阅读说明：本技术 用于治疗与肠道微生物组的扰乱的组成或功能有关的病症的益生元 (Prebiotics for the treatment of disorders associated with disturbed composition or function of the gut microbiome ) 是由 鲁德·阿尔伯斯 玛丽亚·祖玛基 于 2018-09-07 设计创作，主要内容包括：本发明涉及益生元组合物,其用于治疗性或预防性治疗对象中的与肠道微生物组的扰乱的组成或功能有关的病症的方法中的用途中,所述用途包括向所述对象口服施用所述益生元组合物,其中所述组合物包含至少0.1重量％的源自果实、胡萝卜、豌豆、菊苣或甜菜的RG-I多糖的干物质,所述RG-I多糖具有超过15kDa的分子量并且具有由半乳糖醛酸残基和鼠李糖残基组成的主链,所述鼠李糖残基包含在α(1→4)-半乳糖醛酸-α(1→2)-鼠李糖残基中,其中所述RG-I多糖中的半乳糖醛酸残基与鼠李糖残基的摩尔比在20:1至1:1的范围内。(The present invention relates to a prebiotic composition, for use in a method for the therapeutic or prophylactic treatment of a disorder in a subject which is associated with a disturbed composition or function of the gut microbiome, the use comprising orally administering the prebiotic composition to the subject, wherein the composition comprises at least 0.1 wt.% dry matter of RG-I polysaccharides originating from fruit, carrots, peas, chicory or sugar beets, the RG-I polysaccharide has a molecular weight of more than 15kDa and has a backbone consisting of galacturonic acid residues and rhamnose residues, the rhamnose residue is contained in the alpha (1 → 4) -galacturonic acid-alpha (1 → 2) -rhamnose residue, wherein the molar ratio of galacturonic acid residues to rhamnose residues in the RG-I polysaccharide is in the range of 20:1 to 1: 1.)

1. Prebiotic composition for use in a method of therapeutic or prophylactic treatment of a disorder related to disturbed composition or function of the gut microbiome in a subject, said disorder being selected from metabolic disorders and intestinal barrier dysfunction, said use comprising orally administering said prebiotic composition to said subject, wherein the composition comprises at least 0.1 wt.% dry matter of rhamnogalacturonan I (RG-I) polysaccharides derived from fruits, carrots, peas, chicory or sugar beets, the RG-I polysaccharide has a molecular weight of more than 15kDa and has a backbone consisting of galacturonic acid residues and rhamnose residues, the rhamnose residue is contained in the alpha (1 → 4) -galacturonic acid-alpha (1 → 2) -rhamnose residue, wherein the molar ratio of galacturonic acid residues to rhamnose residues in the RG-I polysaccharide is in the range of 20:1 to 1: 1.

2. The prebiotic composition for use of claim 1 wherein the composition is ingested by the subject in an amount to provide at least 1mg RG-I polysaccharide per kg body weight per day for a period of at least 3 days.

3. Prebiotic composition for use according to claim 1 or 2 wherein the RG-I polysaccharide comprises at least 20% by weight of the pectin polysaccharide present in the prebiotic composition.

4. The prebiotic composition for use of any one of the preceding claims wherein the molar ratio of galacturonic acid residues to rhamnose residues in the RG-I polysaccharide is not more than 15:1, preferably not more than 12:1, more preferably not more than 10: 1.

5. The prebiotic composition of any of the preceding claims wherein the molar ratio of arabinose residues to rhamnose residues in the RG-I polysaccharide does not exceed 30: 1.

6. The prebiotic composition of any of the preceding claims wherein the molar ratio of galactose residues to rhamnose residues in the RG-I polysaccharide preferably does not exceed 30: 1.

7. The prebiotic composition for use of any one of the preceding claims wherein less than 85% of the galacturonic acid residues in the RG-I polysaccharide are esterified in the form of methyl esters.

8. The prebiotic composition for use of any one of the preceding claims wherein the RG-I polysaccharide is derived from a plant source selected from the group consisting of carrot, apple, bell pepper, citrus, bilberry, grape, pea, chicory, beet, olive, okra and combinations thereof.

9. The prebiotic composition for use of any one of the preceding claims, wherein the subject has or is at risk of having a metabolic disorder.

10. The prebiotic composition of claim 1 wherein the metabolic disorder is selected from the group consisting of: overweight, obesity, metabolic syndrome, insulin deficiency or insulin resistance related disorders, diabetes, glucose intolerance, abnormal lipid metabolism, hyperglycemia, hepatic steatosis, dyslipidemia, elevated cholesterol, elevated triglycerides.

11. Prebiotic composition for use according to claim 10 wherein the metabolic disorder is selected from the group consisting of overweight, obesity and insulin resistance.

12. The prebiotic composition for use of any one of claims 1-8, wherein the subject has or is at risk of having gut barrier dysfunction.

13. A prebiotic composition comprising:

at least 0.1% by weight of dry matter of rhamnogalacturonan I (RG-I) polysaccharides as defined in claim 1; and

at least 0.1% by weight of dry matter of one or more prebiotics selected from the group consisting of lactulose, inulin, fructooligosaccharides, galactooligosaccharides, lactooligosaccharides, guar gum and acacia gum.

14. A synbiotic composition comprising:

at least 0.1% by weight of dry matter of rhamnogalacturonan I (RG-I) polysaccharides as defined in claim 1; and

one or more probiotic microbial strains in the form of live microorganisms, inactive microorganisms, microbial fragments and combinations thereof.

15. The composition of any one of the preceding claims, wherein the composition is selected from the group consisting of a beverage, a capsule, a tablet, a powder, a bar, and a spread.

Technical Field

The present invention relates to a method for the therapeutic or prophylactic treatment of a disorder related to disturbed composition or function of the gut microbiome, selected from a metabolic disorder and gut barrier dysfunction, in a subject, comprising orally administering to said subject a prebiotic composition comprising a rhamnogalacturonan I (RG-I) polysaccharide derived from fruit, carrot, pea, chicory or sugar beet.

The invention also relates to prebiotic and synbiotic compositions suitable for use in the above methods of treatment.

Background

The relationship between gut microbiota and human health is increasingly recognized. It is now well established that a healthy gut microbiota is the main responsible for the overall health of the host.

The human host provides a habitat and nutrition for the large and diverse ecosystem of microbial communities, which play a key role in digestion, metabolism and regulation of immune functions and have significant effects outside the gastrointestinal tract. The diversity and functional changes of these communities are associated with profound effects on host health and have been associated with a number of disorders including functional bowel disease, inflammatory bowel disease and other immune-mediated diseases (celiac disease, allergy) and metabolic disorders (type 2 diabetes, NASH).

Dysregulation (also known as dysbiosis) is a term applied to a microbial imbalance or maladaptation (e.g., impaired microbiota) on or within the body. For example, bacterial communities that occupy certain surface areas of a host, such as intestinal, skin, or vaginal microbiota, can be modified/altered, often the predominant species is not adequately represented, and often the dominant or contained species increases to undesirable levels. The collection of bacteria in such a community is collectively referred to as the microbiota. Together with other microorganisms (including yeasts, fungi, viruses and parasites present in such niches), the local microbiota is collectively referred to as the microbiome. Dysregulation is not limited to imbalances in microbial populations but may also involve other microorganisms in the microbiome (e.g., viruses, archaea, and fungi).

When the microbiome is well balanced (called normal ecology), the microorganisms occupying a particular niche form a more or less stable community that adapts well to local conditions, has metabolic capacity to live available substrates, effectively handles regulation of stressors and substrate availability, and has regulatory mechanisms in place that contribute to the metastable state of the community and to the long-term health of its host.

Disorders are most commonly studied in gastrointestinal conditions, but it can affect any body cavity, mucosal and skin surfaces that are populated by microflora.

Disorders associated with disease of the host; for example, intestinal disorders are associated with inflammatory bowel disease, chronic fatigue syndrome, obesity, cancer, cardiovascular metabolic conditions, insulin insensitivity, diabetes (prophase), bacterial vaginosis and colitis.

The composition and stability of the microbiota is influenced by the genetic background of the host, environmental conditions or stressors including, for example, diet, lifestyle, use of drugs (e.g., antibiotics), and the developmental stage (age) of the host. 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).

The neonatal microbiota builds up on its own from birth and fluctuates significantly within the first weeks and months of life to approach a core mature microbiota of about 3-4 years of age. This progressive microbial colonization of the gut is critical for the host immunity and the culture and maturation of the enteric nervous system, gut barrier and function, and the metabolic programming of the host that has an impact on short and future life health and disease risk. The neonatal microbiota is influenced by the maternal diet and microbiota, the mode of delivery, infant nutrition (breast feeding or infant formula) and the surrounding environmental conditions.

In contrast, the healthy adult core microbiota is more stable in the sense that: when disturbed by various stressors (e.g., using antibiotics or drugs), it recovers close to its original composition in healthy subjects, which is referred to as the elasticity of the microbiota. Loss of variability, lack or low abundance of beneficial microorganisms, and loss of elasticity are all associated with disease.

The microbiota of an aging or elderly subject differs from that of a healthy adult population by a change in composition, resulting in less bacterial diversity, reduction of beneficial microorganisms and decreased elasticity. All of these changes are related to changes in health status.

The microbiota consists of a wide variety of species that compete for space and resources/nutrients, but can also feed each other their respective fermentation products, resulting in the cooperation of microorganisms that are symbiotic with the mammalian host in a healthy state. High microbial diversity within the gut microbiota is considered beneficial to the health of the host, as diversity makes the microbiota more resilient to factors that interfere with gut microbiota (e.g., antibiotics, dietary changes, new species invasion). It is also important that the cooperation of the microorganisms, i.e. the metastable combination of different microorganisms, which feed on each other's fermentation products and use available substrates (e.g. from mucus, cell shedding and diet) together form a more or less stable ecosystem that thrives in a particular niche.

Typical microbial species found on or in the body are mostly beneficial or harmless. Pathogenic organisms (pathogens) are also part of the "normal" microbiota as long as they remain below a critical level, or even pathogens. The mammalian intestinal microbiota performs a series of helpful and essential functions, such as aiding digestion, providing energy from food, providing specific (micro) nutrients to the host, producing key metabolites such as short chain fatty acids, and culturing (neonates and infants) or maintaining (adult) the host's immune system. They also help protect the body from incoming pathogenic microorganisms or toxic compounds.

Microbial species also secrete many different types of waste byproducts. Using different waste removal mechanisms, the human body manages these by-products effectively, under normal circumstances, with little trouble. Unfortunately, the preponderance of oversized microbial populations and inappropriate microbial species secrete increased amounts of these by-products due to the increased numbers. As the amount of microbial byproducts increases, higher levels of waste byproducts overburdened the body's waste removal mechanisms. An example of this is the formation of ammonia from the fermentation of proteins, which may further ferment compounds that are harmful to the host.

It is the relative predominance or lack of representativeness of a particular microbial species, the low diversity of microbial metabolites and/or the production of interference that results in many of the negative health symptoms observed in subjects with disorders.

The consumption of probiotics and/or prebiotics may have a beneficial effect on the intestinal microbiota.

Probiotics are "living microorganisms that confer a health benefit to the host when administered in sufficient amounts" (definition of the world health organization).

Prebiotics are nondigestible 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.

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 that have the metabolic capability 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 of gases.

Pectin is a structural heteropolysaccharide present in the primary cell wall of terrestrial plants.

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

(i) (ii) a homogalacturonic acid glycan (HG),

(ii) xylogalacturonic acid glycan (XG)

(iii) Celery galacturonic Acid Glycan (AG)

(iv) Rhamnogalacturonan-1 (RG-I) and

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

Figure 1 provides a schematic representation of the structure of a pectin polysaccharide (including the 4 polysaccharide components described above). Note that the polysaccharide components HG, XG and RG-II usually represent only a small fraction of the pectic polysaccharides.

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

Only RG-I comprises a backbone consisting of a linear chain of repeating disaccharide units: 4) a schematic diagram of the structure of- α -D-galacturonic acid- (1,2) - α -L-rhamnose- (1. RG-I) is shown in fig. 2.

The composition and fine structure of the pectic polysaccharides vary widely depending on the plant source and the extraction conditions applied. Homogalacturonan domains may be up to about 100 contiguous D-GalA residues in length. The RG-I domains containing side chains are commonly referred to as 'branched regions' or 'hairy regions', whereas homogalacturonan domains (between the two RG-I domains) are not typically substituted by oligosaccharides.

The GalA residue in RG-I is linked to the Rha residue via positions 1 and 4, while the Rha residue is linked to the GalA residue via the terminal group (anomeric) and 2-OH position. Typically, about 20-80% of the Rha residues are branched at the 4-OH position (depending on plant origin and isolation method), with neutral and acidic side chains. These side chains consist essentially of Ara and Gal residues linked in various ways, constituting polymers known as arabinogalactans I (AG-I) and/or AG-II. AG I consists of a β - (1,4) -linked D-Gal backbone with a substitution at the 3-OH of the α -L-arabinosyl; the Gal backbone may have an intermediate (interpacking) α (1,5) -L-Ara unit. AG-II consists of a highly branched galactan, with predominantly internal β (1,3) -linked D-Gal having short (1,6) -linked chain substitutions on the outside. The latter also have a linkage of (1,3) -and/or α (1,5) -linked L-Ara. The oligosaccharide side chains may be straight or branched chain, and some of these side chains may be terminated with α -L-fucoside, β -D-glucuronide and 4-O-methyl β -D-glucuronide residues.

G. Lo mez et al (biological potential of peptides and peptides from lemon peel waters and sugar beets: A comparative evaluation, Journal of Functional Foods, Vol.20, 2016.1, p.108-121) describe the results of two mixtures of pectic oligosaccharides (SBPOS and LPOS, respectively) obtained using Sugar Beet Pulp (SBP) and Lemon Peel Waste (LPW). The suitability of pectic oligosaccharides, pectins from SBP and LPW, and commercial FOS for eliciting prebiotic effects was compared by in vitro fermentation and fluorescent in situ hybridization using human fecal inocula and 8 different bacterial probes. The combined population of bifidobacteria and lactobacilli increased from 19% to 29%, 34% and 32% in the medium containing LPOS, SBPOS and FOS, respectively. All substrates, especially in the case of LPOS, also increased the count of faecalis (Faecalibacterium) and Roseburia (Roseburia). The highest concentration of organic acids was observed in the medium containing oligosaccharides. According to the authors, this work demonstrated that pectin oligosaccharides exhibit better prebiotic properties than pectin, and exhibit prebiotic properties similar to or better than FOS.

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 Effect of Pectin extracted from different types of Fruit waste (Musa sp.) and sweet lime (Citrus limosum) and watermelon peel (red of Citrus blossom) as well as tomato (tomato Lycopersicum) and guava (Psidium guajava) rotted fruits) on the Growth of Lactic Acid Bacteria (LAB) and bifidobacteria (Lactobacillus casei), Lactobacillus acidophilus (L.acidophilus) and Bifidobacterium bifidum (Bifidobacterium bifidum)) was tested. Pectin was observed to significantly enhance bacterial growth and titratable acidity. The authors concluded that pectin extracted from fruit waste could be used to promote the growth of lactobacilli and bifidobacteria.

Babbar et al (pectin oligosaccharides from agricultural by-products: production, chromatography and health peptides, Crit. Rev. Biotech.2016; 36(4)594-606) mention that pectin-containing agricultural by-products are potential sources of a new class of prebiotics known as Pectin Oligosaccharides (POS). Controlled hydrolysis of pectin-containing agricultural by-products such as sugar beets, apples, olives and oranges by chemical, enzymatic and hydrothermal treatment can be used to produce galacto-oligosaccharides (oligo-galacturonides), galacto-oligosaccharides (galcto-oligosaccharides), rhamnogalacturonan-oligosaccharides (rhamnogalacturonans-oligosaccharides) and the like.

Hoon Kim et al (Effect of arabinoxylan-and rhamnogalacturonan I-rich polysaccharides isolated from raw and barrel leaf on intestinal immunological activity, Journal of Functional Foods, 2017; 35,384-390) prepared four polysaccharide fractions from barley leaves and compared intestinal immunostimulatory activity in vitro. Among these fractions, the high molecular weight fraction (BLE-P) prepared by enzyme extraction showed efficacy in activating bone marrow cell proliferation and stimulating cytokine production in vitro by Peyer's Patch (PP). BLE-P was identified as a mixture of hemicellulose glucuronoaarabinoxylans and pectin rhamnogalacturonans I, accounting for over 80%. BLE-P was then orally administered to mice for 20 days to study the effect on intestinal immunostimulatory activity in vivo. BLEP administration not only increased immunoglobulin A (IgA) production, but also increased levels of IgA associated cytokines such as transforming growth factor-beta and interleukin-10.

US 2014/275233 describes a method for treating a gastrointestinal disorder in a subject comprising orally administering to the subject an effective amount of a composition comprising isolated plant tissue having a glyceollin content of at least 0.25 mg/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.

WO2011/069781 describes a polysaccharide capable of modulating an immune response, the polysaccharide being obtained from a plant of the species Camellia sinensis (Camellia sinensis), wherein the backbone of the polysaccharide comprises alternating rhamnogalacturonan-I domains and alpha (1,4) -linked polygalacturonic acid or alpha (1,4) -linked oligogalacturonide domains, wherein the molar ratio of galacturonic acid residues to rhamnosyl residues in the backbone of the polysaccharide is in the range 2.5:1 to 1:1, and wherein the polysaccharide has a molecular weight of at least 70 kDa.

WO 2012/148277 describes a formulation having a dry matter content of at least 20 wt%, the formulation comprising at least 50 wt% of dry matter of a mixture of pectic polysaccharides comprising at least 20% (by weight of the pectic polysaccharides) of rhamnogalacturonan-I pectin having a molecular weight of more than 40kDa, the mixture of pectic polysaccharides being characterized by:

the degree of methylation of the galacturonic acid residues does not exceed 20%;

the degree of acetylation of galacturonic acid residues does not exceed 20%;

wherein the formulation does not form a gel when diluted with 50mM ammonium bicarbonate in water to a solids content of 2.5 wt.%. The use of the formulation as a medicament for modulating an immune response is also described.

Summary of The Invention

The inventors of the present application have surprisingly found that disorders related to disturbed 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) polysaccharide derived from fruit, carrot, pea, chicory or sugar beet, having a molecular weight of more than 15kDa and having a backbone comprising rhamnogalacturonan I domains and optionally α (1,4) -linked homogalacturonan I domains, wherein the molar ratio of galacturonate residues to rhamnose residues in the backbone is in the range of 20:1 to 1: 1.

Although the inventors do not wish to be bound by theory, it is believed that the RG-I polysaccharide acts as a prebiotic by promoting high microbial diversity within the gut microbiota, by stimulating the growth or activity of beneficial bacteria such as Akkermansia muciniphila (Akkermansia muciniphila) and bifidobacteria, and/or by increasing the resilience of the gut microbiota against interference.

It was further found that intestinal fermentation of RG-I polysaccharide leads to the formation of beneficial short chain fatty acids. Surprisingly, fermentative conversion of RG-I polysaccharides to short-chain fatty acids is accompanied by significantly less gas production than observed with classical prebiotics such as, for example, inulin.

Thus, one aspect of the invention relates to a prebiotic composition, for use in a method for the therapeutic or prophylactic treatment of a disorder related to a disturbed composition or function of the gut microbiome in a subject, said disorder being selected from metabolic disorders and intestinal barrier dysfunction, said use comprising orally administering said prebiotic composition to said subject, wherein the composition comprises at least 0.1 wt.% dry matter of RG-I polysaccharides originating from fruit, carrots, peas, chicory or sugar beets, the RG-I polysaccharide has a molecular weight of more than 15kDa and has a backbone consisting of galacturonic acid residues and rhamnose residues, the rhamnose residue is contained in the alpha (1 → 4) -galacturonic acid-alpha (1 → 2) -rhamnose residue, wherein the molar ratio of galacturonic acid residues to rhamnose residues in the RG-I polysaccharide is in the range of 20:1 to 1: 1.

The RG-I polysaccharides used according to the invention can be isolated from fruits, carrots, peas, chicory or sugar beets by aqueous extraction, optionally in combination with an enzymatic treatment (using e.g.polygalacturonase).

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

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

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

Yet another aspect of the invention relates to synbiotic compositions comprising:

at least 0.1% by weight of dry matter of RG-I polysaccharide; and

one or more probiotic microbial strains in the form of live microorganisms, inactive microorganisms, microbial fragments and combinations thereof.

Detailed Description

One aspect of the present invention relates to a prebiotic composition for use in a method for the therapeutic or prophylactic treatment of a disorder related to disturbed composition or function of the gut microbiome in a subject, the disorder being selected from metabolic disorders and gut barrier dysfunction, the use comprising orally administering the prebiotic composition to the subject, wherein the composition comprises at least 0.1 wt.% of dry matter of a rhamnogalacturonan I (RG-I) polysaccharide derived from fruit, carrot, pea, chicory or sugar beet, the RG-I polysaccharide having a molecular weight of more than 15kDa and having a backbone consisting of galacturonic acid residues and rhamnose residues, the rhamnose residues being comprised in α (1 → 4) -galacturonic acid- α (1 → 2) -rhamnose residues, wherein the molar ratio of galacturonic acid residues to rhamnose residues in the RG-I polysaccharide is between 20:1 and 1:1, in the above range.

The term "disorders associated with disturbed composition or function of gut microbiome" as used herein encompasses gut dysregulated (dysbiological) conditions (disturbed composition) and disorders associated with insufficient fermentation of gut microbiome to produce essential metabolites (disturbed function). Short chain fatty acids (acetate, propionate, and butyrate) are examples of such essential metabolites.

The term "gut dysregulated condition" as used herein refers to a condition that adversely affects the health of a subject and results from a significant deviation from the balanced gut microbiome (normal ecology).

The term "branched polysaccharide" as used herein refers to a polysaccharide comprising a linear backbone of monosaccharide units bonded together by glycoside linkages, wherein at least one monosaccharide unit within the backbone carries a side chain of one or more glycoside-linked monosaccharide units.

The terms "backbone chain" and "backbone" are synonymous.

The term "pectic polysaccharide" as used herein refers to an optionally branched polysaccharide having a molecular weight of more than 15kDa and comprising a backbone consisting of galacturonic acid residues and rhamnose residues, the rhamnose residues being comprised in α (1 → 4) -galacturonic acid- α (1 → 2) -rhamnose residues.

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

The term "domain" as used herein refers to a segment plus any side chains attached to the segment.

The term "rhamnogalacturonan-I segment" or "RG-I segment" refers to a segment consisting of a galacturonic acid (GalA) and rhamnose (Rha) pair, wherein the GalA residues are linked to the Rha residue via positions 1 and 4, and the Rha residue is linked to the GalA residue via the terminal group and 2-OH position, i.e. alternating α (1 → 4) -galacturonic acid- α (1 → 2) -rhamnose residues. The carboxyl group of the galacturonic acid residue in the RG-I region can be esterified. The esterified galacturonic acid can be present as methyl or acetyl ester.

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

The term "rhamnogalacturonan-I polysaccharide" or "RG-I polysaccharide" refers to an optionally branched pectin polysaccharide comprising a backbone containing one or more rhamnogalacturonan-I segments.

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

In addition to the RG-I domain, the RG-I polysaccharide of the invention may also comprise one or more of the following domains:

homogalacturonan (HG),

xylogalacturonan (XG),

apigalacturonan (AG),

rhamnogalacturonan-II (RG-II).

The domains XG, AG and RG-II usually represent only a small part of the RG-I polysaccharide.

The HG domain, XG domain, AG and RG-II domain 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. The carboxyl groups of the galacturonic acid residues in the backbone of these domains can be esterified. The esterified galacturonic acid can be present as methyl or acetyl ester.

The HG domain does not comprise any side chains.

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

The backbone of the AG domain comprises one or more side chains consisting of one or more D-apiose residues.

The backbone of RG-II comprises one or more side chains that are not composed solely of D-xylose or D-celery pool.

The term "fruit" as used herein refers to the structure of seeds in flowering plants.

The term "prebiotic" as used herein refers to a substance that selectively induces the growth or activity of microorganisms that contribute to the well-being of its host.

The term "probiotic" as used herein refers to a microorganism that provides health benefits when administered orally in sufficient amounts. The microorganisms are selected from the group consisting of live microorganisms, inactive microorganisms, fragments of microorganisms, and combinations thereof.

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

The concentration of the 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 suitably determined by high performance anion exchange chromatography in combination with pulsed amperometric detection (HPAEC-PAD).

Molecular size distribution can be determined by high performance size exclusion chromatography using Refractive Index (RI) detection (concentration), light scattering detection (molecular weight detection), UV detection (indicating the presence of protein), and differential pressure detection (intrinsic viscosity detection).

The above mentioned analytical methods are described in: analytical Biochemistry, volume 207, phase 1, 1992, pg 176 (for neutral sugar analysis) and mol.nutr.food res., volume 61, phase 1, 2017,1600243 (for galacturonic acid analysis and molecular size distribution).

All percentages mentioned herein refer to weight percentages unless otherwise indicated.

The subject to which the RG-I polysaccharide-containing composition 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 that is still developing its core maturation microbiota (<4 years) or an elderly person at risk of losing the diversity and elasticity of its core maturation microbiota (>50 years).

Oral administration in the context of the methods of the invention of treatment encompasses self-administration.

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

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, abnormal lipid metabolism, hyperglycemia, hepatic steatosis, dyslipidemia, high cholesterol, elevated triglycerides. The treatment of the invention is particularly suitable for the therapeutic or prophylactic treatment of overweight or obesity and insulin resistance.

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

The intestinal barrier or intestinal mucosal barrier refers to the property of the intestinal mucosa that ensures adequate containment of the undesirable luminal contents in the intestine while maintaining the ability to absorb nutrients. It provides separation between the body and the luminal contents of the intestinal tract preventing uncontrolled transfer of luminal contents into the body. Its role in protecting mucosal tissue and the circulatory system from exposure to proinflammatory pathogens, toxins and antigens is crucial to 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.

According to a preferred embodiment of the invention, the prebiotic composition is for use in the therapeutic or prophylactic treatment of a gut disorder condition.

The subject to be treated for a gut disorder condition according to the invention is preferably a subject suffering from or at risk of suffering from a pathogenic gut disorder condition, most preferably a subject suffering from such a pathogenic gut disorder condition. By "pathogenic" herein is meant that the condition is capable of causing or exacerbating the disease.

According to another preferred embodiment, the prebiotic composition is used to increase the intestinal fermentative production of short chain fatty acids.

The RG-I polysaccharide used according to the invention is preferably derived from a plant source selected from the group consisting of apple, bell pepper, bilberry, carrot, citrus, grape, pea, chicory, beet and olive, okra and combinations thereof. Even more preferably, the RG-I polysaccharide is derived from a plant source selected from the group consisting of apples (e.g. apple pomace), bell peppers, carrots, citrus peels, grapes, chicory, sugar beets (e.g. beet pulp), olives (e.g. olive pulp), okra and combinations thereof. Most preferably, the RG-I polysaccharide is derived from carrot or apple.

The 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, the RG-I polysaccharide constitutes at least 20 wt.%, more preferably at least 40 wt.%, even more preferably at least 50 wt.% and most preferably at least 60 wt.% of the pectin polysaccharides present in the prebiotic composition.

The RG-I polysaccharide has a backbone comprising rhamnogalacturonan-I segments and optionally alpha (1,4) -linked homogalacturonan segments. The molar ratio of galacturonic acid residues to rhamnose residues in the RG-I polysaccharide is in the range of 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, most preferably from 9:1 to 1: 1.

Preferably, the rhamnose residues constitute from 3 to 50%, more preferably from 5 to 50%, and most preferably from 10 to 50% of the monosaccharide residues in the RG-I polysaccharide backbone.

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

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

Galacturonic acid residues typically constitute 10-80%, more preferably 15-70%, and most preferably 20-65% of all monosaccharide residues comprised in the RG-I polysaccharide, i.e. including monosaccharide residues comprised in the side chains.

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

The average molecular weight of the RG-I polysaccharide comprised in the composition of the present invention is preferably above 30kDa, more preferably above 40kDa, and most preferably above 60 kDa.

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 a degree of esterification of 0% to 70%, more preferably 0% to 60%, even more preferably 0% to 55%, most preferably 0% to 50%.

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

The backbone 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 the RG-I polysaccharide may provide small amounts of residues of monomeric fucose, glucose, glucuronic acid, xylose and/or uronic acid. The side chain or chains are preferably selected from galactan side chains, arabinosan side chains and arabinogalactan side chains.

The arabinosan side chains comprise at least one or more alpha (1,5) -linked arabinose residues and are substituted in the 4-OH position of the rhamnose residue in the RG-I domain. The arabinoglycan side chains may be linear or branched. If the side chain is linear, the side chain consists of α (1,5) -linked arabinose residues. If the arabinosan side chain is a branched side chain, one or more alpha-arabinose residues are attached to the 2-OH and/or 3-OH of the alpha (1,5) -linked arabinose. The length of the arabinoglycan side chain (expressed as the number of monomer units) is preferably from 1 to 100 monomer units, more preferably from 1 to 50 units, even more preferably from 1 to 30 units.

The galactan side chains contain at least one or more β (1,4) -linked galactose residues and are substituted at the 4-OH position of the rhamnose residues in the RG-I domain.

The galactan side chains are 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 chains is preferably from 1 to 100 monomer units, more preferably from 1 to 50 units, even more preferably from 1 to 30 units.

The arabinogalactan side chains are substituted at the 4-OH position of the rhamnose residue of the RG-I domain and may be either type I Arabinogalactan (AGI) or type II Arabinogalactan (AGII). AGI consists of a (1 → 4) - β -D-Galp backbone, on which substitution of monomeric Galp units can occur at the O-6 or O-3 position. AGI is further substituted with an α -L-Araf-p residue and/or a short side chain of (1 → 5) - α -L-Araf. AGII consists of an α (1 → 3) - β -D-Galp backbone modified with a (1 → 6) - β -D-Galp secondary chain, which is arabinosylated (arabinosylated).

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

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

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

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

In addition to the RG-I polysaccharide, the prebiotic composition used in the method of the present invention advantageously comprises one or more further prebiotics. Preferably, the composition comprises at least 1 wt% dry matter of one or more prebiotics selected from the group consisting of lactulose, inulin, fructo-oligosaccharides, galacto-oligosaccharides, lacto-oligosaccharides, guar gum and gum arabic, more preferably at least 3 wt% dry matter of one or more prebiotics.

RG-I polysaccharide is considered to be particularly effective in the treatment methods of the present invention if it is no longer entangled in the matrix of the cell wall material. Thus, in a particularly preferred embodiment, the RG-I polysaccharide-containing composition 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 dry matter of RG-I polysaccharide that is readily soluble in water. The concentration of readily water-soluble RG-I polysaccharide in a composition containing RG-I polysaccharide can be determined by combining 100ml of demineralized water (20 ℃) with a sufficient amount of the composition containing RG-I polysaccharide to provide 2.5g of dry matter, followed by stirring for 5 minutes and filtration on a 100 μm filter. The RG polysaccharide in the filtrate is RG-I polysaccharide which is easily soluble in water.

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

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

The prebiotic compositions of the present invention were found to be capable of inducing the growth of intestinal microorganisms which are believed to provide health benefits, particularly akkermansia muciniphila and bifidobacteria. Thus, in another preferred embodiment, the composition comprising RG-I polysaccharide is capable of inducing the growth or activity of akkermansia muciniphila and/or bifidobacteria in the intestinal microbiota of a subject. Most preferably, the composition is capable of inducing growth or activity of akkermansia muciniphila in the gut microbiota of a subject.

It was found that the prebiotic composition of the invention is capable of inducing the production of short chain fatty acids by the gut microbiota, accompanied by significantly less gas production compared to classical prebiotics such as inulin. Thus, in another preferred embodiment, the RG-I polysaccharide containing compositions are capable of enhancing the intestinal fermentative production of short chain fatty acids and reducing the side effects associated with rapid gas production, such as intestinal discomfort, flatulence and reflux.

According to a particularly preferred embodiment, the composition is selected from the group consisting of beverages, oral dosage units, powders, bars (bars) and spreads (spreads).

The beverage is typically a liquid. Preferably, the beverage comprises 80 wt.%, more preferably at least 85 wt.% water. The beverage preferably comprises at least 1.5g/l, more preferably at least 3g/l, and most preferably 5-200g/l of RG-I polysaccharide.

Oral dosage units are preferably capsules or tablets. Oral dosage units preferably have a weight of from 50 to 1500 mg, more preferably from 100 to 800 mg. Oral dosage units typically comprise at least 1%, more preferably at least 20%, and most preferably 40-90% by weight of RG-I polysaccharide.

The composition in powder form is preferably a water-soluble powder which can be used for preparing beverages. Typically, the powder comprises at least 0.5 wt.%, more preferably at least 5 wt.%, and most preferably 10 to 75 wt.% of the RG-I polysaccharide.

The composition in the form of a stick is preferably a stick, preferably a stick weighing from 10 to 200 grams, more preferably from 25 to 100 grams. The strip-forming agent typically comprises at least 0.1 wt.%, more preferably at least 0.5 wt.%, and most preferably 1-20 wt.% of RG-I polysaccharide.

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

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

at least 0.1% by weight of dry matter of RG-I polysaccharide as defined above; and

at least 1% by weight, preferably at least 3% by weight, of dry matter of one or more prebiotics selected from the group consisting of lactulose, inulin, fructooligosaccharides, galactooligosaccharides, lactooligosaccharides, guar gum and acacia gum.

Even more preferably, the product comprises at least 1 wt.% dry matter, more preferably at least 3 wt.% dry matter of one or more prebiotics selected from the group consisting of lactulose, inulin, fructo-oligosaccharides, galacto-oligosaccharides and lacto-oligosaccharides.

Preferred embodiments of the prebiotic composition are the same as described above in relation to the prebiotic composition in the method of the invention for use in therapy.

Yet another aspect of the invention relates to synbiotic compositions comprising:

at least 0.1% by weight of dry matter of RG-I polysaccharide as defined above; and

one or more probiotic microbial strains in the form of live microorganisms, inactive microorganisms, microbial fragments and combinations thereof.

Preferred embodiments of the synbiotic composition are the same as described above for the prebiotic composition in the method of the invention for use in therapy.

The one or more probiotic microbial strains in the synbiotic composition are preferably live microbial strains, more preferably live bacterial strains.

The 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 the genera yeast, debaryomyces (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 the genera bifidobacterium, bacteroides, clostridium (Fusobacterium), melissococcus, propionibacterium, enterococcus, lactococcus, staphylococci, streptococcus digestus, bacillus, pediococcus, micrococcus, leuconostoc, Weissella (Weissella), coprobacterium, Akkermansia (Akkermansia), enterococcus, lactobacillus, allobactum, eubacteria, and combinations thereof.

According to a particularly preferred embodiment, the probiotic microorganism strain or strains is/are selected from the group consisting of Saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus coagulans (Bacillus coagulosus), Bacillus licheniformis (Bacillus licheniformis), Bacillus subtilis (Bacillus subtilis), Bifidobacterium bifidum, Bifidobacterium infantis (Bifidobacterium infantis), Bifidobacterium longum (Bifidobacterium longum), Enterococcus faecium (Enterococcus faecium), Enterococcus faecalis (Enterococcus faecalis), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus digestus (Lactobacillus alimentarius), Lactobacillus casei (Lactobacillus subsp. casei), Lactobacillus casei (Lactobacillus casei), Lactobacillus curvatus (Lactobacillus), Lactobacillus paracasei (Lactobacillus paracasei) Lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus sake (Lactobacillus sake), Lactococcus lactis (Lactobacillus lactis), Micrococcus mutabilis (Micrococcus varians), Pediococcus acidilactici (Pediococcus acidilactici), Pediococcus pentosaceus (Pediococcus pentaticus), Pediococcus halophilus (Pediococcus halophilus), Streptococcus salivarius (Streptococcus salivarius), Streptococcus thermophilus (Streptococcus thermophilus), Staphylococcus carnosus (Staphylococcus carnosus), Staphylococcus carnosus (Staphylococcus xylosus), Staphylococcus epidermidis (Staphylococcus epidermidis), Achromobacter sticklandii, Streptococcus faecalis (Faecalibacterium), human (Lactobacillus plantarum) and Escherichia coli (Escherichia coli).

In case the probiotic microbial strain is applied in an inactive form, the synbiotic composition preferably comprises a concentration of 10⁴-10¹⁰One or more probiotic microbial strains of cfu or an equivalent thereof. More preferably, the synbiotic composition comprises a concentration of 10⁵-10⁹One or more probiotic microbial strains of cfu or an equivalent thereof.

The proportion of akkermansia muciniphila is preferably 10⁵-10¹⁰cfu/g, more preferably 10⁶-10⁹The concentration of cfu/gram is comprised in the probiotic composition.

The Bifidobacterium is preferably 10⁶-10¹⁰cfu/g, more preferably 10⁷-10⁹The concentration of cfu/gram is comprised in the probiotic composition.

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

Examples

Example 1

The RG-I polysaccharide fraction was isolated from pilot-scale dried Sichuan pepper powder (Paprika Mild80-100Atsa Steamtr-Felix reverse S.A.) using the procedure described below.

Pepper material (100kg) was washed three times with 80% aqueous ethanol solution under mild turning, i.e. twice at 80 ℃ for 2 hours, then overnight at room temperature; 12.5% (w/v) each time was used to remove ethanol soluble material. The ethanol-insoluble residue was recovered by centrifugation (1000G for 10min) each time. The ethanol-insoluble residue obtained after 3 washing cycles was dried and 90kg were extracted twice with 1000L of hot water having a temperature of 95 ℃ for 90 minutes. At each time, the supernatant was retained after centrifugation at 1000G for 10 minutes. The collected supernatant was then filtered through cloth and ultrafiltered using a 2KDa molecular weight cut-off membrane to remove small molecular weight material. The dry RG-I-enriched extract was obtained by freeze-drying the retentate, yielding about 5kg of dry RG-I-enriched polysaccharide extract.

Characterization of RG-I polysaccharide-rich extracts

Molecular weight distribution:

the molecular weight distribution of polysaccharide samples was determined by high performance size exclusion chromatography using Refractive Index (RI) detection (concentration), light scattering detection (molecular weight detection), UV detection (indicating the presence of protein) and differential pressure detection (intrinsic viscosity detection). Pullulan (pullulan) molecular weight standards were used for calibration.

Monosaccharide composition:

the polysaccharide samples were dissolved in 0.5M aqueous trifluoroacetic acid and hydrolyzed at 120 ℃ for 2 hours. The sample was then neutralized with NaOH and kept frozen until analyzed by High PH Anion Exchange Chromatography (HPAEC) using Pulsed Amperometric Detection (PAD). Uronic acid in the samples was determined by using an automated colorimetric m-hydroxybiphenyl assay (Achmed & Labavitch, j.food Biochem,1978,361)) on an automated analyzer (Skalar).

Degree of esterification

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

The molecular characteristics of the RG-I polysaccharide fractions are shown in Table 1a and Table 1 b.

TABLE 1a

Monosaccharides (% mol/mol)	Red color pepper
		Rha (rhamnose)	5.0
GalA (galacturonic acid)	70.0
		Ara (arabinose)	9.0
Gal (galactose)	9.0
		Glc (glucose)	3.0
Xyl (xylose)	2.0

TABLE 1b

Ratio of molecules	Red color pepper
		GalA/Rha	14
Ara/Rha	1.8
		Gal/Rha	1.8
Degree of methylation%	39
		Degree of acetylation%	9.0

Example 2

6-week-old, pathogen-free female C57BL/6 mice received ad libitum sterile drinking water and semisynthetic irradiated AIN-93G Diet (Research Diet Services, Wijk bij Duursted, The Netherlands) containing 30mmol/kg calcium (uninfected and infected groups) or The same Diet supplemented with 1% (w/w) of RG-I rich extract from Capsicum annuum.

Mice were randomly assigned to treatment groups and were housed 3 mice per cage for 2 weeks and then individually for one week. Reeves et al describe the composition of the AIN-93G diet (AIN-93purified fractions for laboratories: final report of the American Institute of Nutrition on the recommendation of the AIN-76A cadent fraction. J Nutr (1993)123: 1939-1951).

Feces were collected at baseline before dietary intervention and after 3 weeks of dietary intervention. Genomic DNA was extracted from the stool samples according to the manufacturer's protocol (Zymo research). Microbial identification of isolated gDNA was performed by 16S rRNA gene sequencing. Specific primers are used to PCR amplify genomic regions of interest, 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. The FASTQ sequence file was generated using Illumina Casava pipeline version 1.8.3. The initial quality assessment is based on Illumina homogeneity filtering. Subsequently, the reading containing the PhiX control signal is removed using an internal filtering protocol (baseclean). In addition, reads containing (part of) the aptamer were cut out (up to a minimum read length of 50 bp). The second quality assessment is based on the remaining readings using FASTQC quality control tool version 0.10.0.

Bacterial DNA sequencing

Illumina misseq data was analyzed using a workflow using a Microbial ecological quantification study (Quantitative instruments Into Microbial Ecology) (QIIME, v8) pipeline (Caporo et al, QIIME allowances of high-through mass communication sequencing data, Nature Methods (2010),7(5), 335-. The data was demultiplexed (multiplexed) and unmatched barcodes and small fragments (>50nt) were filtered. Open reference OTU selection was performed in QIIME using the silvera 111 database, chimera detection by USEARCH, and filtering out of OTU. The bilom file and phylogenetic tree file are again generated from the filtered OUT using the silvera 111 database. Further outputs such as filter readings for each sample, PD whole tree diversity measurements, and 1-6 class taxonomic distributions with relative abundance were generated via QIIME.

Effect of RG-I polysaccharide-rich extracts on microbial composition

Illumina 16S rRNA sequencing was performed on individual stool samples from 12 mice per group to further understand microbial composition. 26x10 per sample³To 79x10⁴And (4) reading. The results are shown in tables 2, 3 and 4。

TABLE 2

Indicates significance relative to RG-I baseline

TABLE 3

Indicates significance relative to RG-I baseline

TABLE 4

Indicates significance relative to RG-I baseline

At the phylum level, addition of RG-I-rich polysaccharide extracts to the diet significantly increased the abundance of Actinomycetes and Mycoplasma, whereas the decrease of the abundance of Bacteroides and Proteobacteria (> 0.5% abundance; Wilcoxon, P <0.05) resulted in an increase of the firmicutes/Bacteroides ratio.

At the genus level, redundant analyses showed that the addition of RG-I-rich polysaccharide extracts to the diet had a significant effect on the microbial composition (P < 0.05; Monte Carlo replacement). The inclusion of RG-I rich polysaccharide extracts in the diet resulted in higher abundance of Bifidobacterium (Actinomycetes), Allobaculum (firmicutes), Akkermansia (Microbactria wartii) and unassigned bacterial groups.

Example 3

The indigestible polysaccharides are mainly digested in the human large intestine by the intestinal microbiota. This may lead to the growth of healthy beneficial bacteria, lowering pH, increasing resistance to intestinal pathogens, and regulating the metabolic activity of the microbiota. Beneficial (prebiotic) functional carbohydrates will promote the production of health beneficial metabolites such as short chain fatty acids (SCFA, i.e. acetate, propionate and butyrate) while reducing the production of undesirable metabolites of protein metabolism such as branched SCFA and ammonia.

The established short-term colon incubation model was used to test the effect of plant-derived polysaccharide extracts on the metabolic activity of the microbiota.

At the beginning of the incubation, sugar-depleted basal colon culture medium containing nutrients present in the colon (e.g., host-derived glycans such as mucin) was introduced into a 70mL penicillin bottle already containing the test extract (5g/L final concentration). Sealing the bottle with a rubber stopper by using N₂Anaerobic habitat (anaerobiosis) was obtained by flushing. Subsequently, a human fecal inoculum was prepared by mixing a freshly collected fecal sample with anaerobic phosphate buffer. After homogenization and removal of the particles via centrifugation (2min,500g), the fecal inoculum was added to different bottles. At this point, incubation was initiated for 48 hours, during which time the temperature was controlled at 37 ℃ and continuous mixing was ensured by a shaker (90 rpm). Samples were taken after 6 hours, 24 hours and 48 hours incubation for pH (Senseline F410; ProSense, Oosteerhout, The Netherlands), barometric pressure (hand-held pressure indicator CPH 6200; Wika, Echt, The Netherlands) and SCFA analysis. SCFAs (which are acetate, propionate, butyrate) and branched SCFAs (isobutyrate, isovalerate and isocaproate) were measured as described by De Weirdt et al (Human facet microbial display variable patterns of glycerol methyl isocyanate, FEMS Microbiol. Ecol.2010,74, 601-611).

Sugar-depleted colon medium and inulin, a well-known prebiotic (Beneo, DP. gtoreq.23,. about.100% inulin), were used as negative and positive references, respectively.

Sample A was produced from powdered Capsicum annuum (Paprika poeder, Natural Spices Mijdrecht, the Netherlands) by water extraction (10% w/w, 2 hours at 90 ℃), centrifugation to remove insoluble residues, filtration (40kDa cut-off) to remove small molecules and drying to obtain a powder.

Sample B was produced from dried carrot pomace (residue from carrot juice production (carrot fibre powder, greenfields, Poland)). Sample (I)B by using pectinase: (Ultra mask, Novozymes) was subjected to water extraction (10% w/w at 45 ℃ for 2 hours), heat-inactivated (90 ℃,10 minutes), removal of insoluble residues by decantation, ultrafiltration (40kDa cut-off) and final drying.

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

Sample D was produced from Okra powder (group Okra, My Foods, Blue mountain peak, UK) by extraction with hot water (10% w/w, 2 hours at 90 ℃) and dialysis against water for several hours to remove small molecular weight material. The dialyzed extract was then freeze-dried.

The monosaccharide composition of the above samples was determined as described in example 1. The results are shown in table 5.

TABLE 5

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

TABLE 6

The results show that all 4 plant-derived RG-I polysaccharide extracts are readily fermented by the gut microbiota.

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

All RG-I polysaccharide extracts reduced the production of branched SCFAs and ammonia.

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

Example 4

A pasteurized milk beverage was prepared based on the formulation shown in table 7.

TABLE 7

	By weight%
		Cream	1
Alkalized cocoa powder	0.1
		Sucrose	3
RG-I polysaccharide isolates1	2
		Inulin powder	2
Carrageenan	0.01
		High acyl gellan gum	0.05
Hydroxypropyl starch	0.2
		Carboxymethyl cellulose	0.1
Skimmed milk	The rest part

¹Isolated from dried carrot pomace (see example 3)

Drinking 200ml of this milk drink per day by adults improves gut health and provides additional protection against common cold and flu.

Example 5

Nutritional bars (45g) were prepared based on the formula shown in table 8.

TABLE 8

	By weight%
		Maltodextrin	12.9
Milk protein isolate	9.0
		Soy protein isolate	0.5
Rice flour	5.3
		Oat bran	14.4
Rice potato chips (Rice Crisps)	9.0
		Crystalline fructose	6.8
Evaporation of sugar cane juice syrup	27.7
		Salt (salt)	0.3
Glycerol	1.0
		Almond butter	3.3
RG-I polysaccharide isolates1	4.5
		Inulin powder	1
Vitamin/mineral mixture	3.1
		Vanilla	1.2

¹Isolated from dried carrot pomace (see example 3)

The striping agent was produced as follows: all the wet ingredients were mixed together at 50 ℃ (syrup, glycerin, almond cream and flavoring).

The dry ingredients are mixed together separately, then the wet slurry is added to the dry mixture and the mass is mixed under high shear for 2 to 5 minutes. Prior to packaging, the dough is divided into slabs and cut into strips.

Eating 2 bars per day by adults improves gut health and provides additional protection against common cold and influenza.

Example 6

The dietary supplement was prepared in the form of capsules containing the dry mixtures shown in table 9.

TABLE 9

¹Isolated from dried carrot pomace (see example 3)

Example 7

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, a well-known prebiotic (Beneo, DP. gtoreq.23,. about.100% inulin), were used as negative and positive references, respectively.

Sample A was produced from dried and ground pea shells (ex) Cosucra, Warcoing, Belgium). Dispersing the powder in demineralized water (100g/L), performing enzymatic prehydrolysis with thermostable alpha-amylase (Megazyme) at 90 deg.C for 30 min, and further hydrolyzing with pectinase (2hr 45 deg.C, 0,2 v/v%Ultra mask, Novozymes). Heating at 100 deg.C for 10min to obtain final productThe digestion was stopped, then centrifuged (18.000g,10 min) and the supernatant extensively dialyzed using a 12-14kDa cut-off membrane (Visking, London, UK). The material was then lyophilized.

Sample B was produced from dried and ground beet pulp (from Suiker Unie, Dinteorord, NL) using the same method, but omitting the alpha-amylase pre-incubation step.

Sample C was produced from dried and ground chicory pulp (from Cosucra, Warcoing, Belgium) using the same method, but omitting the alpha-amylase preincubation step.

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

Watch 10

The results of the incubation experiments are shown in table 11.

TABLE 11

These results show that these different plant-derived RG-I polysaccharide extracts are readily fermented by the gut microbiota.

All RG-I polysaccharide extracts increased the production of SCFA. Indeed, all extracts were found to increase SCFA production to levels similar to or exceeding those observed for inulin.

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