Method of producing high and low molecular weight pectic saccharides from potato

Abstract

The invention relates to a method of producing high and low molecular weight pectic saccharides that are enriched in HG and RG-I, said method comprising the steps providing potato pulp as pectin-rich substrate, subjecting the pectin-rich substrate to enzymatic treatment, said enzymatic treatment comprising the use of one pectinase having an endo-polygalacturonase (EC 3.2.1.15) activity, subjecting the hydrolyzed pectic polysaccharides to microfiltration using microfiltration membranes having a molecular cut-off of 0.45 and 0.8 µm, recovering the microfiltration retentate, subjecting the microfiltration permeate to nanofiltration using a membrane having a molecular weight cut-off in the range of 150 – 300 Da, recovering the nanofiltration retentate.

Description

[0001] METHOD OF PRODUCING HIGH AND LOW MOLECULAR WEIGHT PECTIC SACCHARIDES

[0002] FROM POTATO

[0003] TECHNICAL FIELD OF THE INVENTION

[0004] The present invention relates to a method of producing high and low molecular weight pectic saccharides enriched in rhamnogalacturonan-l (RG-I) and homogalacturonan (HG) from potato pulp.

[0005] More particularly, the invention relates to a method of producing such pectic saccharides by enzymatic hydrolysis of pectin-rich substrate that has been obtained from potato material, followed by multi-step of filtration, more specifically microfiltration and nanofiltration, and recovery of the high and low molecular weight pectic saccharides.

[0006] BACKGROUND OF THE INVENTION

[0007] Pectins are complex mixture of colloidal polysaccharides found in the primary cell walls of both monocotyledons and dicotyledons.

[0008] The main polysaccharide structures are homogalacturonan (HG), rhamnogalacturonan-l (RG-I), and substituted galacturonans.

[0009] Structure

[0010] HG is a linear chain of 1 ,4- linked a-D-galactopyranosyluronic acid (GalA) residues in which some of the carboxyl groups are methyl esterified.

[0011] Rhamnogalacturonan (RG-1) has been called the "hairy region" of pectin, and is a family of pectic polysaccharides that contain a backbone of the repeating disaccharide [->4)-a-D-GalA-(l->2)-a-l- Rha-(l->],

[0012] 20-80% of the rhamnosyl (Rha) residues are, depending on the plant source and method of isolation, substituted at C-4 with neutral and acidic oligosaccharide side chains.

[0013] The predominant side chains contain linear and branched a-1 -arabinofuranosy I (Ara), and / or p- D-galactopyranosyl (Gal) residues although their relative proportions and chain lengths may differ depending on the plant source. Some of the side chains may be terminated with a-L- Fucopyranose (Fuc), p-D-Glucuronic acid (GlcA), and 4-O-methyl p-D-GIcA residues.

[0014] The backbone GalA is not typically substituted with oligosaccharides, and the backbone GalA residues are O-acetylated in rhamnogalacturonans from many plants.

[0015] Rhamnogalacturonan-I (RG-1) has been previously isolated from many plants and vegetables including potato.

[0016] Isolated Rhamnogalacturonan-I is typically a polysaccharide with a molecular weight in the 100,000 Da - 1 ,000,000 Da size range.

[0017] When pure, RG-1 is typically soluble in water with low intrinsic viscosity. It has been discussed to have mitogenic, immunostimulation and antitumor activity.

[0018] As the structure, size and composition of RG-1 determines its bioactivity, small changes in structure may confer large changes in activity.

[0019] Potato pulp is as a major low-value product generated primarily upon starch production or potato processing. Despite its interesting phytochemical composition, potato pulp was mainly used as cattle feed.

[0020] Major components of potato pulp are cell wall polysaccharides, which include cellulose, hemicellulose and pectic polysaccharides.

[0021] Pectic polysaccharides (56%) being the major components of potato cell wall have low economic value because of its low gelling properties associated with its high content of neutral sugars.

[0022] Indeed, unlike pectic polysaccharides from other sources, such as citrus and apple, potato pectin consists of high proportion of rhamnogalacturonan I (RG I) (72%) and smaller amount of homogalacturonan (HG).

[0023] Second distinguished structural property of potato pectin arise from the high proportion of neutral p-linked galactan side chains (representing 46% of the total pectin) linked to RG I backbone; while RG I originating from currently commercial sources of pectin are mostly substituted with arabinan side chains. Direct effects

[0024] Furthermore, pectins belong to the soluble viscous dietary fiber category. Their effects on human health and more especially on glucose and cholesterol metabolisms are recognized since a long time and mainly involved a direct effect of these fibers thanks to their specific structure.

[0025] Indeed, they are viscous fibers which slow down the gastric emptying and / or inhibit the carbohydrate degrading enzymes, resulting in a slower and lower postprandial glycemia. This effect is so well documented that an European regulatory health claim was authorized in 2010 for a daily consumption of 10 grams of pectin: “pectins and reduction of post-prandial glycaemic responses” (https: / / efsa.onlinelibrarv.wilev.eom / doi / pdf / 10.2903 / j.efsa.2010.1747) .

[0026] Thanks to its specific structure, pectin are also able to bind different components such as food cholesterol or bile acids during their travel all along gastrointestinal tract, inducing a positive effect on blood cholesterol levels. Here too, the level of proof is so high that EFSA authorized another health claims related to the maintenance of normal cholesterol levels with the consumption of 6 grams of pectin per day: pectins and maintenance of normal blood cholesterol concentrations (https: / / efsa.onlinelibrary.wiley.eom / doi / pdf / 10.2903 / j.efsa.2010.1747).

[0027] If pectins can bind to specific molecules resulting from the digestion of our food, they can also bind and activate some receptors located in the lumen and thus inducing some impacts on our immune system and more especially on the Intestinal Epithelial Cells (lECs).

[0028] Intestinal epithelium constitutes the first physical barrier between microbes, food components in the lumen and the interstitial medium before blood stream. It is reinforced by the mucus layer which is secreted by the globlet cells and constitutes a substrate for gut bacteria growth. An equilibrium is then established as the metabolites resulting from the mucin fermentation modifies the composition and the properties of the mucus layer contributing thus to its barrier function.

[0029] As the mucus layer, the intestinal epithelium can also be reinforced if membrane integrity is strengthened by a higher expression of tight junctions. Pectins were described to have a positive impact on intestinal epithelium and to protect it from damage using DSS-induced colitis animal model.

[0030] The involved mechanism of action in this effect has been described in the literature: as fiber, dietary pectins can reach the colon where they interact dendritic cells involved in innate immunity. These dietary components can be taken up through M cells and are presented to subepithelial dendritic cells, on the Peyer’s Patch. On the surface of these specific region of the intestinal cells, can be found Pattern Recognition Receptors such as Toll-Like Receptor-2 (TLR-2) that are directly involved in the innate immunity. Their activation induces a balance of regulation between pro and anti-inflammatory status to maintain gastrointestinal tract protection against pathogens and tolerance to food antigens.

[0031] The specific structure of pectins allows the effect of this dietary fiber on immunity, more especially the degree of methylation of the pectic structures was involved in this activation as it regulates the electrical charge of the molecule, allowing or not to adhere and penetrate the mucus layer before reaching TLR-2.

[0032] Prebiotic / Indirect effects.

[0033] In addition to the direct health effects of pectins linked to their physico-chemical structures, these soluble dietary fibers are also known for their impact on gut microbiota. According to ISAPP definition from 2017, pectins can be considered as prebiotics as they can be selectively used by gut microorganisms, thus conferring a health benefits to the host.

[0034] The specific impact of pectins on gut microbiota composition and functionality was described in the literature and seems to be different depending on their structure and vegetal origins. The use of pectins or pectic saccharides by gut microbiota leads to the production of specific range of metabolites including Short Chain Fatty Acids (SCFA) that can also impact immune response through the activation of other intestinal receptors: the G-protein coupled receptors GPR109A.

[0035] In spite of the interesting structural properties of pectic polysaccharides and their low cost, so far, they have not been often used as sources for the production of bioactive molecules.

[0036] Improved selective extraction and purification technologies are required if products are to be available on a large enough scale for health applications.

[0037] Without being exhaustive, the following literature lists various processes for isolating pectic polysaccharides form potato :

[0038] WO 2015 / 192247 relates to a process of isolating non-digestible oligosaccharides from potato pulp by extracting rhamnogalacturonan content from the potato pulp, digesting the extracted rhamnogalacturonan content with a multi-enzymatic mixture to yield the non- digestible oligosaccharides from the extracted rhamnogalacturonan content; and isolating the non-digestible oligosaccharides. Also are described pharmaceutical, nutraceutical and food applications for these protein-enriched preparations. WO 2016 / 132130 describes a process for the isolation of modified polysaccharide product from potato, in particular a polysaccharide product that can be used as an immunomodulatory.

[0039] WO 2000 / 043424, WO 2001 / 096405 and WO 2007 / 119366 relates to process for producing pectin involving the step of extracting root crops with hot water under weakly acidic conditions of pH, pectin having good qualities which is obtained by extracting from root crops; and acidic protein foods with the use of the same which are stable in the acidic pH region of the isoelectric point of protein or higher, and finally an extraction process of a pectin having excellent gelling effect from a potato starch pulp with high efficiency (with a chelating agent at a temperature of 90 to 125°C inclusive under condition where the pH value given after the completion of the extraction is adjusted to 5.5 to 11), and to produce a gelatinous food by using the pectin as a gelling agent.

[0040] WO 2005 / 003178 relates to a method for treating pectin-containing plant materials in a manner to obtain fibre-containing pectin products, and subsequently pectin products, having a high molecular weight of the pectin polymer and a homogeneous distribution of the de-esterified sites in the pectin polymer and thereby providing products having improved gel forming and / or viscous giving properties.

[0041] WO 2013 / 148282 provides nutritional supplement compositions. For example, nutritional supplement compositions containing a potato polysaccharide preparation, methods for obtaining potato polysaccharide preparations, methods for making nutritional supplement compositions containing a potato polysaccharide preparation, and methods for increasing or decreasing expression of polypeptides involved with mitochondria activity or function are provided.

[0042] So, it may especially be most interesting to have new pectic saccharides enriched in rhamnogalacturonan-l (RG-I) and homogalacturonan (HG) to propose.

[0043] SUMMARY OF THE INVENTION

[0044] To this end, the applicant company has pursued much laborious study and research which has enabled it to develop products which meet these requirements.

[0045] The applicant company have shown that high and low molecular weight pectic saccharides enriched in RGI and HG with biological functionalities can be obtained by a method comprising:

[0046] - subjecting a pectin-rich substrate that has been obtained from potato pulp to one pectinase having endo-polygalacturonase (EC 3.2.1.15) activity,

[0047] - subjecting the hydrolyzed pectic polysaccharides such obtained to microfiltration and nanofiltration and

[0048] - recovering the microfiltration and nanofiltration fractions. More particularly, the present invention relates to a method of producing high and low molecular weight pectic saccharides that are enriched in HG and RG-I, said method comprising the steps of:

[0049] - providing potato pulp as pectin-rich substrate,

[0050] - subjecting the pectin-rich substrate to enzymatic treatment, said enzymatic treatment comprising the use of one pectinase having an endo-polygalacturonase (EC 3.2.1.15) activity,

[0051] - subjecting the hydrolyzed pectic polysaccharides to microfiltration using microfiltration membranes having a molecular cut-off of between 0.45 and 0.8 pm,

[0052] - recovering the microfiltration retentate,

[0053] - subjecting the microfiltration permeate to nanofiltration using a membrane having a molecular weight cut-off in the range of 150 - 300 Da,

[0054] - recovering the nanofiltration retentate.

[0055] In an embodiment, the process comprises an optional step consisting in treating the pectic polysaccharides, the microfiltration retentate or the nanofiltration retentate with a strongly cationic exchange resin in the H+form to reduce heavy metals and glycoalkaloids, and to produce a final pH below 2.5 at 5% dry matter.

[0056] In such a way, the method is characterized in that the resin treatment reduces heavy metals of the hydrolyzed pectic polysaccharides, the high molecular weight pectic saccharides or the low molecular weight pectic saccharides to: cadmium < 0.01 mg / kg; copper < 0.5 mg / kg; lead < 0.04 mg / kg; nickel < 0.2 mg / kg; arsenic < 0.03 mg / kg; mercury < 0.005 mg / kg; and glycoalkaloids to: alpha-solanine < 25 mg / kg; alpha-chaconine < 25 mg / kg.

[0057] Moreover, this method is characterized in that the products obtained have a pH below 2.5 when suspended at 5 % dry matter.

[0058] The applicant company has first found that contrary to what is taught in the prior art, it is not useful to implement a cocktail of enzymes to achieve the supply of such new pectic saccharides.

[0059] The hydrolyzed pectic polysaccharides present :

[0060] - a molecular weight distribution for the fractions: o lower than 1500 Da, of between 10 and 25 %, preferably between 14 and 21 %, o from 1500 Da to 100 kDa, of between 45 and 65 %, preferably between 52 and 58 %, and o higher than 100 kDa between 20 and 35%, preferably between 22 and 30%,

[0061] - between 17 and 30 % in mol%, preferably between 20 and 27 % in mol% of HG,

[0062] - between 68 and 82 % in mol% preferably between 71 and 79 % in mol% of RG-I having a GalA to Rha ratio between 6.5 and 15.5 mol / mol, preferably between 6.6 and 14.9 mol / mol, and a (Gal plus Ara) to GalA ratio between 1 .8 to 3.8 mol / mol, preferably between 2.0 and 3.5 mol / mol,

[0063] - a methylation degree between 40 and 60 % in mol%, preferably between 43 and 57 % in mol% and an acetylation degree between 40 and 55 % in mol%, preferably between 43 and 50 % in mol%.

[0064] The hydrolyzed pectic polysaccharides is also characterized by its RG-I side chains having: a (Gal plus Ara) to Rha ratio between 10 and 50 mol / mol, preferably between 13 to 47 mol / mol, a Gal to Rha ratio between 8 and 45 mol / mol, more preferably between 10 and 40 mol / mol, an Ara to Rha ratio between 2.5 and 7.5 mol / mol, more preferably between 2.8 and 7.1 mol / mol, a GalA to Gal ratio between 0.1 and 1 .0 mol / mol, preferably between 0.3 and 0.6 mol / mol.

[0065] The applicant company have also found that enzymatic hydrolysis of the pectic polysaccharides using only one aforementioned pectinase followed by microfiltration, yields a high molecular weight pectic saccharide that exhibits biological functionalities.

[0066] So, the present invention relates to the microfiltration retentate containing high molecular weight pectic saccharide having : a molecular weight distribution for the fractions: o lower than 1500 Da, of between 5 and 15 %, preferably between 7 and 12 %, o from 1500 Da to 100 kDa, of between 40 and 65 %, preferably between 47 and 56 %, and o higher than 100 kDa, of between 25 and 50%, preferably between 32 and 46%, between 15 and 28 % in mol%, preferably between 16 and 25 % in mol% of HG, between 70 and 85 % in mol%, preferably between 73 and 83 % in mol% of RG-I having a GalA to Rha ratio between 4.0 and 6.0 mol / mol, preferably between 4.1 and 5.5 mol / mol, and a (Gal plus Ara) to Gal A ratio between 1.5 to 4.0 mol / mol, preferably between 1.9 and 3.5 mol / mol, a methylation degree between 15 and 50 % in mol% preferably between 17 and 46 % in mol% and an acetylation degree between 35 and 95 % in mol%, preferably between 39 and 90 % in mol%.

[0067] The terminology "degree of acetylation" refers to the number of acetyl residues per galacturonic acid residue, expressed as a molar percentage.

[0068] The terminology "degree of methylation" refers to the number of methyl residues per galacturonic acid residue, expressed as a molar percentage.

[0069] The concentration of different polysaccharides and their monosaccharide composition can be determined by analytical techniques known to the skilled person.

[0070] All percentages mentioned herein, unless otherwise stated, refer to the percentage in % by dry weight.

[0071] The RG-I side chains of the high molecular weight pectic saccharides of the invention are characterized by: a (Gal plus Ara) to Rha ratio between 9 to 15 mol / mol, preferably between 10 and 14 mol / mol, a Gal to Rha ratio between 5 and 13 mol / mol, more preferably between 7 and 11 mol / mol, an Ara to Rha ratio between 2.0 and 4.0 mol / mol, more preferably between 2.4 and 3.5 mol / mol, a GalA to Gal ratio between 0.2 and 1 .0 mol / mol, preferably between 0.4 and 0.7 mol / mol.

[0072] The high molecular weight pectic saccharides of the invention may be also characterized by their total sugar composition and their free sugars compositions, as will be exemplified below.

[0073] As it will be exemplified below, its total sugar composition is mainly :

[0074] Ara: between 8 and 14 %w / w, preferably 9 and 12 %w / w,

[0075] Rha: between 3.0 and 5.0 %w / w, preferably between 3.7 and 4.5 %w / w,

[0076] Gal: between 32 and 48 %w / w, more preferably between 36 and 44 %w / w, GalA: between 16 and 30 %w / w, more preferably between 18 and 28 %w / w.

[0077] Then, the following nanofiltration step that eliminate notably DP1 - DP2 (DP for Degree of Polymerization) of the microfiltration permeate yields a low molecular weight pectic saccharide that exhibits other biological functionalities.

[0078] So, the present invention relates to the nanofiltration retentate containing low molecular weight pectic saccharide having : a molecular weight distribution for the fractions: o lower than 1500 Da between 5 and 30%, preferably between 11 and 25% o from 1500 Da to 100 kDa between 50 and 80%, preferably between 54 and 79%, and o higher than 100 kDa between 0 and 35%, preferably between 1 and 30%, between 20 and 35 % in mol%, preferably between 22 and 33 % in mol% of HG between 65 and 80 % in mol%, preferably between 66 and 78 % in mol% of RG-I having a GalA to Rha ratio between 5 and 30 mol / mol, preferably between 7 and 26 mol / mol, and a (Gal plus Ara) to GalA ratio between 1 .5 and 3.5 mol / mol, preferably between 1 .8 and 3.2 mol / mol. a methylation degree between 20 and 55 % in mol%, preferably between 23 and 53 % in mol% and an acetylation degree between 10 and 50 % in mol%, preferably between 11 and 48 % in mol%.

[0079] The RG-I side chains of the low molecular weight pectic saccharides of the invention are characterized by: a (Gal plus Ara) to Rha ratio between 15 and 60 mol / mol, preferably between 18 and 55 mol / mol, a Gal to Rha ratio between 10 and 50 mol / mol, more preferably between 15 and 48 mol / mol, an Ara to Rha ratio between 2.8 and 7.5 mol / mol, more preferably between 3.1 and 7.3 mol / mol, a GalA to Gal ratio between 0.3 and 0.7 mol / mol, preferably between 0.4 and 0.6 mol / mol.

[0080] The low molecular weight pectic saccharides of the invention may be also characterized by their total sugar composition and their free sugars compositions, as will be exemplified below.

[0081] As it will be exemplified below, its total sugar composition is mainly :

[0082] Ara: between 4.0 and 10.0 %w / w, preferably 4.8 and 8.1 %w / w,

[0083] Rha: between 0.5 and 3.0 %w / w, preferably 0.9 and 2.8 %w / w,

[0084] Gal: between 44 and 57 %w / w, more preferably between 46 and 55 %w / w, GalA: between 20 and 36 %w / w, more preferably between 23 and 34 %w / w.

[0085] A final subject of the present invention relates to the use of the high or low molecular weight pectic saccharides according to the invention, or able to be obtained according to the method defined above, in nutritional formulation, food product, dietary supplement, beverage or pharmaceutical product.

[0086] More particularly, as will be exemplified below, the low molecular weight pectic saccharides may be advantageously used in neutral and acid beverages. Thanks to their complex structure, pectins and pectic saccharides are considered as doubly bioactive ingredients that can modulate immunity through two different mechanisms of action: direct activation of intestinal immunity and indirect impact through their prebiotic properties.

[0087] Then, the high molecular weight pectic saccharides of the invention may be used as long-lasting substrate for gut microbiota, allowing several cross-feeding interactions and a larger set of bioactive metabolites production.

[0088] The low molecular weight pectic saccharides of the invention increase the expression of the MUC3 protein involved in the mucus layer that protects and reinforces the intestinal epithelium.

[0089] The high molecular weight pectic saccharides and of the low molecular weight pectic saccharides of the invention may then be used as immuno-modulators.

[0090] DETAILED DESCRIPTION OF THE INVENTION

[0091] Accordingly, a first aspect of the invention relates to a method of producing high and low molecular weight pectic saccharides that are enriched in HG and RG-I, said method comprising the steps of:

[0092] - providing potato pulp as pectin-rich substrate,

[0093] - subjecting the pectin-rich substrate to enzymatic treatment, said enzymatic treatment comprising the use of one pectinase having an endo-polygalacturonase (EC 3.2.1.15) activity,

[0094] - subjecting the hydrolyzed pectic polysaccharides to microfiltration using a microfiltration membrane having a molecular cut-off of 0.45 and 0.8 pm,

[0095] - recovering the microfiltration retentate,

[0096] - subjecting the microfiltration permeate to nanofiltration using a membrane having a molecular weight cut-off in the range of 150 - 300 Da,

[0097] - recovering the nanofiltration retentate.

[0098] The first step of the process following the present invention consists in providing potato pulp as pectin-rich substrate.

[0099] Potato pulp is a major waste product of the potato-processing industry. Approximately 0.75 kg of potato pulp is generated to produce 1 .0 kg of starch.

[0100] On a dry weight basis, the non-starch components of potato pulp include fibers (88.3%), proteins (6.3%), ashes (5.0%) and fat (0.4%).

[0101] The moisture (8.7 - 87.0%w / w) and starch content (13.1 - 37.0% by dry weight) of potato pulp varies depending on the starch extraction method and any further drying process.

[0102] The pectin-rich substrate used in the present method preferably contains at least 25 % by dry weight, more preferably at least 28 % by dry weight (the pectin content is determined by summing the contents of Gal, Ara, Rha and GalA sugars, quantified using the analytical methods described below).

[0103] The second step of the process following the present invention consists in subjecting the pectin- rich substrate to enzymatic treatment.

[0104] The enzymatic treatment employs one specific pectinase, more particularly an endopolygalacturonase (EC 3.2.1.15).

[0105] The pure galacturonase may be the ISOZYM PG200 form MCN Biotech or the Polygalacturonase PF from the company Soufflet Biotechnologies.

[0106] The polygalacturonase enzyme activity must be known in advance and quantified by a method known to the person skilled in the art, such as the codified method used in oenology (COEI-1- ACTPGA).

[0107] The result are expressed in the unit uPG in relation to the mass quantity of commercial solution (uPG / g) as described in https: / / www.oiv.int / index.php / standards / international-oenoloqical- codex / part-i-monographs / enzymes / polygalacturonase-activity

[0108] For the treatment, the enzyme dose is in the range of 20 to 55 uPG by grams of pulp on dry matter, preferably of 25 to 50 uPG by grams of pulp on dry matter.

[0109] The pectin-rich substrate is subjected to one enzymatic treatment (enzyme dose in the range of 20 to 55 uPG by grams of pulp on dry matter, preferably of 25 to 50 uPG by grams of pulp on dry matter) in the form of an aqueous liquid containing the pectin-rich substrate and having a dry matter content of 2 to 20 % In weight, preferably of 5 to 15 % in weight.

[0110] The enzymatic treatment of the method according to the invention is preferably carried out at a pH in the range of 2.5 to 6.0, preferably in the range of 3.5 to 5.5.

[0111] The enzymatic treatment of the pectin-rich substrate with one or more pectinases is preferably carried out at a temperature in the range of 40° to 55°C, more preferably of 50°C.

[0112] The duration of the enzymatic treatment preferably is between 2 to 3 hours.

[0113] According to a particular mode of realization of the invention, a liquid / solid separation step is carried out to recover the hydrolyzed pectic polysaccharides fraction.

[0114] This operation can be carried out using various technologies, with a potential combination of these technologies (to maximize recovery yields), for example: decanter centrifuge, disc stack centrifuge, belt press, hydraulic filter press, rotary vacuum filter. A thermal treatment is also performed, with the objective to inactivate the enzyme.

[0115] The operation is carried out by applying a defined temperature / treatment time scale, e.g. 70°C for 2 h, or 90°C for 30min.

[0116] This operation can also be carried out using a high-pressure thermal process, well known by the man skilled in the Art.

[0117] The hydrolyzed pectic polysaccharides present : a molecular weight distribution for the fractions: o lower than 1500 Da, of between 10 and 25 %, preferably between 16 and 19 %, o from 1500 Da to 100 kDa, of between 45 and 65 %, preferably between 52 and 58 %, and o higher than 100 kDa between 20 and 35%, preferably between 26 and 29%, between 20 and 30 % in mol%, preferably between 22 and 27 % in mol% of HG, between 70 and 75 % in mol% preferably between 71 and 74 % in mol% of RG-I having a GalA to Rha ratio between 6.5 and 7.5 mol / mol, preferably between 6.6 and 7.4 mol / mol, and a (Gal plus Ara) to GalA ratio between 1.8 to 3.0 mol / mol, preferably between 2.0 and 2.6 mol / mol, a methylation degree between 40 and 60 % in mol%, preferably between 44 and 57 % in mol% and an acetylation degree between 45 and 50 % in mol%, preferably between 47 and 49 % in mol%.

[0118] The HG domains do not contain any sidechains. The carboxyl groups of galacturonic acid residues within the backbone of HG domains may be esterified. Esterified galacturonic acid may occur in the form of the methyl ester or acetyl ester.

[0119] The backbone of RG-I contains side chains that are composed of Galactose (Gal), Arabinose (Ara), Rhamnose (Rha) and GalA (Galacturonic Acid), here in the following proportion: a (Gal plus Ara) to Rha ratio between 10 and 20 mol / mol, preferably between 13 and 19 mol / mol, a Gal to Rha ratio between 8 and 20 mol / mol, more preferably between 10 and 16 mol / mol, an Ara to Rha ratio between 2.5 and 3.5 mol / mol, more preferably between 2.8 and 3.4 mol / mol, a GalA to Gal ratio between 0.3 and 1 .0 mol / mol, preferably between 0.5 and 0.6 mol / mol.

[0120] The hydrolyzed pectic polysaccharides such obtained may be also characterized by their total sugar composition and their free sugars compositions. As it will be exemplified below, its total sugar composition is mainly :

[0121] Ara: between 7.0 and 9.0 %w / w, preferably 7.5 and 8.4 %w / w,

[0122] Rha: between 1.5 and 4.0 %w / w, preferably between 2.1 and 3.2 %w / w,

[0123] Gal: between 35 and 55 %w / w, more preferably between 37 and 50 %w / w,

[0124] GalA: between 20 and 30 %w / w, more preferably between 22 and 28 %w / w.

[0125] The third step of the process following the invention consists in subjecting the hydrolyzed pectic polysaccharides to microfiltration.

[0126] The microfiltration treatment employs using a membrane having a molecular cut-off of between 0.45 and 0.8 pm, chosen in the group consisting in ceramic tubular membrane, ceramic disc membrane, organic spiral wound membrane, flat sheet membrane, hollow fiber membrane chosen alone or in combination. Thus, microfiltration can be carried out in one or two successive stages using membranes with the different cut-off values.

[0127] The fourth step of the process following the invention consists in recovering the microfiltration retentate, that could be optionally evaporated and dried by any techniques known by the man skilled in the Art.

[0128] The recovered microfiltration retentate contains the high molecular weight pectic saccharide of the invention.

[0129] This retentate may suitably be concentrated and then dried using one or more drying techniques selected from spray drying, freeze drying, air drying, roller drying, flatbed drying, belt drying and drum drying.

[0130] The high molecular weight pectic saccharide such obtained has: a molecular weight distribution for the fractions: o lower than 1500 Da, of between 5 and 15 %, preferably between 7 and 12 %, o from 1500 Da to 100 kDa, of between 40 and 65 %, preferably between 47 and 56 %, and o higher than 100 kDa, of between 25 and 50%, preferably between 32 and 46%, between 15 and 28 % in mol%, preferably between 16 and 25 % in mol% of HG, between 70 and 85 % in mol%, preferably between 73 and 83 % in mol% of RG-I having a GalA to Rha ratio between 4.0 and 6.0 mol / mol, preferably between 4.1 and 5.5 mol / mol, and a (Gal plus Ara) to Gal A ratio between 1.5 to 4.0 mol / mol, preferably between 1.9 and 3.5 mol / mol, a methylation degree between 15 and 50 % in mol% preferably between 17 and 46 % in mol% and an acetylation degree between 35 and 95 % in mol%, preferably between 39 and 90 % in mol%. The RG-I side chains of the high molecular weight pectic saccharides of the invention are characterized by: a (Gal plus Ara) to Rha ratio between 9 and 15 mol / mol, preferably between 10 and 14 mol / mol, a Gal to Rha ratio between 5 and 13 mol / mol, more preferably between 7 and 11 mol / mol, an Ara to Rha ratio between 2.0 and 4.0 mol / mol, more preferably between 2.4 and 3.5 mol / mol, a GalA to Gal ratio between 0.2 and 1 .0 mol / mol, preferably between 0.4 and 0.7 mol / mol.

[0131] The high molecular weight pectic saccharides of the invention may be also characterized by their total sugar composition and their free sugars compositions, as will be exemplified below.

[0132] As it will be exemplified below, its total sugar composition is mainly :

[0133] Ara: between 8 and 14 %w / w, preferably 9 and 12 %w / w,

[0134] Rha: between 3.0 and 5.0 %w / w, preferably between 3.7 and 4.5 %w / w,

[0135] Gal: between 32 and 48 %w / w, more preferably between 36 and 44 %w / w, GalA: between 16 and 30 %w / w, more preferably between 18 and 28 %w / w.

[0136] The fifth step of the process following the invention consists in subjecting the final microfiltration permeate to nanofiltration using a membrane having a molecular weight cut-off in the range of 150 - 300 Da.

[0137] The recovered nanofiltration retentate contains the low molecular weight pectic saccharide of the invention.

[0138] This retentate may suitably be concentrated and then dried using one or more drying techniques selected from spray drying, freeze drying, air drying, roller drying, flatbed drying, belt drying and drum drying.

[0139] The low molecular weight pectic saccharide such obtained has: a molecular weight distribution for the fractions: o lower than 1500 Da between 5 and 30%, preferably between 11 and 25% o from 1500 Da to 100 kDa between 50 and 80%, preferably between 54 and 79%, and o higher than 100 kDa between 0 and 35%, preferably between 1 and 30%, between 20 and 35 % in mol%, preferably between 22 and 33 % in mol% of HG between 65 and 80 % in mol%, preferably between 66 and 78 % in mol% of RG-I having a GalA to Rha ratio between 5 and 30 mol / mol, preferably between 7 and 26 mol / mol, and a (Gal plus Ara) to GalA ratio between 1 .5 and 3.5 mol / mol, preferably between 1 .8 and 3.2 mol / mol. a methylation degree between 20 and 55 % in mol%, preferably between 23 and 53 % in mol% and an acetylation degree between 10 and 50 % in mol%, preferably between 11 and 48 % in mol%.

[0140] The RG-I side chains of the low molecular weight pectic saccharides of the invention are characterized by: a (Gal plus Ara) to Rha ratio between 15 and 60 mol / mol, preferably between 18 and 55 mol / mol, a Gal to Rha ratio between 10 and 50 mol / mol, more preferably between 15 and 48 mol / mol, an Ara to Rha ratio between 2.8 and 7.5 mol / mol, more preferably between 3.1 and 7.3 mol / mol, a GalA to Gal ratio between 0.3 and 0.7 mol / mol, preferably between 0.4 and 0.6 mol / mol.

[0141] The low molecular weight pectic saccharides of the invention may be also characterized by their total sugar composition and their free sugars compositions, as will be exemplified below.

[0142] As it will be exemplified below, its total sugar composition is mainly :

[0143] Ara: between 4.0 and 10.0 %w / w, preferably 4.8 and 8.1 %w / w,

[0144] Rha: between 0.5 and 3.0 %w / w, preferably 0.9 and 2.8 %w / w,

[0145] Gal: between 44 and 57 %w / w, more preferably between 46 and 55 %w / w,

[0146] GalA: between 20 and 36 %w / w, more preferably between 23 and 34 %w / w.

[0147] A final subject of the present invention relates to the use of the high or low molecular weight pectic saccharides according to the invention, or able to be obtained according to the method defined above, in nutritional formulation, food product, dietary supplement, beverage or pharmaceutical product.

[0148] More particularly, as will be exemplified below, the low molecular weight pectic saccharides may be advantageously used in neutral and acid beverages.

[0149] Thanks to their complex structure, pectins and pectic saccharides are considered as doubly bioactive ingredients that can modulate immunity through two different mechanisms of action: direct activation of intestinal immunity and indirect impact through their prebiotic properties.

[0150] Then, the high molecular weight pectic saccharides of the invention may be used as long-lasting substrate for gut microbiota, allowing several cross-feeding interactions and a larger set of bioactive metabolites production. The low molecular weight pectic saccharides of the invention increase the expression of the MUC3 protein involved in the mucus layer that protects and reinforces the intestinal epithelium.

[0151] The high molecular weight pectic saccharides and of the low molecular weight pectic saccharides of the invention may then be used as immuno-modulators.

[0152] Figures:

[0153] Figure 1 : Cytokines secreted by PBMC when exposed to high or low Mw pectic oligosaccharides in basal (A) or challenged (B) conditions, a b means a and b are significantly different with p < 0.05.

[0154] Figure 2: MUC3 relative expression level in Caco2 / HT29MTX cells when exposed to high or low Mw pectic oligosaccharides, a b means a and b are significantly different with p < 0.05.

[0155] Figure 3: SCFAs production using SIFR® technology, d means a, b, c and d are significantly different with p < 0.05.

[0156] Figure 4: Bacterial cell load, a b c d means a, b, c and d are significantly different with p < 0.05.

[0157] The examples which follow make it possible to better understand the present invention, without however limiting the scope thereof.

[0158] EXAMPLES

[0159] ANALYTICAL METHODS

[0160] Molecular mass distribution:

[0161] Throughout the present application, the Mw, Mn, and PDI were determined by high pressure sizeexclusion chromatography (HPSEC).

[0162] This method permits to separate molecules in solution by their size, which correlates to their molecular weight and are compared to Pullulan standards with known molecular weight.

[0163] Pullulan standards (Grade P-5, P-10, P-20, P-50, P-100, P-200, P-400 and P-800) are purchased from Showa Denko K.K. (Japan); glycerin, glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose are purchased from Sigma Aldrich. 0.5 % standard solutions are prepared by dissolving 50 mg of each standard and 50 mg of glycerin (output marker) in 10.0 mL of eluent solvent and then filtered through a 0.45 micron filter.

[0164] Samples of the carbohydrate compositions are first de-ashed with 1 .0 g LEWATIT S4228 and PUROLITE C 150 MBH mixed resins and then diluted with eluent solvent to about 2.5 %, 0.25 % of glycerin (output marker) is added, followed by filtration through a 0.45 micron filter.

[0165] 100 pL of either the standard solutions or sample solutions are injected into a HPLC system equipped with a refractive index detector and 3 SHODEX SEC columns (OHpak SB-805 HQ, SB- 803 HQ, SB-802 HQ connected in series). The chromatography is performed with following parameters:

[0166] - Injecting volume: 100 pL

[0167] - Flowrate: 0.5 mL / min

[0168] - Column temperature: 35°C

[0169] - Eluent: sodium nitrate 0.1 M + sodium azide 0.02 % filtered on a 0.02 micron filter

[0170] - Eluting time: 80 min

[0171] The resulting chromatograms are analyzed offline using Empower software. The liberated mono-, di-, oligo- and polysaccharides size is determined according to the eluting time comparing to glycerin, glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and Pullulans standard eluting time.

[0172] From the GPC distribution table, it is determined the cumulative percentage of saccharides having these following range of molecular weights:

[0173] - Fraction lower than 1500 Da

[0174] - Fraction from 1500 Da to 100 kDa

[0175] - Fraction higher than 100 kDa.

[0176] Total neutral sugars composition:

[0177] The sugars are determined by gas chromatography in the form of silylated methoxime derivatives and then quantified using the internal calibration method. Galactitol is used as the internal standard.

[0178] The analysis is carried out using the following equipment:

[0179] - A gas chromatograph (type VARIAN 3400) equipped with: o A split-splitless injector fitted with a split liner, o A flame ionisation detector, o An integrator-recorder or any computer system for processing the detector signal.

[0180] - A DB-1 GC capillary column o Length: 40 metres o Internal diameter: 0.18 millimetres o Film thickness: 0.40 microns

[0181] Chromatographic conditions:

[0182] - Column temperature: 100°C to 250°C at 3°C / min, then 250 to 325°C at 15°C / min. Hold for 16 minutes.

[0183] - Helium pressure: 40 psi (in constant flow mode)

[0184] - Injection mode: split

[0185] - Split flow rate: 100 mL / min

[0186] - Injector temperature (split / splitless): 325°C

[0187] - FID detector temperature: 330°C

[0188] - Injected volume: 1 pL According to the following protocol:

[0189] 1) Internal standard solution

[0190] Prepare a solution of galactitol at approximately precisely 5 mg / mL in osmosis water.

[0191] 2) Hydrochloric hydrolysis

[0192] In duplicate: in a 15 ml hydrolysis tube, weigh approximately 50 to 500 mg of sample precisely, add 2 mL of the internal standard solution, add 3 mL of water and 5 mL of the 4N HCI solution. Stir for 1 min on a vortex mixer. Place the tube in a thermostated dry bath regulated at 100°C for 1 hour, vortexing from time to time.

[0193] 3) Demineralisation and concentration

[0194] After cooling, transfer the entire hydrolysis to a 50 ml beaker. Add 6 to 8 g of a 50 / 50 mixture of AG 4-X4 anion resin and AG 50W-X8 cation resin. Leave under magnetic stirring for 5 minutes. Filter the contents of the beaker and recover the juice in the rinsed hydrolysis tube. Repeat the demineralization step until a neutral pH is obtained.

[0195] 4) Preparing the derivative

[0196] Place approximately 1 mL of the demineralised hydrolysis in a 2 mL well, add approximately 1 mL of pyridine and evaporate to dryness under a stream of nitrogen. Take up with 20 mg methoxylamine hydrochloride and 1 mL pyridine. Leave for 40 min at 70°C. Add 0.5 mL BSTFA and leave for 30 min at 70°C. Cool to room temperature before injecting.

[0197] 5) Calculation

[0198] Read the area of the peaks corresponding to each sugar and the internal standard.

[0199] The total sugar content expressed in g per 100 g of raw product is given by the equation:

[0200] Sj P 100

[0201] With:

[0202] Si = area of the peak(s) of sugar i

[0203] Se= Area of the internal standard peak

[0204] Pe= weight of internal standard introduced into the beaker (in mg) (corrected weight - Purity / Dry matter).

[0205] P = weight of sample introduced into the beaker (in mg)

[0206] Kt= response coefficient of sugar i

[0207] 6) System calibration: Standard solution:

[0208] Use the classic ‘methoximated sugars’ solution containing each of the sugars at approximately precisely 100mg of each sugar in 200ml of water.

[0209] - Determination of response coefficients:

[0210] In a 15 ml hydrolysis tube, add 5 mL of the ‘methoximated sugars’ solution, 2 mL of the internal standard solution, add 3 mL of water and 5 mL of the 4N HCI solution. Stir for 1 min with a vortex mixer. Place the tube in a thermostated dry bath regulated at 100°C for 1 hour, vortexing from time to time. Then, as for the samples, proceed with demineralization and derivatization as in 3 and 4.

[0211] Record the area of the peak(s) corresponding to each sugar and the internal standard. The response coefficient KLfor each species i is given by the following equation:

[0212] With :

[0213] Si = area of the sugar i peak(s)

[0214] Se= Surface area of the internal standard peak

[0215] Pe= weight of internal standard introduced into the hydrolysis tube (in mg) (corrected weight - Purity / Dry matter)

[0216] P = weight of sugar i introduced into the hydrolysis tube (in mg)

[0217] Free neutral sugars composition:

[0218] The sugars are determined by gas chromatography in the form of silylated methoxime derivatives and then quantified using the internal calibration method. Methylalpha-D-glucopyranoside is used as the internal standard.

[0219] The analysis is carried out using the following equipment:

[0220] - A gas chromatograph (type VARIAN 3400) equipped with: o A split-splitless injector fitted with a split liner, o A flame ionisation detector, o An integrator-recorder or any computer system for processing the detector signal.

[0221] A DB-1 GC capillary column o Length: 40 metres o Internal diameter: 0.18 millimetres o Film thickness: 0.40 microns

[0222] Chromatographic conditions:

[0223] - Column temperature: 100°C - 3°C / min 300°C - Hold 12.66 min

[0224] - Injector temperature (split / splitless): 300°C

[0225] - FID detector temperature: 300°C

[0226] - Carrier gas: Helium - Injection mode: split

[0227] - Split ratio: 40

[0228] - Pressure mode: constant flow - flow = 1 .00 mL / min (44 psi initial)

[0229] - Make up (nitrogen): 25 mL / min

[0230] - Air: 300ml / min - H2: 30 mL / min

[0231] - Injected volume: 1 pL

[0232] According to the following protocol:

[0233] 1) Internal standard solution

[0234] Prepare a solution of methyl alpha-D-glucopyranoside at approximately precisely 0.3 mg / mL in osmosis water.

[0235] 2) Preparing the derivative

[0236] In a tare box, weigh approximately, precisely 100 to 200mg of sample, add 10 mL of the internal standard solution. Leave under magnetic stirring until completely dissolved. In a 2 mL tube, place 1 mL of the solution, evaporate to dryness under a stream of nitrogen. Add approximately 20mg of methoxylamine hydrochloride. Add 1 mL of pyridine. Cap and hold for 40 minutes at 70°C in Reacti-Therm. Add 0.5 mL of BSTFA and maintain 30 min at 70°C in Reacti-Therm.

[0237] 3) Calculation and system calibration are carried out in the same way as for total sugars.

[0238] Total and free acid sugars composition:

[0239] Throughout the present application, the contents of free uronic acids or total uronic acids are determined using HPAEC-PAD.

[0240] Free and total uronic acids methods are that described in the publication of 2004, Journal of Agricultural and Food Chemistry 4652 “New Method for a Two-Step hydrolysis and chromatographic analysis of pectin neutral sugar chains" with some adaptations. The chemical hydrolysis with 5 mL of 0.2 M TFA is carried out at 80°C for 6 hours instead of 72 hours. For the analysis of free uronic acids, the sample is diluted in water. After homogenization, the sample is filtered through a ww PTFE 0.45 micron filter and injected. Here, the free and total uronic acids are assimilated to free and total galacturonic acid.

[0241] Bound neutral and acidic sugars composition:

[0242] For the following structural characterization, it must be considered only the pectic saccharides bounded in the pectic structure, without monosaccharides that can be present in the product composition.

[0243] For each (neutral or acid) sugar, the bound sugar content is determined by subtracting the free sugar content from the total sugar content. For e.g., the mass content of bound galactose is expressed as the subtraction of the mass content of free galactose from the mass content of total galactose. This method of calculation is similar for galacturonic acid, rhamnose, arabinose, mannose, xylose, fucose and ribose. Due to the eventual presence of starch in product composition, the calculation must be adapted for the glucose. The mass content of bound glucose is calculated by subtracting the mass content of glucose from starch and the mass content of free glucose from the mass content of total glucose.

[0244] The mass glucose content from starch is expressed as percentage the mass content of starch divided by the factor 0.9.

[0245] Homogalacturonan (HG) and rhamnogalacturonan-l (RG-I) composition:

[0246] The composition in chains HG and chains RG-I, expressed as molar, is determined according to methods described by (M'sakni, N., & al, European Polymer Journal, 2006, 42) and by (Yang, J.- S., & et al., Food Chemistry, 2018, 244) with some calculations adaptation.

[0247] For characterization of structural pectic polysaccharides, the molar content of bound sugars must be expressed by considering only the pectic saccharides, without other compounds (like proteins, starch, ashes...).

[0248] In this restricted matrix, the molar content of each bound (neutral and acid) sugar is expressed as percentage as the molar content of corresponding bound sugar divided by the sum of molar contents of all bound sugars (i.e., galacturonic acid, galactose, glucose, rhamnose, arabinose, mannose, xylose, fucose, and ribose).

[0249] The molar amount of each bound sugar is determined from the corresponding mass amount and the molar weight. For e.g., the molar content of bound galactose is expressed as the mass content of bound galactose divided by the molar weight of galactose (i.e., 180.16 g / mol).

[0250] According to these molar contents, the homogalacturonan (HG) and rhamnogalacturonan-l (RG- I) composition is determined by these formulas:

[0251] • The molar content in chains HG is: HG (mol%) = GalA (mol%) - Rha (mol%)

[0252] • The molar content is chains RG-I is: RG-I (mol%) = [GalA (mol%) - HG (mol%)] + Rha (mol%) + Gal (mol%) + Ara (mol%)

[0253] • The molar GalA to Rha ratio is: GalA (mol%) I Rha (mol%)

[0254] • The molar (Gal plus Ara) to GalA ratio is: [Gal (mol%) + Ara (mol%)] I GalA (mol%)

[0255] • The molar Ara to Rha ratio is: Ara (mol%) / Rha (mol%)

[0256] • The molar GalA to Gal ratio is: GalA (mol%) I Gal (mol%)

[0257] Quantification of total and free methanol and total and free acetate:

[0258] Quantification of total and free methanol and total and free acetate was carried out by proton NMR at 60°C in D2O, using an internal standard (Na benzoate = 20 mg on dry basis / g).

[0259] The samples were run twice, the first time in solution in D2O and the second time with NaOD added. The purpose of the sodium hydroxide is to saponify the sample and release the methyl and acetyl compounds bound to it. The difference between the total acetate level in the saponified sample and the free acetate level in the crude sample gives us the previously fixed acetate level. The reasoning is the same for the methyl function. The procedure is as follows:

[0260] - Weigh approximately 10 mg of sample into the NMR tube and add an ampoule of deuterium oxide (0.75 mL), then add approximately 30 mg of internal standard solution into the tube. Stir until dissolved by placing it in an ultrasonic bath.

[0261] - Fit the spinner onto the tube and place it in the magnet.

[0262] - Perform the acquisition, without solvent suppression, with a relaxation time of at least 10 s, without rotation, after the appropriate instrument settings (field, lock phase and shims), at a temperature of 60°C. The spectral window must be at least between 0 and 9 ppm, with the water peak calibrated at 4.40 ppm.

[0263] - The spectrum is processed after Fourier transformation, phase correction and baseline subtraction in manual mode (without exponential multiplication, LB=GB=0).

[0264] - For the second analysis, in the same tube, add 100 pL of 2N NaOD solution. Stir, then place the tube in a water bath for a few minutes (to colour the solution). Repeat the acquisition under the same conditions as before.

[0265] Quantification of free species or species released after saponification (in g / 100g on wet basis):

[0266] The free species found or released after saponification (methanol and sodium acetate) in pectic saccharides are expressed in g per 100g on wet basis. To obtain these results, integration must be carried out on the spectrum analyzed in water and on the spectrum analyzed with the addition of NaOD. MeOHXPEx WEx CEx MMe0Hx 100

[0267] Methanol = EXPMCOHXMEX

[0268] PE. number of protons from the internal standard (PE= 5)

[0269] PMeoH- number of protons from methanol (PMeoH=3)

[0270] WEmass, in grams, of weighed internal standard solution

[0271] CEconcentration, in milligrams on dry basis / gram of Na benzoate solution MEmolar mass, in grams per mole, of Na benzoate (ME=144 g / mol) MMe0H:molar mass, in grams per mole, of methanol (MMeOH=32g / mol) Ws: mass, in milligrams, of the sample to be analysed.

[0272] SE: Surface area of Na benzoate=100

[0273] SMe0H:Surface area of methanol x 100 _

[0274] PEnumber of protons from the internal standard (PE= 5)

[0275] PAcnumber of protons from acetate (PAc= 3)

[0276] WE: mass, in grams, of weighed internal standard solution.

[0277] CE: concentration, in milligrams on dry basis / gram of Na benzoate solution MEmolar mass, in grams per mole, of Na benzoate (ME=144 g / mol) MAC: molar mass, in grams per mole, of acetate (Myk.=82g / mol)

[0278] Ws: mass, in milligrams, of the sample to be analysed.

[0279] SE: Surface area of Na benzoate=100

[0280] SMe0H: Surface area of acetate

[0281] Quantification of fixed species (in q / 100q on wet basis):

[0282] The content of bound methanol is determined by subtracting the free methanol content from the total methanol content.

[0283] Bound methanol (in % by wet weight) = Total methanol after saponification (in % by wet weight) - Free methanol (in % by wet weight)

[0284] The content of bound sodium acetate is calculated in the same way.

[0285] Bound acetate (in % by wet weight) = Total acetate after saponification (in % by wet weight) - Free acetate (in % by wet weight).

[0286] Degrees of esterification and acetylation:

[0287] The degree of esterification is expressed as percentage as the molar amount of bound methanol divided by the molar amount of bound galacturonic acid.

[0288] In the same way, the degree of acetylation is expressed as percentage as the molar amount of bound sodium acetate divided by the molar amount of bound galacturonic acid.

[0289] Starch content:

[0290] Throughout the present application, the content of starch was determined according to the following method:

[0291] - Add test sample to the glass jars with metal lids, containing around 750 mg of starch.

[0292] - Add 60 to 100 mL distilled water and homogenize.

[0293] - Adjust the pH of the aqueous suspension to be between 6-7.

[0294] - Keep the jar for 3 minutes in a boiling water bath while shaking the jar. Then transfer to the oven at 130°C for an hour leaving the jar closed.

[0295] - At the exit of the oven leave them on the bench for 10 min before cooling them in a cold- water bath and depressurize the jar.

[0296] - Add 5 mL of 1 ,2 M Sodium Acetate solution pH 4,6 and adjust the pH a 4,6 if necessary.

[0297] - Add 500 pL of amyloglucosidase (E-AMGDF from Megazyme).

[0298] - Place the jar in a 60°C water bath for 2 hours.

[0299] - After cooling, transfer the test portion to a 500 mL volumetric flask and make up to volume with distilled water. Homogenize and filter.

[0300] - Carry out the enzymatic determination of glucose by the hexokinase method to obtain total released glucose.

[0301] - Analyze the free glucose in the sample and deduct it from the total glucose result obtained after hydrolysis. Calculate the amount of starch (expressed in %): glucose concentration in g / L of the sample (total released glucose - free glucose) x 0,9 (conversion factor from glucose to starch) x 0,5 (volume of volumetric flask in ml / 1000) x (100 / weight of wet product in g).

[0302] Measurement of pH in solution at 5% DS:

[0303] Throughout the present application, the measurement of pH in solution at 5% DS is determined according to the following method.

[0304] - On a precision scale (0.1g), tare a glass beaker with a capacity of 150ml.

[0305] - Precisely introduce 5g of product into the glass beaker.

[0306] - Add 95g of distilled water whose resistivity is higher than 500 000 ohms and whose pH is between 5-7.

[0307] - Stir with a magnetic bar at minimum 1500rpm for 30minutes to homogenize the preparation.

[0308] - Perform the measurement at between 20°C to 25°C with a pH meter equipped with a pH electrode.

[0309] Glycoalkaloids content (alpha-chaconine and alpha-solanine):

[0310] Throughout the present application, the content in glycoalkaloids is determined according to the following method, on a powder sample (i.e. after any drying step, such as atomization, freeze- drying, etc.).

[0311] Equipment:

[0312] A high-performance liquid chromatography such as Waters Alliance 2695 (or equivalent) equipped with an autosampler, coupled with a UV detector such as the Waters 2487 (or equivalent) and a recorder-integrator or any computer system allowing to process the UV signal.

[0313] Chromatographic conditions:

[0314] Column: C18 CDS Spherisorb 5pm x 4,6mm x 250mm or equivalent

[0315] Wavelength: 202nm (resolution 1 ,2nm)

[0316] Mobile phase: 55% solution A + 45% solution B (v / v) and adjustment to pH 7.6 with concentrated phosphoric acid

[0317] Solution A: weight 3.8g of K2HPO4 in a 1000ml flash and complete to the gauge line with ultrapure water (a resistivity of 18.2 MQ.cm)

[0318] Solution B: Acetonitrile (>99.9%, gradient grade, suitable for HPLC)

[0319] Pump flow: 0.7 ml / min

[0320] Column temperature: 30°C

[0321] Injected volume: 20 pl

[0322] Sample temperature: 15 °C

[0323] Acquisition time: 35 min Preparation of the extraction solution:

[0324] In a glass beaker, introduce 0.5g of sodium bisulphite (99>% purity) with a precision scale 0,01 mg. Add precisely 5mL of glacial acetic acid (purity at 100%).

[0325] Then add 95mL of ultrapure water (resistivity of 18.2 Mfl.cm) and homogenize.

[0326] Preparation of the calibration range:

[0327] Prepare a calibration range of alpha-chaconine and alpha-solanine from 1.5 mg / kg to 34 mg / kg for each compound into the mobile phase (take into account the purity and dry matter of the standards).

[0328] This solution is stable for a maximum of 6 months.

[0329] Sample preparation:

[0330] In a glass beaker, weigh precisely 1g of product then add 100mL the extraction solution

[0331] Stir magnetically for 1 hour.

[0332] Then filter on Filtres Acrodisc GF 25mm with GF 0.45|jm GHP.

[0333] SPE cartridge packaging (model SPE OASIS HLB Vac 60mg or eguivalent):

[0334] Put the cartridges on the Manifolds system (or eguivalent).

[0335] Pass 5 ml of acetonitrile through the cartridge by gravimetry until the cartridge is dry.

[0336] Then pass 5ml of the extraction solution into the cartridge by gravimetry. Do not leave the cartridge dry.

[0337] Pass 30ml of filtrate (in several times and continuously without ever letting the cartridge dry out) by gravity.

[0338] Once the 30mL has been eluted, the filtration is completed when the cartridge is dry.

[0339] Rinse with 10 ml of the Acetonitrile / Water mixture (15% / 85% v / v) by gravimetry until the cartridge is dry.

[0340] Put the SPE cartridges on a support to collect the filtrate (beaker or test tube) Extract glycoalkaloids with 4 mL of mobile phase in a tube by gravity.

[0341] Dry the column and agitate the Ultra using a vortex

[0342] Then inject into the HPLC

[0343] Expression of results:

[0344] The concentration of alpha-solanine or alpha-chaconine in mg / kg is given by the formula:

[0345] Q x 100 x 4

[0346] P x X Where:

[0347] Q = amount of alpha-chaconine or alpha-solanine read on the curve (mg / kg)

[0348] P = sample weight in g

[0349] X = filtrate volume (ml)

[0350] Heavy metals content (cadmium, copper, lead, nickel, arsenic, mercury):

[0351] Throughout the present application, the content of heavy metals is determined by ICP-MS on a powder sample (i.e. after any drying step, such as atomization, freeze-drying, etc.).

[0352] The method is that described in the publication of 2011 , Journal of Food Composition and Analysis 24 “Simultaneous analysis of 21 elements in foodstuffs by ICP-MS after closed-vessel microwave digestion: Method validation” without any adaptations.

[0353] Example 1. Preparation of the hydrolyzed pectic polysaccharides according to the invention

[0354] Four samples (A1 , B1 , C1 and D1) of hydrolyzed pectic polysaccharides were produced according to the method in accordance with the present invention.

[0355] Potato pulps from the potato-processing industry present the following composition.

[0356] Note: nd for undetectable and / or unquantifiable

[0357] To produce the Sample A1 , 115 kg of potato pulp is loaded in the “ploughshare mixer type DVT- 300 / 1 MZ” from the supplier Lodige and under stirring, demineralized water is added in the mixer to get a dry substance of 10 %.

[0358] Still under stirring, the pectin-rich substrate is pre-heated until 50°C. When the temperature is reached, the enzymatic treatment is started by adding the pectinase (ISOZYM PG200) in the mixer at a dose of 30 uPG by grams of pulp on dry matter.

[0359] The pH of pectin-rich substrate is controlled in beginning of the enzymatic hydrolysis, a pH value of 5.2 is obtained.

[0360] Then the reaction is carried out for 2h still under stirring and maintaining the temperature at 50°C. When the treatment time is up, the hydrolyzed pulp feeds, in batch mode, a “hydraulic filter press type HPX207” from the supplier Bucher Unipektin.

[0361] The machine parameters are adjusted so that:

[0362] • the insoluble residue pulp has a dry substance of 26 % and,

[0363] • the filtrate has an insoluble content below 2 % v / v (lab centrifuge spin test performed at 5000 g for 5 min), and,

[0364] • the dry substance yield for the filtrate is 27 % in weight.

[0365] The total filtration time gets 150 min.

[0366] As soon as the filtration is finished, the filtrate is loaded in a double jacket tank, and heats at 80°C for 30 min in order to inactivate the pectinase.

[0367] Then the hydrolyzed pectic polysaccharides are recovered, freeze-dried, and labelled Sample A1 .

[0368] To produce the Sample B1 , 350 kg of potato pulp is loaded in a double jacket tank equipped with a turbine agitator, and under stirring, decarbonated water is added in the tank to get a dry substance of 6.5 %.

[0369] Still under stirring, the pectin-rich substrate is pre-heated until 50°C. When the temperature is reached, the enzymatic treatment is started by adding the pectinase (ISOZYM PG200) in the tank at a dose of 25 uPG by grams of pulp on dry matter.

[0370] The pH of pectin-rich substrate is controlled in beginning of the enzymatic hydrolysis, a pH value of 5.3 is obtained.

[0371] Then the reaction is carried out for 2 h still under stirring and maintaining the temperature at 50°C. When the treatment time is up, the hydrolyzed pulp feeds in continuous a “rotary vacuum filter type precoat” from the supplier NIVOBA with 2 m2filtration surface.

[0372] Beforehand, the precoat layer is formed on filter with 100 kg of potato starch.

[0373] The machine parameters are adjusted so that:

[0374] • the insoluble residue pulp has a dry substance of 17 % and,

[0375] • the filtrate has an insoluble content below 2 % v / v (lab centrifuge spin test performed at 5000 g for 5 min), and,

[0376] • the dry substance yield for the filtrate is 22 % in weight.

[0377] The total filtration time gets 120 min.

[0378] As soon as the filtration is finished, the filtrate feds a UHT skid at 130°C in continuous for contact time of 5min in order to inactivate the pectinase.

[0379] Then the hydrolyzed pectic polysaccharides are recovered, freeze-dried, and labelled Sample B1 . To produce the Sample C1 , 300 kg of potato pulp is loaded in a double jacket tank equipped with a turbine agitator, and under stirring, decarbonated water is added in the tank to get a dry substance of 5.5 %.

[0380] Still under stirring, the pectin-rich substrate is pre-heated until 50°C.

[0381] The pH of pectin-rich substrate is controlled and is adjusted at 3.5 by adding hydrochloric acid solution at concentration of 5 % in weight.

[0382] When the temperature is reached, the enzymatic treatment is started by adding the pectinase (ISOZYM PG200) in the tank at a dose of 50 uPG by grams of pulp on dry matter.

[0383] Then the reaction is carried out for 3 h still under stirring and maintaining the temperature at 50°C. When the treatment time is up, the hydrolyzed pulp feeds in continuous a “centrifuge decanter type Z2” from the supplier Flottweg.

[0384] The machine parameters are adjusted so that:

[0385] • the insoluble residue pulp has a dry substance of 18 % and,

[0386] • the filtrate has an insoluble content below 2 % v / v (lab centrifuge spin test performed at 5000 g for 5 min), and,

[0387] • the dry substance yield for the overflow is 25 % in weight.

[0388] The total filtration time gets 50 min.

[0389] As soon as the filtration is finished, the overflow is loaded in a double jacket tank, and heats at 80°C for 30 min in order to inactivate the pectinase.

[0390] Then the hydrolyzed pectic polysaccharides are recovered, freeze-dried, and labelled Sample C1 .

[0391] To produce the Sample D1 , 2700 kg of potato pulp is loaded in a double jacket tank equipped with a two-stage pitched blade turbine agitator, and under stirring, decarbonated water is added in the tank to get a dry substance of 4.5 %.

[0392] Still under stirring, the pectin-rich substrate is pre-heated until 50°C.

[0393] The pH of pectin-rich substrate is controlled and is adjusted at 3.5 by adding hydrochloric acid solution at concentration of 5 % in weight.

[0394] When the temperature is reached, the enzymatic treatment is started by adding the pectinase (ISOZYM PG200) in the tank at a dose of 50 uPG by grams of pulp on dry matter.

[0395] Then the reaction is carried out for 3 h still under stirring and maintaining the temperature at 50°C. When the treatment time is up, the hydrolyzed pulp feeds in continuous a “centrifuge decanter type Z3” from the supplier Flottweg.

[0396] The machine parameters are adjusted so that:

[0397] • the insoluble residue pulp has a dry substance of 21 % and,

[0398] • the filtrate has an insoluble content below 1 % v / v (lab centrifuge spin test performed at 5000 g for 5 min), and,

[0399] • the dry substance yield for the overflow is 37 % in weight.

[0400] The total filtration time gets 105 min.

[0401] As the same time of decantation, the overflow feeds in a UHT system (model 6600) from the supplier GEA, and heats at 110°C for 15 s in order to inactivate the pectinase. Then the product is concentrated at dry substances of 9 % and is heat-treated at 130 °C for 3.5 min.

[0402] Then the hydrolyzed pectic polysaccharides are recovered, dried at dry substances of 96.0% using a multi-stage spray dryer equipped with a spray nozzle, and labelled Sample D1 .

[0403] Example 2. Preparation of the high molecular weight pectic saccharides according to the invention

[0404] Four samples (A2, B2, C2 and D2) of high molecular weight saccharides were produced according to the method in accordance with the present invention.

[0405] To produce the Sample A2, the same experimental procedure is followed than the production of Sample A1 , until the pectinase inactivation step included.

[0406] From this point, the heat-treated filtrate which contains the hydrolyzed pectic polysaccharides is cooled until 45°C in double jacket tank and then feeds a “rotary dynamic cross-flow filter type DYNAMOS 1 ” from the supplier TMCI PADOVAN.

[0407] The machine is equipped with 8 ceramic discs of molecular cut-off of 0.5 pm from the supplier TMCI PADOVAN.

[0408] The microfiltration is operated in batch mode, by recycling the retentate in the feed tank and by going out the permeate.

[0409] The machine parameters are adjusted so that:

[0410] • the filtration temperature is controlled at 45°C and,

[0411] • the transmembrane pressure is regulated at 1.1 bar and,

[0412] • the average dry substance of permeate is 0.8 % and,

[0413] • the yield for the retentate is 34 % in weight.

[0414] Then the retentate which contains the high molecular weight pectic saccharides is recovered, dried at dry substances of 94.0% using a single-stage spray dryer equipped with atomizer wheel, and labelled Sample A2.

[0415] To produce the Sample B2, the same experimental procedure is followed than the production of Sample B1 , until the pectinase inactivation step included.

[0416] From this point, the heat-treated filtrate is cooled until 65°C in double jacket tank and then feeds a “tangential microfiltration type SIVALAB” from the supplier SIVA.

[0417] The machine is equipped with two carters of a tubular ceramic membrane from the supplier TAMI Industries.

[0418] The membrane characteristics are: INSIDE CeRAM series, molecular cut-off of 0.45 pm, 23 channels, membrane diameter of 25mm, membrane length of 1178 mm.

[0419] The microfiltration is operated in batch mode, by recycling the retentate in the feed tank and by going out the permeate.

[0420] The machine parameters are adjusted so that: • the filtration temperature is controlled at 65°C and,

[0421] • the transmembrane pressure is regulated at 1 .6 bar and,

[0422] • the average dry substance of permeate is 2.3 % and,

[0423] • the yield for the retentate is 14 % in weight.

[0424] Then the retentate which contains the high molecular weight pectic saccharides is recovered, dried at dry substances of 93.0% using a single-stage spray dryer equipped with atomizer wheel, and labelled Sample B2.

[0425] To produce the Sample C2, the same experimental procedure is followed than the production of Sample C1 , until the pectinase inactivation step included.

[0426] From this point, the heat-treated overflow is cooled until 60°C in double jacket tank and then feeds a “tangential microfiltration type 12-E-042” from the supplier TIA (“Techniques Industrielles Appliquees”, France).

[0427] The machine is equipped with one carter of 7 tubular ceramic membranes from supplier TAMI Industries.

[0428] The membrane characteristics are: INSIDE CeRAM series, molecular cut-off of 0.8 pm, 8 channels, membrane diameter of 25 mm, membrane length of 1020 mm.

[0429] The microfiltration is operated in batch mode, by recycling the retentate in the feed tank and by going out the permeate.

[0430] The permeate is loaded in a double jacket tank and maintained at a temperature of 60°C.

[0431] The machine parameters are adjusted so that:

[0432] • the filtration temperature is controlled at 60°C and,

[0433] • the transmembrane pressure is regulated at 1 .0 bar and,

[0434] • the average dry substance of permeate is 1 .3 % and,

[0435] • the yield for the retentate is 20 % in weight.

[0436] Then the retentate from microfiltration of 0.8 pm is recovered.

[0437] The permeate from microfiltration of 0.8 pm feeds a “tangential microfiltration type SIVALAB” from the supplier SIVA.

[0438] The machine is equipped with two carters of a tubular ceramic membrane from the supplier TAMI Industries.

[0439] The membrane characteristics are: INSIDE CeRAM series, molecular cut-off of 0.45 pm, 23 channels, membrane diameter of 25 mm, membrane length of 1178 mm.

[0440] The microfiltration is operated in batch mode, by recycling the retentate in the feed tank and by going out the permeate.

[0441] The machine parameters are adjusted so that:

[0442] • the filtration temperature is controlled at 60°C and,

[0443] • the transmembrane pressure is regulated at 2.0 bar and,

[0444] • the average dry substance of permeate is 1 .3 % and,

[0445] • the yield for the retentate is 5 % in weight. Then the retentate from microfiltration of 0.45 pm is recovered and mixed with the retentate from microfiltration of 0.8 pm. The retentate mix which contains the high molecular weight pectic saccharides is dried at dry substances of 94.0% using a single-stage spray dryer equipped with atomizer wheel, and labelled Sample C2.

[0446] To produce the Sample D2, the same experimental procedure is followed than the production of Sample D1 , until the pectinase inactivation step included.

[0447] From this point, the heat-treated overflow is cooled until 60°C and stored in double jacket tank and then feeds a “tangential microfiltration type PILOTE MF BK6553” from the supplier PIGNAT. The machine is equipped with two carters of 19 tubular ceramic membranes from supplier TAMI Industries.

[0448] The membrane characteristics are: INSIDE CeRAM series, molecular cut-off of 0.8 pm, 8 channels, membrane diameter of 25 mm, membrane length of 1178 mm.

[0449] The microfiltration is operated in batch mode, by recycling the retentate in the feed tank and by going out the permeate.

[0450] The permeate is loaded in a double jacket tank and maintained at a temperature of 60°C.

[0451] The machine parameters are adjusted so that:

[0452] • the filtration temperature is controlled at 60°C and,

[0453] • the transmembrane pressure is regulated at below 1 .0 bar and,

[0454] • the average dry substance of permeate is 1 .6 % and,

[0455] • the final volumetric concentration factor (VCF) is 12.

[0456] Then the retentate from microfiltration of 0.8 pm is recovered.

[0457] The permeate from microfiltration of 0.8 pm feeds a “tangential microfiltration type K07” from the supplier TIA (“Techniques Industrielles Appliquees”, France).

[0458] The machine is equipped with one carter of 19 tubular ceramic membranes from the supplier TAMI Industries.

[0459] The membrane characteristics are: INSIDE CeRAM series, molecular cut-off of 0.45 pm, 23 channels, membrane diameter of 25 mm, membrane length of 1020 mm.

[0460] The microfiltration is operated in batch mode, by recycling the retentate in the feed tank and by going out the permeate.

[0461] The machine parameters are adjusted so that:

[0462] • the filtration temperature is controlled at 60°C and,

[0463] • the transmembrane pressure is regulated at below 1 .0 bar and,

[0464] • the average dry substance of permeate is 1 .4 % and,

[0465] • the yield for the permeate is 98 % in weight.

[0466] Then the retentate from microfiltration of 0.45 pm is recovered and mixed with the retentate from microfiltration of 0.8 pm. The retentate mix which contains the high molecular weight pectic saccharides is concentrated at dry substances of 11 % and is heat-treated at 130 °C for 3.5 min. The high molecular weight pectic saccharides are dried at dry substances of 95.0% using a multistage spray dryer equipped with a spray nozzle, and labelled Sample D2. Example 3. Preparation of the low molecular weight pectic saccharides according to the invention

[0467] Four samples (A3, B3, C3 and D3) of low molecular weight saccharides were produced according to the method in accordance with the present invention.

[0468] To produce the Sample A3, the same experimental procedure is followed than the production of Sample A2, until the microfiltration step included with membrane cut-off of 0.5 pm.

[0469] From this point, the permeate is recovered, loaded in a double jacket tank and cooled at a temperature of 40°C.

[0470] Then, the permeate feeds a “tangential nanofiltration type 23-E-028” from the supplier TIA (“Techniques Industrielles Appliquees”, France). The machine is equipped with one carter of one organic spiral wounded membrane from supplier SUEZ.

[0471] The membrane characteristics are: DL series, molecular cut-off of 150-300 Da, membrane diameter of 40.0 inches, spacer mil of 50mm, membrane reference “DL2540C50”.

[0472] The nanofiltration is operated in batch mode, by recycling the retentate in the feed tank and by going out the permeate.

[0473] The machine parameters are adjusted so that:

[0474] • the filtration temperature is controlled at 40°C and,

[0475] • the transmembrane pressure is regulated at 30 bar and,

[0476] • the final average dry substance of retentate is 10.8 % and,

[0477] • the yield for the retentate is 13 % in weight.

[0478] Then the retentate from nanofiltration which contains the low molecular weight pectic saccharides is recovered, dried at dry substances of 94.0% using a single-stage spray dryer equipped with atomizer wheel, and labelled Sample A3.

[0479] To produce the Sample B3, the same experimental procedure is followed than the production of Sample B2, until the microfiltration step included with membrane cut-off of 0.45pm.

[0480] From this point, the permeate is recovered, loaded in a double jacket tank and cooled at a temperature of 35°C.

[0481] Then, the permeate feeds a “tangential nanofiltration type 23-E-028” from the supplier TIA (“Techniques Industrielles Appliquees”, France).

[0482] The machine is equipped with one carter of one organic spiral wounded membrane from supplier SUEZ.

[0483] The membrane characteristics are: DL series, molecular cut-off of 150-300 Da, membrane diameter of 40.0 inches, spacer mil of 50mm, membrane reference “DL2540C50”.

[0484] The nanofiltration is operated in batch mode, by recycling the retentate in the feed tank and by going out the permeate.

[0485] The machine parameters are adjusted so that:

[0486] • the filtration temperature is controlled at 35°C and, • the transmembrane pressure is regulated at 30 bar and,

[0487] • the final average dry substance of retentate is 8.9% and,

[0488] • the yield for the retentate is 15% in weight.

[0489] Then the retentate from nanofiltration which contains the low molecular weight pectic saccharides is recovered, dried at dry substances of 95.0% using a single-stage spray dryer equipped with atomizer wheel, and labelled Sample B3.

[0490] To produce the Sample C3, the same experimental procedure is followed than the production of Sample C2, until the microfiltration step included with membrane cut-off of 0.45pm.

[0491] From this point, the permeate is recovered, loaded in a double jacket tank and cooled at a temperature of 35°C.

[0492] Then, the permeate feeds a “tangential nanofiltration type 23-E-028” from the supplier TIA (“Techniques Industrielles Appliquees”, France).

[0493] The machine is equipped with one carter of one organic spiral wounded membrane from supplier SUEZ.

[0494] The membrane characteristics are: DL series, molecular cut-off of 150-300 Da, membrane diameter of 40.0 inches, spacer mil of 50mm, membrane reference “DL2540C50”.

[0495] The nanofiltration is operated in batch mode, by recycling the retentate in the feed tank and by going out the permeate.

[0496] The machine parameters are adjusted so that:

[0497] • the filtration temperature is controlled at 50°C and,

[0498] • the transmembrane pressure is regulated at 30 bar and,

[0499] • the final average dry substance of retentate is 24.0% and,

[0500] • the yield for the retentate is 6% in weight.

[0501] Then the retentate from nanofiltration which contains the low molecular weight pectic saccharides is recovered, dried at dry substances of 95.0% using a single-stage spray dryer equipped with atomizer wheel, and labelled Sample C3.

[0502] To produce the Sample D3, the same experimental procedure is followed than the production of Sample D2, until the microfiltration step included with membrane cut-off of 0.45pm.

[0503] From this point, the permeate is recovered, loaded in a double jacket tank and cooled at a temperature of 55°C.

[0504] Then, the permeate feeds a “tangential nanofiltration type unit SW” from the supplier TETRA PAK. The machine is equipped with one carter of four organic spiral wounded membranes from supplier SUEZ.

[0505] The membrane characteristics are: Duracon* NF2 series, molecular cut-off of 150-300 Da, membrane diameter of 80.0 inches, spacer mil of 50mm, membrane reference “NF2 8040C50”.

[0506] The nanofiltration is operated in batch mode, by recycling the retentate in the feed tank and by going out the permeate.

[0507] The machine parameters are adjusted so that: • the filtration temperature is controlled at 55°C and,

[0508] • the transmembrane pressure is regulated at 15 bar and,

[0509] • the final average dry substance of retentate is 8.0% and,

[0510] • the yield for the retentate is 12% in weight.

[0511] 5 Then the retentate from nanofiltration which contains the low molecular weight pectic saccharides is recovered and is heat-treated at 130 °C for 1.0 min. The low molecular weight pectic saccharides are dried at dry substances of 93.0% using a multi-stage spray dryer equipped with a spray nozzle, and labelled Sample D3. 0 Example 4. Characterization of the hydrolyzed, high and low molecular weight pectic saccharides according to the invention

[0512] The chemical structures of the hydrolyzed pectic polysaccharides, the low molecular weight and high molecular weight pectic saccharides following the invention are given in the following table. 5

[0513] For comparison purpose, are given the chemical structures of the following polysaccharides:

[0514] • SOYAFIBE-S-DN (two samples), SOYAFIBE-S-CA300 and SOYAFIBE-S-DA100, as water-soluble soybean hemicellulose, from the company Fuji Oil, and produced from okara (soybean pulp residue) 0 • GENU Pectin PAN as high ester pectin, from the company CP Kelco, and produced from sugar beet pulp residue.

[0515] • Rhamnogalacturonan I (Potato) of reference P-RHAM1 and Galactan (Potato) of reference P-GALPOT, analytical standards used in research, from the company MEGAZYME, and produced from potato fiber. 5 Note: nd for undetectable

[0516] The hydrolyzed pectic polysaccharides, the low molecular weight and high molecular weight pectic saccharides following the invention have a high content in rhamnogalacturonan-l chains and a low content in homogalacturonan chains.

[0517] Comparatively to comparative polysaccharides, the hydrolyzed pectic polysaccharides and the high molecular weight pectic saccharides have simultaneously a higher methylation degree and higher acetylation degree.

[0518] Both present simultaneously an intermediary range of ratios for GalA / Rha, (Gal+Ara) / GalA and Ara I Rha.

[0519] The low molecular weight pectic saccharides have a particular intermediary range of methylation degree and acetylation degree. In comparison to all (poly)-saccharides, the low molecular weight pectic saccharides present simultaneously very high ratios for GalA / Rha, (Gal+Ara) / Rha and Gal / Rha.

[0520] The composition and molecular weights of the hydrolyzed pectin rich substrate, the low molecular weight and high molecular weight pectic saccharides following the invention are given is the following tables.

[0521] Molecular weights (Da)

[0522] Concerning the low molecular weight pectic saccharides, the fraction of saccharides having a molecular weight higher than 100 kDa is lower in comparison to all (poly)-saccharides.

[0523] Comparatively to all (poly)-saccharides, the hydrolyzed pectin rich substrate presents simultaneously an intermediary range of values for the fraction lower than 1500 Da and the fraction higher than 100 kDa. The high molecular weight pectic saccharides are simultaneously poor in saccharides having a molecular lower than 1500 Da, and have an intermediary range of values for the fraction between 1500 Da and 100 kDa, and for the fraction higher than 100 kDa.

[0524] Total sugars composition (% by dry weight)

[0525] Note: nd for undetectable

[0526] The hydrolyzed pectic polysaccharides, the low molecular weight and high molecular weight pectic saccharides following the invention are simultaneously rich in galactose and in galacturonic 5 acid, and poor in fucose, in rhamnose, in xylose and in arabinose. Comparatively to hydrolyzed pectic polysaccharides and the high molecular weight pectic polysaccharides, the low molecular weight pectic saccharides are richer in galactose and in galacturonic acid, and poorer in arabinose and rhamnose.

[0527] Free sugars composition (% by dry weight) O Note: nd for undetectable

[0528] The hydrolyzed pectic polysaccharides and the high molecular weight pectic polysaccharides have very low content in monosaccharides.

[0529] Only, the low molecular weight pectic polysaccharides have quantifiable content in 5 monosaccharide of galacturonic acid. Example 5. Preparation of the pectic saccharides with low qlycoalkaloids and heavy metals content

[0530] Three samples (E1 , E2, E3) of pectic saccharides were produced according to the method in accordance with the present invention.

[0531] First of all, the strong cation resin (Lewatit® S 1668 from the supplier Lanxess) in Na+ionic form is conditioned in H+ ionic form. The required volume of resin is hydrated for 24h in demineralized water in an appropriate glass beaker.

[0532] Then the resin beads are poured into an appropriate jacketed glass column. A solution of hydrochloric acid at 5%(v / v) feeds the column at 25°C and at a flowrate of 2 BV / h until 3 BV of solution is through the column.

[0533] Then, the resin beads are rinsed with demineralized water at a flowrate of 2 BV / h until the pH is superior to 5. Finally, the resin beads are pulled back to the column.

[0534] To produce the Sample E1 containing the hydrolyzed pectic polysaccharides, 1 L of aqueous solution is prepared by dispersing 50g of Sample D1 powder into demineralized water to achieve a dry substance of 5%.

[0535] The solution is evenly spread into 4 glass beakers of 300mL. The beakers are positioned into a drybath (2mag Stirring Drybath 15-250 equipped with “Magnetic motion heat mix control”). Under stirring, the solution is heated at 70°C.

[0536] When the temperature is achieved and still under stirring, pour 50mL of strong cation resin (Lewatit® S 1668 from the supplier Lanxess, conditioning in H+ ionic form) in each beaker. Then the contact time is to 5 hours, keeping the temperature at 70°C, under stirring.

[0537] When the treatment time is up, the solution from each beaker is filtered through a folded paper filter (240mm, filtration threshold of 8pm, paper weight of 97g / m2), using a 100mL measuring cylinder and funnel.

[0538] The four supernatants which contain hydrolyzed pectic polysaccharides are recovered, mixed, freeze-dried, and labelled Sample E1.

[0539] To produce the sample E2 containing the high molecular weight pectic saccharides, the same experimental procedure is followed than the production of Sample E1 but using the powder Sample D2 instead of Sample D1.

[0540] To produce the sample E3 containing the low molecular weight pectic saccharides, the same experimental procedure is followed than the production of Sample E1 but using the powder Sample D3 instead of Sample D1. Example 6. Characterization of the hydrolyzed, high and low molecular weight pectic saccharides with low glycoalkaloids and heavy metals according to the invention

[0541] The characterization of the hydrolyzed, high and low molecular weight pectic saccharides following the invention are given in the following table.

[0542] After treatment with strong cation resin, the content in heavy metals and in glycoalkaloids is drastically decreased for all pectic saccharides. In the way, the pH of solution containing pectic saccharides is reduced.

[0543] Example 1. Determination of the bioactivity of the high or low molecular weight pectic saccharides according to the invention

[0544] Different in vitro models were chosen to investigate if the two pectic saccharides extracted from potato of the present invention (see the Table below) exhibit the same double bioactivity and how the structure of these molecules (i.e. Mw) could impact these effects.

[0545] 1 - Caco2 + PBMC model = immune response / inflammation model (Ambiotis).

[0546] Cell Isolation and culture: Frozen Caco2 cells were thawed, seeded in 96 well plates and grown for 18 days. Then, pectic compounds were added on the apical side. After a contact time of 24 hours, the basolateral supernatants were collected. Frozen PBMC were thawed, seeded in 96 well plates and the previously collected supernatant were added on the PBMC cultures so as negative (PBS blank) and positive (dexamethasone) controls. The impact of the pectic saccharides were tested in basal or challenged conditions.

[0547] The challenge was performed using a lipopolysaccharides (LPS) stimulation (1 pg / mL) during 18h. Then, the supernatants of the LPS-stimulated or not PBMC were collected and frozen until the analysis.

[0548] Cytokines quantification: Cytokines (IL1 b, IL1 RA, IL10 and TNFa) were analyzed with a milliplex assay kit and measured on the apparatus Luminex MAGPIX.

[0549] Gene expression analysis: mRNAs of cells were extracted by using specific extraction system, Turbocapture mRNA (QIAGEN). Then a reverse transcription was made to obtain cDNA (Fermentas) with the Maxima first strand synthesis kit. PCR were realized on StepOne apparatus (Applied Biosystems). For qPCR, primers has been designed by Ambiotis.

[0550] For calculating relative quantification of target gene mRNA expression, we will use the following procedure :

[0551] ACt gene of interest= CtSample- CtVehicle.

[0552] ACtp-house keeping gene= CtSample-CtVehicle.

[0553] Then, AACt represented the difference between ACt house keeping gene and ACt gene of interest calculated by the formula AACt=ACtphouse keeping gene -ACt gene of interest.

[0554] Finally, the N-fold differential expression of gene of interest mRNA samples compared to the vehicle will be express as 2AACt.

[0555] The results are expressed in fold induction compared to the control and are obtained by using the AACt method where the Ct correspond to the number of cycles necessary to generate a fluorescent signal above the predefined threshold (the more the targets, the less cycles required to reach this threshold).

[0556] Two cut-offs have been defined to allow interpretation:

[0557] - Ct>30: few targets (data can be interpreted but with caution)

[0558] - Ct>33: not enough targets (data should not be interpreted)

[0559] Also a result is considered to be significant when the fold-change is at least two, i.e. more than 2 or less than 0.5 compared to the control.

[0560] 2 - Caco2 + HT29 (Prodiqest): mucin production for immunity Caco-2 and HT29-MTX cells:

[0561] Caco-2 cells (HTB-37; American Type Culture Collection) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing glucose and glutamine and supplemented with HEPES and 20% (v / v) heat inactivated (HI) fetal bovine serum (FBS). HT29-MTX-E12 cells (Sigma-Aldrich) were maintained in McCoy’s 5A medium supplemented with HEPES, sodium pyruvate, L- Glutamine, 10% HI-FBS and antibiotics.

[0562] Cells were seeded at a ratio of 75% Caco-2 cells and 25% HT29-MTX cells in 0.1 % gelatine coated 24 well plates and cultured for 14 days in DMEM supplemented with HEPES, 20% HI-FBS and antibiotics, with three medium changes / week.

[0563] Cells were incubated at 37°C in a humidified atmosphere of air / CO2 (95:5, v / v). qPCR for detection of mucins:

[0564] After 14 days of culturing, Caco-2 / HT29-MTX co-cultures were treated for 48h with the sterile- filtered (0.22 pm) CoAP samples, pure products High Mw pectic saccharides and low Mw pectic saccharides.

[0565] After 24h incubation, cells were lysed for RNA isolation with lysis buffer containing TCEP (ISOLATE II RNA mini kit, GC Biotech), according to the manufacturer’s instructions and stored at -80°C.

[0566] RNA was further isolated and cDNA was prepared using the high-capacity cDNA reverse transcription kit (Applied Biosystems) in a Thermal cycler Biometra (Westburg). qPCRs were performed on a Quantstudio 5 (Applied Biosystems) with the Sensifast SYBR LO- ROX mix (Bioline). Primers were used for the detection of MUC3.

[0567] Statistics:

[0568] To assess differences between the complete medium (CM) control and the different concentrations of pectic saccharides products on gene expression, an ordinary one-way ANOVA with Dunnett’s multiple comparisons test was performed against the CM control.

[0569] Statistically significant differences are indicated on the graphs as: a = b = c means they are significantly different with p<0.05. All statistics were performed using GraphPad Prism version 10.1.1 for Windows (GraphPad Software, San Diego, CA, USA). 3 - Prebiotic activity (fermentation models: Crypto): bacteria modulation and SCFA production

[0570] Pectic saccharides colonic fermentation using SIFR® ex vivo model

[0571] The SIFR® technology was developed to study the human gut microbiota in a highly biorelevant manner across numerous parallel test conditions (both treatments and test subjects). See Van Den Abbeele, P.; Deyaert, S.; Thabuis, C.; Perreau, C.; Bajic, D.; Wintergerst, E.; Joossens, M.; Firrman, J.; Walsh, D.; Baudot, A. Bridging Preclinical and Clinical Gut Microbiota Research Using the Ex Vivo SIFR® Technology. Front. Microbiol. 2023, 14, 1 131662. https: / / doi.ora / 10.3389 / fmicb.2023.1 131662.

[0572] Briefly, individual bioreactors were processed in parallel in a bioreactor management device (Cryptobiotix, Ghent, Belgium).

[0573] Each bioreactor contained 5 mL of a nutritional medium-faecal inoculum blend supplemented with 5 g test compound / L (i.e low Mw pectic saccharides, high Mw pectic saccharides or appel pectin (Sigma)), then sealed individually, before being rendered anaerobic.

[0574] Blend M0003 was used for the preparation of the nutritional medium (Cryptobiotix, Ghent, Belgium).

[0575] After preparation, bioreactors were incubated under continuous agitation (140 rpm) at 37°C (MaxQ 6000, Thermo Scientific, Thermo Fisher Scientific, Merelbeke, Belgium).

[0576] Following a 48h incubation, the pressure was measured in the bioreactors’ headspace, and liquid samples were subsequently collected for the analysis of key fermentation parameters and microbial composition and metabolites.

[0577] Fresh faecal samples were collected according to a procedure approved by the Ethical Committee of the University Hospital Ghent (reference number BC-09977).

[0578] This procedure required participants to sign an informed consent in which they donated their faecal sample for the current study.

[0579] The selection criteria for the 6 donor samples used herein were as follows: 25-65 years of age, no antibiotic use in the past 3 months, no gastrointestinal disorders (cancer, ulcers, IBD), no use of probiotic, nonsmoking, alcohol consumption <3 units / d and BMI < 30.

[0580] Fundamental Fermentation Parameters SCFA (acetate, propionate and butyrate) were extracted from the samples with diethyl ether, after addition of 2-methyl hexanoic acid as an internal standard. Briefly, 0.5 mL samples were diluted in distilled water (1 :3), acidified with 0.5 mL of 48% sulfuric acid, after which an excess of sodium chloride was added along with 0.2 mL of internal standard (2-methylhexanoic acid) and 2 mL of diethyl ether.

[0581] Upon homogenization and subsequent separation of the water and diethyl ether layer, diethyl ether extracts were collected and analyzed using a Trace 1300 chromatograph (Thermo Fisher Scientific, MerelMerelbeke, Belgium) equipped with a tabilwax-DA capillary GC column, a flame ionization detector, and a split injector using nitrogen gas as the barrier and makeup gas.

[0582] The injection volume was 1 mL and the temperature profile was set from 110°C to 240°C. The carrier gas was nitrogen, and the temperatures of the injector and detector were 240 and 250°C, respectively.

[0583] The sample pH was measured using an electrode (Hannah Instruments Edge HI2002, Temse, Belgium).

[0584] Microbiota Analysis:

[0585] For the total cell count analysis, liquid samples were diluted in anaerobic phosphate-buffered saline (PBS), after which cells were stained with SYTO 16 at a final concentration of 1 pM and counted via a BD FACS Verse flow cytometer (BD, Erembodegem, Belgium).

[0586] Data were analyzed using FlowJo, version 10.8.1.

[0587] Statistical Analysis:

[0588] All univariate and multivariate analyses were performed using GraphPad Prism (v9.3.1 ; www.graphpad.comm, accessed on 23 November 2022). Treatment effects were compared with the NSC using a repeated measures ANOVA (based on paired testing) and p-values were corrected with Benjamini-Hochberg’s method (FDR = 0.05).

[0589] RESULTS

[0590] 1- Direct effects in the Caco2 / PBMC model

[0591] In basal conditions (Figure 1.A), mimicking individuals with healthy status, both high and low Mw pectic saccharides induced an increase in the secretion of the pro4nflammatory cytokines IL-1 p and Tnf-a showing a moderate stimulation of a resting immune system compared to medium control. In addition, a medium increase in IL-10 secretion was also induced by both pectic saccharides compared to control underlying a counterbalance to the induced inflammation.

[0592] In LPS-challenged conditions (Figure 1 .B), mimicking individuals facing bacterial or viral infection inducing an inflammatory status, both high and low Mw pectic saccharides induced a decrease in the secretion of the pro-inflammatory cytokines IL-1 p, Tnf-a and IL-10 compared to challenged LPS-induced control showing an anti-inflammatory effect in these challenged conditions.

[0593] 2- Direct effects in the Caco2 / HT29 MTX model

[0594] Using Caco2 / HT29MTX model to mimic colic epithelium, it was observed that when exposed to raw low Mw pectic saccharides, the expression of the gene coding for MUC3 protein was significantly increased compared to control without pectic saccharides (Figure 2).

[0595] This was not the case for the raw high Mw pectic saccharides compared to control without pectic saccharides. MUC3 is a protein involved in the mucus layer that protects and reinforces the intestinal epithelium.

[0596] 3- Indirect effect through the prebiotic activity: bacteria modulation and SCFA production

[0597] Using 6 donors with gut microbiota that were dominated by various bacteria (different enterotypes), both high and low Mw pectic saccharides were fermented using the SIFR® technology.

[0598] Both prototypes induced a decrease in the medium pH due to the production of fermentative metabolites such as Short Chain Fatty Acids (SCFAs) compared to our negative control without fiber after 48h of fermentation.

[0599] Concerning propionate and butyrate, the fermentative capacity of both prototypes was equivalent.

[0600] Nevertheless, for acetate and consequently for the total SCFA production, the high Mw prototypes demonstrated a significantly higher metabolite production (figure 3).

[0601] Compared to the apple pectin reference, both high and low Mw pectic saccharides demonstrated a slightly lower fermentation activity for acetate and total SCFA, but not for propionate and butyrate production.

[0602] The fermentation capacity of the different pectic samples can also be evaluated through the bacterial cell growth that was promoted in the fermenters inoculated with different gut microbiota. Compared to the control containing no fiber, both pectic saccharides samples induced a significant higher bacterial growth, but it is to be noted that high Mw demonstrated a higher impact on cell growth than the low Mw.

[0603] Interestingly, the supernatants resulting from the above ex vivo experiments were also tested in the cellular tests previously described and did not exert any significant effect neither on cytokines production / expression nor on mucin production (data not shown).

[0604] Conclusion:

[0605] As shown, the low Mw pectic saccharides was able to significantly induce a higher expression of MUC3 in Caco2 / HT29MTX co-culture, showing a specific ability of the unfermented low Mw pectic saccharides.

[0606] High Mw did not exert this activity, neither did the fermented pectic saccharides samples (data not shown).

[0607] So the immuno-modulation properties of the pectic saccharides of the invention are linked to their low methylation degree, explaining the positive results for both high and low Mw, when their impact on gut barrier integrity as unfermented sample was mainly linked to the low Mw pectic saccharides.

[0608] This could be linked to the fact that a low Mw could allow a better interaction with epithelial receptors involved in the regulation of mucin genes.

[0609] In addition to these direct effects linked to their specific structures, pectins and pectic saccharides also demonstrated prebiotic effects thanks to their solubility and their capacity to be selectively used by gut bacteria, thus conferring a health benefit to the host.

[0610] Considering these indirect effects linked to microbiota interaction, molecular weight seems to be the determinant parameter of this activity as we demonstrated in the ex vivo trial.

[0611] Ester residues were also strongly pointed as structurally determinant for the immuno-stimulant properties as their removal using saponification suppressed their efficiency towards cytokines secretion.

[0612] A putative explanation of the impact of the Mw could rely on the fact that the high Mw offer a long- lasting substrate for gut microbiota, allowing thus several cross-feeding interactions and a larger set of bioactive metabolites production. Using potato pectic saccharides, we showed here that 2 different structural parameters could be used to understand the structure / function relationship of pectins and pectic saccharides: methylation degree for direct immuno-modulation and Mw for some direct effects such as mucin production with low Mw and some indirect effects linked to microbiota interactions.

[0613] Considering this last prebiotic property, a high Mw seems to allow a more complex and long- lasting fermentation in the colon resulting in a high SCFA production and a longer contact of these metabolites with the intestinal cells.

[0614] Example 8. Use of low molecular weight pectic saccharides according to the invention for the preparation of neutral and acid beverage without protein

[0615] The low molecular weight pectic saccharides produced following example 3 has been added to neutral and acidic beverages without any protein or stabilizer.

[0616] Viscosity and color have been measured to assess the beverage behavior and its stability after the heat treatment.

[0617] The recipes are the following.

[0618] Note : the content of low Mw pectin saccharides is defined considering a serving size of beverage of 1000 grams per day and an intake of potato fiber of 5 grams per day.

[0619] Preparation

[0620] All the ingredients are mixed in water at 75°C, during 5 minutes at 6000 rpm with a PRIMIX mixer. The mix is packed into heat resistant plastic bag to be sterilized at 121 °C during 15min in a Retort machine, with a target F-value of 12. Viscosity, pH and color are measured before the heat treatment and at D+1 . Equipment

[0621] Viscosity analysis: Brookfield viscometer:

[0622] Sample temperature 10°C

[0623] Sample volume 16ml

[0624] Rotation speed: < 10 cP = 60rpm ; > 10 cP = 0.3 - 6.0 rpm

[0625] After starting the rotation, wait 2.5min to wait until the score is stable, then check the score.

[0626] Color analysis L*A*B:

[0627] The Bench-top Colorimeter CR-5 from Konica Minolta is used to measure the colour of solid, pasty granular and liquid samples either in reflectance or transmittance. The system parameters L * a * b * are :

[0628] - The clarity L * which takes values between 0 (black) to 100 (reference white);

[0629] - The parameter a * represents the value on a green — red axis.

[0630] - The parameter b * represents the value on a blue — yellow axis.

[0631] Color difference AE (delta E) is calculated between chosen sample color L1a1 b1 and reference color L2a2b2.

[0632] The result is calculated with Konica's Colibri® Color software, where "CIE 2000" formula is used (closest to the human eye perception).

[0633] The smallest AE, the most similar color the 2 products are. The color difference is usually perceptible if AE is > 2; il is considered that the color difference is perceptible by the human eye if AE is > 1 ,5.

[0634] Results In neutral conditions (trials 1 and 2), the addition of the Low Mw pectic saccharides of the invention results in a pH decrease, from 7.61 to 4.69, before the heat treatment.

[0635] The viscosity remains stable after the heat treatment at Day + 1 , despite the lower pH in trial 2, showing the stabilizing effect of the Low Mw pectic saccharides in the formula. The color is beiger when adding potato fiber in the formula.

[0636] In presence of citric acid in the formula (trials 3 and 4), the initial mix pH is approximately at 3.50 in both trials.

[0637] After the heating treatment, viscosity also remains stable, even in a lower range of pH (pH below 4), showing that the Low Mw pectic saccharides do not disturb the stability in acid beverages.

[0638] The color is beiger when adding Low Mw pectic saccharides in acidic beverage, and this difference is especially higher after heating.

Claims

CLAIMS1 . Method of producing high and low molecular weight pectic saccharides that are enriched in homogalacturonan (HG) and rhamnogalacturonan-l (RG-I), said method comprising the steps of:- providing potato pulp as pectin-rich substrate,- subjecting the pectin-rich substrate to enzymatic treatment, said enzymatic treatment comprising the use of one pectinase having an endo-polygalacturonase (EC 3.2.1.15) activity,- subjecting the hydrolyzed pectic polysaccharides to microfiltration using microfiltration membranes having a molecular cut-off of 0.45 and 0.8 pm,- recovering the microfiltration retentate,- subjecting the microfiltration permeate to nanofiltration using a membrane having a molecular weight cut-off in the range of 150 - 300 Da,- recovering the nanofiltration retentate.

2. Method of claim 1 comprising an optional step consisting in treating the pectic polysaccharides, the microfiltration retentate or the nanofiltration retentate with a strongly cationic exchange resin in the H+form to reduce heavy metals and glycoalkaloids, and to produce a final pH below 2.5 at 5% dry matter.

3. Method of claim 1 to 2, characterized in that the hydrolyzed pectic polysaccharides have:- a molecular weight distribution for the fractions: o lower than 1500 Da, of between 10 and 25 %, preferably between 14 and 21 %, o from 1500 Da to 100 kDa, of between 45 and 65 %, preferably between 52 and 58 %, and o higher than 100 kDa between 20 and 35%, preferably between 22 and 30%,- between 17 and 30 % in mol%, preferably between 20 and 27 % in mol% of HG,- between 68 and 82 % in mol% preferably between 71 and 79 % in mol% of RG-I having a GalA to Rha ratio between 6.5 and 15.5 mol / mol, preferably between 6.6 and 14.9 mol / mol, and a (Gal plus Ara) to GalA ratio between 1.8 to 3.8 mol / mol, preferably between 2.0 and 3.5 mol / mol,- a methylation degree between 40 and 60 % in mol%, preferably between 43 and 57 % in mol% and an acetylation degree between 40 and 55 % in mol%, preferably between 43 and 50 % in mol%.

4. Method of claim 1 to 3, characterized in that the RG-I side chains of the hydrolyzed pectic polysaccharides have:- a (Gal plus Ara) to Rha ratio between 10 and 50 mol / mol, preferably between 13 to 47 mol / mol,- a Gal to Rha ratio between 8 and 45 mol / mol, more preferably between 10 and 40 mol / mol,- an Ara to Rha ratio between 2.5 and 7.5 mol / mol, more preferably between 2.8 and 7.1 mol / mol,- a GalA to Gal ratio between 0.1 and 1.0 mol / mol, preferably between 0.3 and 0.6 mol / mol.

5. Method of claims 1 to 4, characterized in that the microfiltration retentate contains high molecular weight pectic saccharides having: a molecular weight distribution for the fractions: o lower than 1500 Da, of between 5 and 15 %, preferably between 7 and 12 %, o from 1500 Da to 100 kDa, of between 40 and 65 %, preferably between 47 and 56 %, and o higher than 100 kDa, of between 25 and 50%, preferably between 32 and 46%, between 15 and 28 % in mol%, preferably between 16 and 25 % in mol% of HG, between 70 and 85 % in mol%, preferably between 73 and 83 % in mol% of RG-I having a GalA to Rha ratio between 4.0 and 6.0 mol / mol, preferably between 4.1 and 5.5 mol / mol, and a (Gal plus Ara) to Gal A ratio between 1.5 to 4.0 mol / mol, preferably between 1.9 and 3.5 mol / mol, a methylation degree between 15 and 50 % in mol% preferably between 17 and 46 % in mol% and an acetylation degree between 35 and 95 % in mol%, preferably between 39 and 90 % in mol%.

6. Method of claims 1 to 5, characterized in that the RG-I side chains of the high molecular weight pectic saccharides have: a (Gal plus Ara) to Rha ratio between 9 and 15 mol / mol, preferably between 10 and 14 mol / mol, a Gal to Rha ratio between 5 and 13 mol / mol, more preferably between 7 and 11 mol / mol, an Ara to Rha ratio between 2.0 and 4.0 mol / mol, more preferably between 2.4 and 3.5 mol / mol, a GalA to Gal ratio between 0.2 and 1 .0 mol / mol, preferably between 0.4 and 0.7 mol / mol.

7. Method of claims 1 to 6, characterized in that the total sugar composition of the high molecular weight pectic saccharides contain:Ara: between 8 and 14 %w / w, preferably 9 and 12 %w / w,Rha: between 3.0 and 5.0 %w / w, preferably between 3.7 and 4.5 %w / w,Gal: between 32 and 48 %w / w, more preferably between 36 and 44 %w / w, GalA: between 16 and 30 %w / w, more preferably between 18 and 28 %w / w.

8. Method of claims 1 to 7, characterized in that the nanofiltration retentate contains low molecular weight pectic saccharides having: a molecular weight distribution for the fractions: o lower than 1500 Da between 5 and 30%, preferably between 11 and 25% o from 1500 Da to 100 kDa between 50 and 80%, preferably between 54 and 79%, and o higher than 100 kDa between 0 and 35%, preferably between 1 and 30%, between 20 and 35 % in mol%, preferably between 22 and 33 % in mol% of HG between 65 and 80 % in mol%, preferably between 66 and 78 % in mol% of RG-I having a GalA to Rha ratio between 5 and 30 mol / mol, preferably between 7 and 26 mol / mol, and a (Gal plus Ara) to GalA ratio between 1 .5 and 3.5 mol / mol, preferably between 1 .8 and 3.2 mol / mol. a methylation degree between 20 and 55 % in mol%, preferably between 23 and 53 % in mol% and an acetylation degree between 10 and 50 % in mol%, preferably between 11 and 48 % in mol%.

9. Method of claims 1 to 8, characterized in that the RG-I side chains of the low molecular weight pectic saccharides have: a (Gal plus Ara) to Rha ratio between 15 and 60 mol / mol, preferably between 18 and 55 mol / mol, a Gal to Rha ratio between 10 and 50 mol / mol, more preferably between 15 and 48 mol / mol, an Ara to Rha ratio between 2.8 and 7.5 mol / mol, more preferably between 3.1 and 7.3 mol / mol, a GalA to Gal ratio between 0.3 and 0.7 mol / mol, preferably between 0.4 and 0.6 mol / mol.

10. Method of claim 1 to 8, characterized in that the total sugar composition of the low molecular weight pectic saccharides has:Ara: between 4.0 and 10.0 %w / w, preferably 4.8 and 8.1 %w / w,Rha: between 0.5 and 3.0 %w / w, preferably 0.9 and 2.8 %w / w,Gal: between 44 and 57 %w / w, more preferably between 46 and 55 %w / w, GalA: between 20 and 36 %w / w, more preferably between 23 and 34 %w / w.1 1 . Method of claim 1 to 9, characterized in that the resin treatment reduces heavy metals of the hydrolyzed pectic polysaccharides, the high molecular weight pectic saccharides or the low molecular weight pectic saccharides to: cadmium < 0.01 mg / kg; copper < 0.5 mg / kg; lead < 0.04 mg / kg;nickel < 0.2 mg / kg; arsenic < 0.03 mg / kg; mercury < 0.005 mg / kg; and glycoalkaloids to: alpha-solanine < 25 mg / kg; alpha-chaconine < 25 mg / kg.

12. The method of claim 11 , characterized in that the products have a pH below 2.5 when suspended at 5 % dry matter.

13. High molecular weight pectic saccharides having: a molecular weight distribution for the fractions: o lower than 1500 Da, of between 5 and 15 %, preferably between 7 and 12 %, o from 1500 Da to 100 kDa, of between 40 and 65 %, preferably between 47 and 56 %, and o higher than 100 kDa, of between 25 and 50%, preferably between 32 and 46%, between 15 and 28 % in mol%, preferably between 16 and 25 % in mol% of HG, between 70 and 85 % in mol%, preferably between 73 and 83 % in mol% of RG-I having a GalA to Rha ratio between 4.0 and 6.0 mol / mol, preferably between 4.1 and 5.5 mol / mol, and a (Gal plus Ara) to Gal A ratio between 1.5 to 4.0 mol / mol, preferably between 1.9 and 3.5 mol / mol, a methylation degree between 15 and 50 % in mol% preferably between 17 and 46 % in mol% and an acetylation degree between 35 and 95 % in mol%, preferably between 39 and 90 % in mol%.

14. High molecular weight pectic saccharides of claim 13, characterized in that their RG-I side chains has: a (Gal plus Ara) to Rha ratio between 9 and 15 mol / mol, preferably between 10 and 14 mol / mol, a Gal to Rha ratio between 5 and 13 mol / mol, more preferably between 7 and 11 mol / mol, an Ara to Rha ratio between 2.0 and 4.0 mol / mol, more preferably between 2.4 and 3.5 mol / mol, a GalA to Gal ratio between 0.2 and 1 .0 mol / mol, preferably between 0.4 and 0.7 mol / mol.

15. High molecular weight pectic saccharides of claim 13, characterized in that their total sugar composition contains:Ara: between 8 and 14 %w / w, preferably 9 and 12 %w / w,Rha: between 3.0 and 5.0 %w / w, preferably between 3.7 and 4.5 %w / w,Gal: between 32 and 48 %w / w, more preferably between 36 and 44 %w / w, GalA: between 16 and 30 %w / w, more preferably between 18 and 28 %w / w.

16. High molecular weight pectic saccharides of claim 13, characterized by: heavy metal contents below: cadmium < 0.01 mg / kg; copper < 0.5 mg / kg; lead < 0.04 mg / kg; nickel < 0.2 mg / kg; arsenic < 0.03 mg / kg; mercury < 0.005 mg / kg; glycoalkaloids: alpha-solanine < 25 mg / kg; alpha-chaconine < 25 mg / kg; and a pH below 2.5 in a 5% dry matter suspension.

17. Low molecular weight pectic saccharides having: a molecular weight distribution for the fractions: o lower than 1500 Da between 5 and 30%, preferably between 11 and 25% o from 1500 Da to 100 kDa between 50 and 80%, preferably between 54 and 79%, and o higher than 100 kDa between 0 and 35%, preferably between 1 and 30%, between 20 and 35 % in mol%, preferably between 22 and 33 % in mol% of HG between 65 and 80 % in mol%, preferably between 66 and 78 % in mol% of RG-I having a GalA to Rha ratio between 5 and 30 mol / mol, preferably between 7 and 26 mol / mol, and a (Gal plus Ara) to GalA ratio between 1 .5 and 3.5 mol / mol, preferably between 1 .8 and 3.2 mol / mol. a methylation degree between 20 and 55 % in mol%, preferably between 23 and 53 % in mol% and an acetylation degree between 10 and 50 % in mol%, preferably between 11 and 48 % in mol%.

18. Low molecular weight pectic saccharides of claim 17, characterized in that their RG-I side chains have: a (Gal plus Ara) to Rha ratio between 15 and 60 mol / mol, preferably between 18 and 55 mol / mol, a Gal to Rha ratio between 10 and 50 mol / mol, more preferably between 15 and 48 mol / mol, an Ara to Rha ratio between 2.8 and 7.5 mol / mol, more preferably between 3.1 and 7.3 mol / mol, a GalA to Gal ratio between 0.3 and 0.7 mol / mol, preferably between 0.4 and 0.6 mol / mol.

19. Low molecular weight pectic saccharides of claim 17, characterized in that their total sugar composition contains:Ara: between 4.0 and 10.0 %w / w, preferably 4.8 and 8.1 %w / w,Rha: between 0.5 and 3.0 %w / w, preferably 0.9 and 2.8 %w / w,Gal: between 44 and 57 %w / w, more preferably between 46 and 55 %w / w, GalA: between 20 and 36 %w / w, more preferably between 23 and 34 %w / w.

20. Low molecular weight pectic saccharides of claim 17, characterized by: heavy metal contents below: cadmium < 0.01 mg / kg; copper < 0.5 mg / kg; lead < 0.04 mg / kg; nickel < 0.2 mg / kg; arsenic < 0.03 mg / kg; mercury < 0.005 mg / kg; glycoalkaloids: alpha-solanine < 25 mg / kg; alpha-chaconine < 25 mg / kg; and a pH below 2.5 in a 5% dry matter suspension.

21. Use of the high molecular weight pectic saccharides of any of claims 13 to 15, or able to be obtained according to the method of claim 1 , in nutritional formulation, food product, dietary supplement, beverage or pharmaceutical product.

22. Use of the low molecular weight pectic saccharides of any of claims 17 to 19, or able to be obtained according to the method of claim 1 , in nutritional formulation, food product, dietary supplement, beverage or pharmaceutical product.

23. Use of the low molecular weight pectic saccharides of any of claims 17 to 19, or able to be obtained according to the method of claim 1 , in neutral and acid beverages.

24. Use of the high molecular weight pectic saccharides of any of claims 13 to 15, or able to be obtained according to the method of claim 1 as long-lasting substrate for gut microbiota, allowing several cross-feeding interactions and a larger set of bioactive metabolites production.

25. Use of the low molecular weight pectic saccharides of any of claims 17 to 19, or able to be obtained according to the method of claim 1 to increase the expression of the MUC3 protein involved in the mucus layer that protects and reinforces the intestinal epithelium.

26. Use of the high molecular weight pectic saccharides of any of claims 13 to 15, or able to be obtained according to the method of claim 1 and ofthe low molecular weight pectic saccharides of any of claims 17 to 19, or able to be obtained according to the method of claim 1 as immunomodulators.

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