosaccharide residues contained in the RG-I polysaccharide.

[0082] Galactose residues typically represent 4 to 42%, more preferably 8 to 40%, and most preferably 10 to 38% of all monosaccharide residues contained in the RG-I polysaccharide.

[0083] The molar ratio of galacturonic acid residues to rhamnose residues in the RG-I polysaccharide is preferably within the range of 5:1 to 1:1, more preferably 4.8:1 to 1:1, even more preferably 4.5:1 to 1:1, even more preferably 4.2:1 to 1:1, and most preferably 4:1 to 1.1:1.

[0084] Preferably, the combination of galacturonic acid residues, rhamnose residues, arabinose residues and galactose residues together constitute at least 88 mol %, more preferably at least 90 mol %, and most preferably at least 92 mol % of the monosaccharide residues in the RG-I polysaccharide.

[0085] The arabinan side chain comprises at least one or more alpha(1,5)-linked arabinose residues, which are substituted at the 4-OH position of the rhamnose residue in the RG-I domain. The arabinan side chain can be linear or branched. When the side chain is linear, it consists of alpha(1,5)-linked arabinose residues. When the arabinan side chain is branched, one or more alpha-arabinose residues are linked to the O-2 and / or O-3 of the alpha(1,5)-linked arabinose.

[0086] The galactan side chain contains at least one or more beta(1,4)-linked galactose residues, which are substituted at the O-4 position of the rhamnose residue in the RG-I domain.

[0087] The arabinogalactan side chain is substituted at the O-4 position of the rhamnose residue in the RG-I domain, and can be type I arabinogalactan (AGI) or type II arabinogalactan (AGII). AGI is composed of an alpha(1→4)-beta-D-Galp backbone, which can be substituted at the O-6 or O-3 position with a monomeric Galp unit. AGI is further substituted with alpha-L-Araf-p residues and / or a (1→5)-alpha-L-Araf short chain. AGII is composed of an alpha(1→3)-beta-D-Galp backbone modified with an arabinosylated (1→6)-beta-D-Galp secondary chain.

[0088] Arabinose and rhamnose residues are preferably present in the RG-I polysaccharide in a molar ratio of less than 4:1, more preferably less than 3:1, and most preferably less than 2:1.

[0089] Galactose and rhamnose residues are preferably present in the RG-I polysaccharide in a molar ratio of less than 4:1, more preferably less than 3.2:1, and most preferably less than 2.5:1.

[0090] The molar ratio of the combination of arabinose and galactose residues to rhamnose residues in the RG-I polysaccharide is preferably less than 7:1, more preferably less than 5:1, and most preferably less than 4:1.

[0091] The combination of galacturonic acid residues and rhamnose residues preferably constitutes at least 30 mol %, more preferably 35 to 90 mol %, and most preferably 40 to 75 mol % of the monosaccharide residues contained in the RG-I polysaccharide.

[0092] The RG-I polysaccharide for this use preferably has the following monosaccharide composition: 20-60 mol % galacturonic acid residues, where individual galacturonic acids may be methylated and / or acetyl esterified; · 8-50 mol% rhamnose residues; · 0-40 mol% arabinose residues; · 0-40 mol% galactose residues; a molar ratio of galacturonic acid residues to rhamnose residues ranging from 5:1 to 1:1; and · galacturonic acid residues, rhamnose residues, arabinose residues and galactose residues together constitute at least 85 mol % of the monosaccharide residues in the RG-I polysaccharide; It has.

[0093] In one particularly preferred embodiment, the RG-I polysaccharide has the following monosaccharide composition: 21 to 55 mole % galacturonic acid residues, where individual galacturonic acids may be methylated and / or acetyl esterified; · 9-35 mol% rhamnose residues; 5-35 mol% arabinose residues; 5-40 mol% galactose residues; It has.

[0094] The RG-I polysaccharide is preferably obtained by partial enzymatic hydrolysis of pectin. In a particularly preferred embodiment, the RG-I polysaccharide is obtained by enzymatic hydrolysis of pectin using one or more pectinases selected from pectinase (EC 4.2.2.10), pectate lyase (EC 4.2.2.2), endopolygalacturonase (EC 3.2.1.15), and exopolygalacturonase (EC 3.2.1.67 and EC 3.2.1.82). Most preferably, the RG-I polysaccharide is obtained by enzymatic hydrolysis of pectin using one or more pectinases selected from pectin lyase (EC 4.2.2.10) and endopolygalacturonase (EC 3.2.1.15).

[0095] The prebiotic composition according to the invention preferably contains trace amounts of one or more of the above-mentioned pectinases, which may be present in the product in active and / or inactive form.

[0096] In a preferred embodiment, the RG-I polysaccharide is obtained by enzymatic hydrolysis of pectin using endo- and / or exo-polygalacturonase in combination with pectinesterase (EC 3.1.1.11).

[0097] In another preferred embodiment, the RG-I polysaccharide is obtained by enzymatic hydrolysis of pectin using pectin lyase and / or pectate lyase.

[0098] RG-I polysaccharides typically have a molecular weight of at least 15 kDa, more preferably between 20 kDa and 300 kDa, more preferably between 40 kDa and 300 kDa.

[0099] The RG-I polysaccharide preferably has a degree of acetylation of at least 20%, more preferably 30 to 110%, even more preferably 35 to 90%, and most preferably 40 to 70%.

[0100] The RG-I polysaccharide preferably has a degree of methylation of 50% or less, more preferably 40% or less, and most preferably 10 to 30%.

[0101] In another preferred embodiment, the ratio of the degree of acetylation (DA) of the RG-I polysaccharide to the degree of methylation (DM) of the RG-I polysaccharide is preferably 1 or greater, more preferably 2 or greater, more preferably 3 or greater, and most preferably 5 or greater.

[0102] Although pectin polysaccharides generally do not contain unsaturated galacturonic acid residues, hydrolysis of pectin polysaccharides with pectin lyase and / or pectate lyase inevitably produces polysaccharide fragments containing terminal unsaturated, non-reducing galacturonic acid residues. Preferably, at least 10%, more preferably at least 25%, and most preferably at least 50% of the terminal unsaturated, non-reducing galacturonic acid residues in RG-I polysaccharide are unsaturated galacturonic acid residues. Unsaturated galacturonic acid can be readily identified, for example, by measuring UV absorbance at 235 nm.

[0103] In one particularly preferred embodiment, the pectic polysaccharides (including RG-I polysaccharide) in the prebiotic composition have the following monosaccharide composition: 20-60 mol % galacturonic acid (GalA) residues, where individual galacturonic acids may be methylated and / or acetyl esterified; 8–50 mol% rhamnose (Rha) residues; 0-40 mol% arabinose (Ara) residues; 0-40 mol% galactose (Gal) residues and · 2 × [Rha] + [Ara] + [Gal] ≥ 50 mol%; [GalA]-[Rha]<50 mol% where [Rha], [Ara], [Gal] and [GalA] represent the molar concentrations in mole % of rhamnose, arabinose, galactose and galacturonic acid, respectively. It has.

[0104] The monosaccharide composition is more preferably determined under the following conditions: · 2 × [Rha] + [Ara] + [Gal] ≥ 60 mol%; [GalA]-[Rha]<30 mol% Meet the following.

[0105] The prebiotic composition is preferably a nutritional formulation, a food, a dietary supplement (eg, a pill, tablet, gummy or powder) or a beverage.

[0106] Advantageously, the combination of RG-I polysaccharide with one or more moderately to highly specific or highly specific fibers allows for the specific stimulation of a subset of taxa of symbiotic bacteria without stimulating the wide variety of taxa present in the symbiotic bacteria as occurs with less specific fibers.

[0107] In a preferred embodiment, the use comprises oral administration of a prebiotic fiber before, simultaneously with, or after oral administration of a prebiotic composition, wherein the prebiotic fiber is selected from beta-glucan, arabinoxylan, type 2 resistant starch, and combinations thereof. More preferably, the use comprises oral administration of a prebiotic fiber before, simultaneously with, or after oral administration of 0.1 to 20 grams of a prebiotic composition to a subject per day for at least three days, preferably for at least six days, more preferably for at least eight days, even more preferably for at least ten days, even more preferably for at least 20 days, and most preferably for at least 30 days. In a particularly preferred embodiment, the use comprises oral administration of 0.2 to 10 grams, most preferably 0.3 to 5 grams, of prebiotic fiber per day for the aforementioned periods. As used herein, the phrase "oral administration of a prebiotic fiber before, simultaneously with, or after oral administration of a prebiotic composition" refers to administration of a prebiotic fiber at a time interval of -8 hours to +8 hours relative to the time of oral administration of the prebiotic composition (0 hours).

[0108] The prebiotic composition preferably comprises at least 0.1% by weight of a dry matter of a prebiotic fiber selected from beta-glucan, arabinoxylan, type 2 resistant starch and combinations thereof.

[0109] The prebiotic composition preferably contains 0.2 to 75% by weight, more preferably 0.3 to 50% by weight, even more preferably 0.4 to 25% by weight, and most preferably 0.5 to 10% by weight of dry matter of prebiotic fibre.

[0110] The prebiotic composition is preferably a solid dosage unit having a weight of 200 to 4,000 mg and containing 100 to 2,000 mg of RG-I polysaccharide and 100 to 2,000 mg of prebiotic fiber.

[0111] The prebiotic composition is preferably a packaged aqueous liquid having a packaged volume of 10 to 250 mL and containing 100 to 2,000 mg of RG-I polysaccharide and 100 to 2,000 mg of prebiotic fiber.

[0112] The prebiotic fiber is preferably selected from beta-glucan, arabinoxylan, and combinations thereof, and more preferably the prebiotic fiber is beta-glucan.

[0113] A second aspect of the present invention is at least 0.1% by weight of a dry matter of rhamnogalacturonan I (RG-I) polysaccharide as defined herein; at least 0.1% by weight of a dry matter of a prebiotic fiber selected from beta-glucan, arabinoxylan, type 2 resistant starch, and combinations thereof; The present invention relates to a prebiotic composition comprising:

[0114] In a preferred embodiment, the prebiotic composition is the same as described herein for the prebiotic composition for use.

[0115] The present invention will now be further illustrated by the following non-limiting examples. [Example]

[0116] [Example 1] (Human clinical trials) (Research Design) A single-center, randomized, double-blind, placebo-controlled, dose-response study with a parallel design was conducted with three arms: 0, 0.3 g / day cRG-I, and 1.5 g / day cRG-I. The study design, adverse events, safety monitoring, subject disposition, and primary outcome measures are described in Lutter et al. (The Dietary Intake of Carrot-Derived Rhamnogalacturonan-I Accelerates and Augments the Innate Immune and Antiviral Interferon Response to Rhinovirus Infection and Reduces Duration and Severity of Symptoms in Humans in a Randomized Trial, Nutrients 2021, 13, 4395, doi:10.3390 / nu13124395).

[0117] Stool samples were collected from subjects (see Table 1) before the start of cRG-I supplementation and after 8 weeks of cRG-I supplementation. The subjects remained healthy during this period. Stool samples were stored at -80°C until the microbiota composition was analyzed.

[0118] [Table 1]

[0119] (nutritional supplement) cRG-I is a natural extract from carrot (Daucus carota subsp. sativus) and was supplied by Nutrileads (Wageningen, The Netherlands). cRG-I is a water-soluble, indigestible, fermentable fiber rich in the RG-I domain of pectin. The carbohydrate content of the extract is approximately 73% by weight. The extraction method and extract characteristics (composition and structure) are described in McKay et al. (Development of an Affordable, Sustainable, and Efficacious Plant-Based Immunomodulatory Food Ingredient Based on Bell Pepper or Carrot RG-I Pectic Polysaccharides, Nutrients 2021, 13, 963).

[0120] The monosaccharide composition of cRG-I (% mol / mol) was 14.3 rhamnose, 34.8 arabinose, 19.6 galactose, 0.8 fucose, 4.3 glucose, 0.9 mannose, 0.7 xylose, and 25.0 galacturonic acid.

[0121] Based on these figures, the percentages of monosaccharides (mol / mol) contained in RG-I and HG, respectively, can be estimated as follows: · %RG-I=2×[Rha]+[Ara]+[Gal]=2×14.3+34.8+19.6=83% · %HG=[GalA]-[Rha]=25.0-14.3=10.7%

[0122] The percentage (mol / mol) of monosaccharides contained in each backbone RG-I and HG can be estimated as follows: · %RG-I=2×[Rha] / ([Rha]+[GalA])=100%×2×14.3 / / (14.3+25.0)=72.8% %HG=100%-%RG-I=27.2%

[0123] The dietary supplements were prepared by mixing maltodextrin and caramel coloring, such as 0, 0.3, and 1.5 g of cRG-I extract, 3, 2.7, and 1.5 g of maltodextrin (MALDEX 170, Tereos, Belgium), and 0.5 g of caramel coloring 1 (Natural spices, Midrecht, Netherlands), for each dose, to obtain identical powders and sachets with 3.5 g of powdered supplement of identical volume and appearance for the no-dose, low-dose, and high-dose groups, respectively.

[0124] Participants were instructed to take the dietary supplement, supplied as a powder in a sachet, once daily with their preferred food and beverage during their first meal (preferably breakfast).

[0125] (Analysis of microbial composition in fecal samples) DNA extraction: DNA was extracted from stool samples using a NucleoSpin® 96 Soli kit (Macherey-Nagel). Bead-beating was performed on a Vortex-Genie 2 for 5 minutes at level 9 in horizontal orientation. A minimum of one negative control was included per batch of samples from DNA extraction and throughout the laboratory process (including sequencing). The ZymoBIOMICS® Microbial Community Standard (Zymo Research) was also included in the analysis.

[0126] PCR: PCR was performed with Illumina adapter-tagged forward primer SD-Bact-0341-bS-17 and reverse primer SD-Bact-0785-aA-21 (Klindworth et al., 2013). These are common bacterial 16S rDNA primers targeting the V3-V4 region. The PCR program was 98°C for 30 seconds, 25x (98°C for 10 seconds, 55°C for 20 seconds, 72°C for 20 seconds), and 72°C for 300 seconds. Amplification was confirmed by running the product on an agarose gel. Indexes were added in subsequent PCRs using the Nextra Index Kit V2 (Illumina) with a PCR program of 98°C for 30 seconds, 8x (98°C for 10 seconds, 55°C for 20 seconds, 72°C for 20 seconds), and 72°C for 300 seconds. Index incorporation was confirmed by running the product on an agarose gel.

[0127] Normalization and sequencing: Nested PCR products were pooled based on band intensity, and the resulting libraries were cleaned with magnetic beads. The DNA concentration of the pooled libraries was measured fluorometrically. Sequencing was performed on an Illumina MiSeq desktop sequencer using the MiSeq Reagent Kit V3 (Illumina) for 2 × 300 bp paired-end sequencing.

[0128] Bioinformatics analysis: For bioinformatics analysis of sequence data, we used the 64-bit version of USEARCH 10.0 (Edgar 2013), mothur 1.38 (Schloss et al. 2009), and in-house scripts. After tag identification and trimming, sequences were trimmed with QS10 and fused, requiring a minimum overlap of 20 bp and a merged length of 400–500 bp. Sequences with perfect matches to primers, homopolymers longer than 10, or more than one expected error and ambiguous bases were discarded. Primer sequences were trimmed, sequences were rigorously dereplicated, and clusters with fewer than five were discarded. Sequences were clustered at 97% sequence identity using the most abundant, rigorously dereplicated read as the centroid and discarding suspected chimeras based on internal comparisons using the cluster_otus command in USEARCH. Taxonomic assignment of OTUs was performed using SINTAX with a cutoff value of 0.8 (Edgar 2016) on the RDP training set v16 (Cole et al. 2014). All analyses were performed on purified data.

[0129] Diversity indices: Diversity indices were calculated based on the algorithms described in the Vegan package in R (https: / / cran.r-project.org / web / packages / vegan / vegan.pdf) and by Oksanen J et al. (https: / / rdrr.io / cran / vegan / man / vegdist.html).

[0130] (intra-individual variability) A diversity index per subject was calculated comparing the microbiota composition in the stool samples of subjects before the initiation of RG-I supplementation versus the microbiota composition in the stool samples of subjects after 8 weeks of daily RG-I supplementation.

[0131] Bray-Curtis dissimilarity was used as a diversity index and was calculated using the R software package mentioned above. Bray-Curtis dissimilarity is described in Bray, JR, and Curtis, JT (1957) An ordination of the upland forest communities of Southern Wisconsin. Ecol. Monogr. 27, 325–349. doi:10.2307 / 1942268. Bray-Curtis dissimilarity is limited to a range of 0–1, where 0 means that the bacterial flora composition is the same in both stool samples (i.e., all species are common), and 1 means that no species are common between the two stool samples.

[0132] Figure 3 shows the results of the intra-individual Bray-Curtis dissimilarity for the three treatment groups. Both groups A and B, corresponding to 1.5 and 0.3 g of cRG-I supplementation per day, had lower mean intra-individual Bray-Curtis dissimilarity compared to the control group C, which did not consume cRG-I.

[0133] (Inter-individual variability) The generalized UniFrac distance per subject was calculated by comparing the microbiota composition of a subject's stool sample with the microbiota composition of other subjects in the same subject group (A, B, or C) and at the same time point (before or after supplementation). For each subject, the average generalized UniFrac distance was calculated for both time points (before or after supplementation) and compared with each other. This was done for each subject.

[0134] Generalized UniFrac distances were used as a diversity index and were calculated using the R software package mentioned above. Generalized UniFrac distances were described in Chen et al., "Associating microbiome composition with environmental covariates using generalized UniFrac distances," Bioinformatics, Volume 28, Issue 16, 15 August 2012, Pages 2106-2113, https: / / doi.org / 10.1093 / bioinformatics / bts342. Generalized UniFrac distances were calculated with α = 0.5 (α is a constant that determines how much weight is given to changes in abundance when calculating UniFrac distances). Smaller values ​​are associated with higher similarity, and larger values ​​indicate higher dissimilarity.

[0135] Figure 4 shows that the mean generalized UniFrac distance from one subject to another within the same group was lower at the end of the cRG-I supplementation period than before the start of cRG-I supplementation. Groups A and B, both of which received 1.5 and 0.3 g of cRG-I per day, showed a mean change in generalized UniFrac distance of -0.004 comparing before and after cRG-I supplementation, whereas in control group C, which did not consume cRG-I, the mean difference was +0.002 when comparing UniFrac distances before and after control supplementation. This difference was statistically significant for groups A (paired t-test, p = 0.022) and B (paired t-test, p = 0.034), but not for group C (paired t-test, p = 0.42). Inter-individual variability thus decreased in groups A and B after cRG-I supplementation, whereas inter-individual variability remained unchanged in the control group.

[0136] [Example 2] (Ex vivo data) (stool sample) Fresh stool samples were collected from healthy donors aged 25–65 years, with no antibiotic use in the past 3 months, no gastrointestinal disorders (cancer, ulcer, IBD), no probiotic use, no smoking, alcohol consumption less than 3 units / day, and a BMI less than 30.

[0137] (setting) An ex vivo SIFR® study was performed by stimulating colonic fermentation of the test products by the intestinal flora of human adults (n=24) from stool samples. Colonic fermentation samples were collected after 48 hours of incubation with the test products.

[0138] There were five study arms using different test products (Table 2). Blank = background medium + bacterial flora (no product) cRG-I tested at a dose of cRG-I_L = 0.3 g / day cRG-I_H = cRG-I tested at a dose of 1.5g / day Inulin tested at a dose of IN = 1.5 g / day Xanthan tested at a dose of XA = 1.5 g / day

[0139] [Table 2]

[0140] (Ex Vivo SIFR® Technology) Individual bioreactors were processed in parallel using a proprietary bioreactor management device (Cryptobiotix, Ghent, Belgium). Each bioreactor contained 5 mL of the nutrient medium-fecal inoculum blend supplemented with 0.3 or 1.5 g prebiotic / L and was then individually sealed before being made anoxic. Blend M0003 was used to prepare the nutrient medium (Cryptobiotix, Ghent, Belgium). M0003 is a growth medium (pH 6.5) developed by Cryptobiotix. This medium also contains sources of carbon, nitrogen, phosphorus, sulfur, vitamins, and minerals in amounts sufficient to support bacterial growth. After preparation, the bioreactors were incubated at 37°C for 48 hours under continuous agitation (140 rpm) in a MaxQ® 6000 stackable shaker with incubation / cooling (Thermo Scientific, Thermo Fisher Scientific, Merelbeke, Belgium). Upon measuring the gas pressure in the headspace, liquid samples were collected for subsequent analysis.

[0141] (Microbial composition analysis) Upon DNA extraction, library preparation and sequencing were performed on an Illumina MiSeq platform using v3 chemistry. Primers 341F (50-CCT ACG GGN GGC WGC AG-30) and 785Rmod (50-GAC TAC HVG GGT ATC TAA KCC-30) were used to amplify the 16S rRNA gene V3-V4 hypervariable region. Results were analyzed at various taxonomic levels (phylum, family, and OTU levels). For taxonomic analysis, percentage data from sequencing were normalized by the total amount of cells present in each sample (detected via flow cytometry) to provide more representative insight into the impact of interventions on the gut microbiota.

[0142] (Inter-individual variability) Weighted UniFrac distances were calculated within each set of 24 samples from 24 donors: blank and cRG-I / IN / XA treatment at 48 hours. Assuming that a given donor's sample was always compared to 23 other donors, a total of 276 comparisons were made within each experimental group.

[0143] Weighted UniFrac distances were calculated using the software package QIIME v1.9.0. Weighted UniFrac distances are described in Lozupone et al., "Quantitative and Qualitative β Diversity Measures Lead to Different Insights into Factors That Structure Microbial Communities," Appl Environ Microbiol. 2007 Mar;73(5):1576-1585, doi:10.1128 / AEM.01996-06. Identical groups yield a weighted UniFrac distance of 0, and completely different groups yield a value of 1.

[0144] Figure 5 shows the results of interindividual weighted UniFrac distance for the various experimental groups. Compared with the blank group, two doses of cRG-I significantly decreased the weighted UniFrac distance in a dose-response manner. Meanwhile, compared with the blank group, both IN and XA significantly increased the weighted UniFrac distance. When comparing the IN and XA groups with the cRG-I group at equal dosages (1.5 g / day), the cRG-I group was significantly different from both the IN and XA groups. Interindividual variability decreased in the presence of cRG-I, but increased in the presence of inulin or xanthan.

Claims

1. 1. A prebiotic composition for use in stabilizing a healthy gut microbiota in a subject by selective prebiotic stimulation of at least one taxonomic group of commensal bacteria, comprising: the prebiotic composition comprises at least 0.1% by weight of a dry matter of rhamnogalacturonan I (RG-I) polysaccharide having a molecular weight greater than 10 kDa and having a backbone composed of galacturonic acid and rhamnose residues, the rhamnose residues being comprised in alpha(1→4)-galacturonic-alpha(1→2)-rhamnose residues, and the molar ratio of galacturonic acid to rhamnose residues in the RG-I polysaccharide is in the range of 20:1 to 1:1; said use comprising oral administration of 0.1 to 5 grams of RG-I polysaccharide per day to a subject for at least three days; The use, reducing the intraindividual variability of the gut microbiota in a subject; and / or reducing inter-individual variation in gut microbiota within a group of subjects; Prebiotic composition for use.

2. 2. The prebiotic composition for use according to claim 1, wherein said use comprises oral administration of 0.2 to 2 grams of RG-I polysaccharide per day to a subject for at least three days.

3. 3. The prebiotic composition for use according to claim 1 or 2, wherein the use comprises oral administration of a prebiotic fiber before, simultaneously with or after oral administration of the prebiotic composition, and the prebiotic fiber is selected from beta-glucan, arabinoxylan, type 2 resistant starch and combinations thereof.

4. 4. The prebiotic composition for use according to any one of claims 1 to 3, wherein the prebiotic composition comprises at least 0.1% by weight of dry matter of a prebiotic fibre selected from beta-glucan, arabinoxylan, type 2 resistant starch and combinations thereof.

5. A prebiotic composition for use according to any one of claims 1 to 4, wherein the RG-I polysaccharides represent at least 20% by weight of the pectic polysaccharides present in the prebiotic composition.

6. 6. A prebiotic composition for use according to any one of claims 1 to 5, wherein the molar ratio of galacturonic acid residues to rhamnose residues in the RG-I polysaccharide does not exceed 15:

1.

7. The RG-I polysaccharide has the following monosaccharide composition: 20 to 60 mole % galacturonic acid residues, each of which may be methylated and / or acetyl esterified; 8-50 mol % rhamnose residues; 0-40 mole % arabinose residues; 0 to 40 mole % galactose residues; a molar ratio of galacturonic acid residues to rhamnose residues ranging from 5:1 to 1:1; galacturonic acid residues, rhamnose residues, arabinose residues and galactose residues together constitute at least 85 mole % of the monosaccharide residues in the RG-I polysaccharide; A prebiotic composition for use according to any one of claims 1 to 6, comprising:

8. The pectic polysaccharide has the following monosaccharide composition: 20 to 60 mole % galacturonic acid (GalA) residues, where individual galacturonic acids may be methylated and / or acetyl esterified; 8-50 mol % rhamnose (Rha) residues; 0-40 mol % arabinose (Ara) residues; 0 to 40 mol% galactose (Gal) residues and 2 × [Rha] + [Ara] + [Gal] ≥ 50 mol%; [GalA]-[Rha]<50 mol% where [Rha], [Ara], [Gal] and [GalA] represent the molar concentrations in mole % of rhamnose, arabinose, galactose and galacturonic acid, respectively. A prebiotic composition for use according to any one of claims 1 to 7.

9. A prebiotic composition for use according to any one of claims 1 to 8, wherein the RG-I polysaccharide has a degree of acetylation of at least 20% and a degree of methylation of not more than 50%.

10. A prebiotic composition for use according to any one of claims 1 to 9, wherein the RG-I polysaccharide is derived from carrot, apple or chicory.

11. At least 0.1% by weight of the dry matter of rhamnogalacturonan I (RG-I) polysaccharide according to any one of claims 1 to 10; and At least 0.1% by weight of a dry matter of prebiotic fiber selected from beta-glucan, arabinoxylan, type 2 resistant starch, and combinations thereof. A prebiotic composition comprising:

12. 12. The prebiotic composition of claim 11, which is a nutritional formulation, food, dietary supplement or beverage.

13. 13. Prebiotic composition according to claim 11 or 12, wherein the prebiotic fibre is a beta-glucan and / or an arabinoxylan, preferably a beta-glucan.

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