Method for isolation of polysaccharides

Abstract

The invention relates to a method for production of a preparation from a vegetable material, said preparation being enriched in a polysaccharide with a backbone comprising rhamnogalacturonan-I cores, said method comprising the steps: mixing the vegetable material with a polar alcoholic solvent; and separating a solid residue obtained in step a) from the solvent; and mixing the solid residue obtained in step b) with a buffered aqueous solution having a pH between 7 and 8. Also provided is a preparation containing rhamnogalacturonan-I pectins that can be obtained by this method and the use of such a preparation to modulate immune response.

Claims

1. A method of preparing a food or pharmaceutical composition, comprising incorporating into the composition 0.5-25 wt. % of polysaccharide preparation isolated from a vegetable material selected from apple, carrot, bell pepper, tomato, onion, or combinations thereof enriched in rhamnogalacturonan-1 (RG-1) polysaccharide with a backbone comprising rhamnogalacturonan-I cores and optionally alpha(1,4)-linked homo-galacturonic acid stretches, wherein the molar ratio of galacturonyl acid residues to rhamnosyl residues in the backbone of the RG-1 polysaccharide ranges from 10:1 to 1:1, and wherein the RG-1 polysaccharide has a molecular weight between 40 kDa and 2,000 kDa; wherein the pectic polysaccharides contained in the polysaccharide preparation comprise at least 20 wt. % of the RG-I polysaccharide, wherein the polysaccharide preparation does not form a gel when diluted with an aqueous solution of 50 mM ammonium bicarbonate to a solids content of 2.5 wt. %, and wherein at least 90 wt. % of the polysaccharide preparation dissolves when 25 g of the polysaccharide preparation is added to 1 liter of distilled water having a temperature of 20 C.

2. The method according to claim 1, wherein at least 95 wt. % of the polysaccharide preparation dissolves when 25 g of the polysaccharide preparation is added to 1 liter of distilled water having a temperature of 20 C.

3. The method according to claim 1, wherein less than 30% of the galacturonyl acid residues in the polysaccharide is methylated or acetylated.

4. The method according to claim 1, wherein the polysaccharide preparation has a ratio [Ara]/[Rha] of less than 20, wherein [Ara] represents the molar concentration of alpha-(1,5)-linked arabinosyl residues and [Rha] represents the molar concentration of rhamnosyl residues.

5. The method according to claim 1, wherein the vegetable material is selected from selected from apple, carrot, bell pepper, or combinations thereof.

6. The method according to claim 1, wherein the polysaccharide preparation has a water activity of less than 0.6.

7. The method according to claim 1, wherein the polysaccharide preparation is obtained by: (a) mixing the vegetable material with a polar alcoholic solvent to provide a solid residue; (b) separating the solid residue obtained in step (a) from the solvent; (c) mixing the solid residue obtained in step (b) with a buffered aqueous solution having a pH between 7 and 8; and (d) isolating the polysaccharide preparation from the buffered aqueous solution; (e) optionally separating the solid residue from the aqueous solution of step (c); and (f) optionally concentrating the aqueous solution from step (d) to provide the isolated polysaccharide preparation.

8. The method according to claim 7, wherein the polar alcoholic solvent is ethanol.

9. The method according to claim 7, wherein the buffered aqueous solution comprises a weak acid or a weak base having a pK.sub.a in the range of 6.0 to 8.8.

10. The method according to claim 7, wherein the mixing in step (c) is performed at a temperature between 30 C. and 100 C. and at atmospheric pressure.

11. The method according to claim 10, wherein the temperature is between 60 C. and 100 C.

12. The method according to claim 7, wherein cell walls in the vegetable material have been destructed.

13. The method according to claim 7, wherein the vegetable material is mixed with a total amount of the polar alcoholic solvent that is at least 8 times higher than the dry weight of the vegetable material.

14. The method according to claim 7, wherein the vegetable material is mixed with the polar alcoholic solvent and optionally water to produce a mixture containing the polar alcoholic solvent and the water in a weight ratio that is within the range of 1:1 to 19:1.

15. The method according to claim 7, wherein the polar alcoholic solvent is a C1-4 alcohol.

16. The method according to claim 7, wherein the solid residue is mixed with the aqueous buffering solution in a weight ratio of 2:100 to 25:100.

17. The method according to claim 7, wherein the molar ratio of galacturonyl acid residues to rhamnosyl residues in the backbone of the polysaccharide ranges from 20:1 to 1:1.

18. The method according to claim 7, wherein the polysaccharide has a molecular weight between 50 kDa and 2,000 kDa.

19. The method according to claim 18, wherein the polysaccharide has a molecular weight between 70 kDa and 2,000 kDa.

20. The method according to claim 1, wherein the polysaccharide preparation is obtained by: (a) mixing the vegetable material with 85% ethanol at 80 C. for 2.5 h to provide a solid residue; (b) separating the solid residue obtained in step (a) from the ethanol; (c) mixing the solid residue obtained in step (b) with NaHCO.sub.3 solution at 100 C. for 60 min; (d) separating the solid residue from the NaHCO.sub.3 solution as obtained from step (c); and (d) isolating the polysaccharide preparation from the NaHCO.sub.3 solution; (f) optionally repeating steps (c) and (d); and (g) centrifuging the NaHCO.sub.3 solution in step (d) to provide the isolated polysaccharide preparation.

21. The method according to claim 1, wherein less than 20% of the galacturonyl acid residues in the polysaccharide are methylated or acetylated.

Description

DESCRIPTION OF FIGURES

(1) FIG. 1: Immune modulating effect of extract obtained from bell pepper, from example 2; whole blood cell assay. Concentration of extract in microgram per milliliter (x-axis) versus percentage phagocytosis (y-axis).

(2) FIG. 2: Immune modulating effect of extract obtained from carrot, from example 2; whole blood cell assay. Concentration of extract in microgram per milliliter (x-axis) versus percentage phagocytosis (y-axis).

(3) FIG. 3: Immune modulating effect of extract obtained from apple, from example 2; whole blood cell assay. Concentration of extract in microgram per milliliter (x-axis) versus percentage phagocytosis (y-axis).

EXAMPLES

(4) The following non-limiting examples describe extraction and preparation of polysaccharides obtained according to the method of the invention.

Example 1: Preparation of Vegetable Extracts

(5) Materials and Methods

(6) Vegetable materials used as source of the polysaccharides were dried powders: bell pepper (paprika, Capsicum annuum); Paprika Mild 80-100 (Asta StFelix Reverte, S. A., Librilla (Murcia), Spain). carrot (Daucus carota subsp. sativus); ex. R. Steinicke GmbH, Breitbrunn, Germany. apple powders (Malus domestica): ex Mahevi Oy, Polyijrvi, Finland.

(7) From the vegetable materials first alcohol insoluble residues (AIRs) were prepared by ethanol washings from these vegetable materials. The AIR materials were subjected to a set of extraction experiments in respectively water, sodium bicarbonate, or dilute hydrochloric acid.

(8) Each vegetable material was first extracted with 80% aqueous ethanol twice (80 C., about 2 h) and then overnight at room temperature; each time using 12.5% (w/v). The insoluble residue was dried and this material is called alcohol insoluble residue, AIR. The AIR materials were used in all extraction experiments described here. Extractions were performed with the defined solution for specified time, and the liquid phase was recovered after centrifugation.

(9) All extracts prepared in this sample series were dialyzed using a MWCO 6000-8000 dialysis tube to aid comparison between treatments carrying different salts. Samples (5 g insoluble residue) were twice dialyzed against about 1 liter of 50 mM ammonium bicarbonate for ca. 3-4 h and then against fresh 50 mM ammonium bicarbonate overnight, and then lyophilized.

(10) Beside the yield, extracts were also analyzed by GPC in Superdex200 column to obtain the molecular weight profile of the extracts. In addition, the extracts were subjected to proton-NMR analysis, and the substitution pattern and composition of each extract were deduced from the spectra.

(11) The mass % of pectin in the fractions was determined by gel-filtration chromatography on Superdex200 columns and the fraction>70 kD was found to be mainly RG-1 as confirmed by .sup.1H NMR and monosaccharide hydrolysis/HPLC. The mass % of pectin could be corrected for the presence of non-pectin type monosaccharides like glucose which is indicative of the presence of starch.

(12) Separation of Polysaccharides by Molecular Weight

(13) Polysaccharides obtained by extraction are separated by gel filtration chromatography (GPC) on a column of Superdex 200 (130 cm; GE Healthcare). The polysaccharide samples are dissolved in 0.1 M ammonium bicarbonate and aliquots of 5 to 15 mg were run on the column in the same buffer. Eluting components are detected by absorption at 214 nm.

(14) The polysaccharides are pooled into three fractions: M.sub.W>110 kDa, 70-110 kDa and 40-70 kDa. The pooling limits are determined by comparing to elution positions of Dextran standards 40 kDa, 70 kDa and 110 kDa (ex GE Healthcare).

(15) Substitution and Composition Analysis of Polysaccharides

(16) The composition of polysaccharide fractions as obtained from the extraction process are determined using NMR analysis. Prior to NMR analysis, dry polysaccharide samples (1-10 mg) are dissolved in deuterium oxide (D.sub.2O) and dried in a vacuum centrifuge. Samples are then dissolved in 600 microliter of D.sub.2O, Spectra are collected on a Varian Unity 500 NMR spectrometer at 296 K and referenced to internal acetone standard (2.225 ppm).

(17) Referring to Table 3, Table 6, and Table 9, the polysaccharide substitution level and the molar composition of the building units are analysed by using the NMR data as follows:

(18) Rhamnogalacturonan Substitution Level

(19) The ratio of 4-substituted rhamnosyl units and nonsubstituted units is estimated by integrating the splitted rhamnose CH.sub.3 signals. The CH.sub.3 protons in 4-substituted rhamnosyl units resonate around 1.32 ppm, while those of the nonsubstituted rhamnosyl units at 1.25 ppm.

(20) Molar Ratio of Galactans and Arabinans

(21) The molar amount of galactans and arabinans is analysed by integrating the observed beta-1,4-galactan H-1 signal (4.64 ppm) and the arabinan H-1 signals (-5Ara H-1, 5.09 ppm; -3,5Ara H-1 5.12 ppm; terminal Ara-alpha-1-3 5.15 ppm; terminal Ara-alpha-1-2 5.18 ppm; -2,3,5Ara H-1 5.26 ppm. These integration values are compared to the integrated rhamnose CH.sub.3 signals.

(22) Methylation and Acetylation of Galacturonic Acid Residues

(23) The galacturonic acid O-acetyl group CH.sub.3 signals reside around 2.07-2.18 ppm, and are integrated as no interfering signals are present in this area. The O-acetylation level is normalized to 100% being 1 acetyl group per galacturonic acid residue. The galacturonic acid methyl esterification level is estimated from the integrated galacturonic acid H-4 signals. The H-4 signal of methyl esterified galacturonic acid unit resides at 4.47 ppm, while the H-4 signal of nonesterified galacturonic acid is at 4.42 ppm.

(24) Polygalacturonic Acid/Rhamnose Ratio

(25) The ratio of polygalacturonic acid to rhamnose is analysed as follows: the total amount of galacturonic acid H-4 signals is integrated between 4.42 and 4.47 ppm, and the amount of rhamnose is obtained by integration of the CH.sub.3 signals (1.25-1.32 ppm). The H-4 signal of the RG-I specific GalA-alpha-1-2 unit is located in the same 4.42-4.47 signal, and its portion has to be deducted from the total H-4 signal. This value is the same as rhamnose amount, as RG-I is a 1:1 polymer of Rha and GalA. The remaining H-4 signal represents the share of polygalacturonic acid type GalA-alpha-1-4H-4 signal.

(26) Monosaccharide Analysis (Acid Hydrolysis+HPLC)

(27) Polysaccharide samples were dissolved in 0.5 M aqueous trifluoroacetic acid and hydrolyzed at 120 C. for 2 hours. Samples were then neutralized with NaOH, and kept frozen until analyzed by high-pH anion-exchange chromatography (HPAEC) in DX600 Ion Chromatography System (Dionex, Sunnyvale, USA) using pulsed-amperometric detection (PAD). Samples were analyzed in two different columns, CarboPacMA-1 and CarboPacPA-1 (Dionex, Sunnyvale, USA) to obtain reliable analysis of the reported monosaccharide species. The MA-1 column runs were carried out using an isocratic elution with 180 mM NaOH. PA-1 runs were carried out with the following linear gradients: minute 0-5, 50 mM NaOH, minute 5-10 from 50 to 100 mMNaOH, minute 10-35 from 0 to 200 mM NaAc in 100 mM NaOH, and minute 35-40 from 200 to 400 mM NaAc in 100-240 mM NaOH.

(28) Extraction Methods

(29) The following extractions of the AIR from the vegetable materials were performed: Water extractions for 30, 60 and 90 min at 100 C. and at 70 C. Unless indicated otherwise, 10% (w/vol) suspensions were used in these experiments. The extraction with water is known in the prior art, these are comparative experiments. Extractions with sodium bicarbonate (pH 7.5-8) were typically carried out at 7.5% (wt/vol), unless indicated otherwise. The rationale to study this extraction method was that the slightly alkaline solution could promote the degradation of the methyl esterified polygalacturonic acid chain through beta-eliminative cleavage. This would yield enriched RG-I product, as beta-elimination would be much slower for the naturally nonesterified RG-I. These are extractions according to the method of the invention. Acid extractions were performed by hydrochloric acid at pH 2.5 and 5% (wt/vol) unless indicated otherwise, for the specified time at 70 C., after which solutions were cooled and neutralized by sodium bicarbonate. Pectin used as a gelling material in food industry is produced by acid extraction of e.g. citrus peels, followed by alcohol precipitation. It was therefore studied here whether acid extraction could produce higher amounts of RG-I also, and whether the RG-I would be stable in the acidic solution. The extraction with acid is known in the prior art, these are comparative experiments.
Extraction Efficiencies

(30) The extraction yields, composition analysis and molecular weight distribution of the extracts obtained from the three vegetable materials, extracted using water, acid or bicarbonate are given in Table 1 to Table 9. The molecular weight distribution was measured by GPC on Superdex 200. Fractions corresponding to >110 kDa (HMW), 70-110 kDa (MMW) and 40-70 kDa (LMW) were collected and the yields measured. The remainder of the extract is considered to have a molecular weight less than 40 kDa, and this is calculated by subtracting the weight percentages of the 3 fractions from 100%. Yields are presented as weight/weight percent compared to extract injected to column. Typically 10-15 mg of extract was dissolved in about 400 microliter of running buffer. Non-dissolved material was removed before injection, and not taken into account in the calculation.

(31) TABLE-US-00002 TABLE 1 Yield of extraction of bell pepper using water, acid, or bicarbonate. AIR yield starting extraction after material volume extraction yield Code Treatment [g] [mL] [g] [%] P-W1 water 100 C., 30 min 5.00 50 0.41 8.2 P-W2 water 100 C., 60 min 5.03 50 0.44 8.7 P-W3 water 100 C., 90 min 5.03 50 0.45 9.0 P-W4 water 70 C., 30 min 5.00 50 0.48 9.5 P-W5 water 70 C., 60 min 5.05 50 0.53 10.4 P-W6 water 70 C., 90 min 5.05 50 0.46 9.1 P-A1 HCl 70 C., 30 min 5.02 100 0.33 6.7 P-A2 HCl 70 C., 60 min 5.00 100 0.38 7.6 P-A3 HCl 70 C., 90 min 5.02 100 0.48 9.5 P-B1 NaHCO.sub.3 100 C., 5.04 66 0.63 12.5 30 min P-B2 NaHCO.sub.3 100 C., 5.04 66 0.67 13.4 60 min P-B1 NaHCO.sub.3 100 C., 5.04 66 0.59 11.6 30 min

(32) TABLE-US-00003 TABLE 2 Molecular weight distribution of bell pepper extracts. M.sub.W >110 kDa M.sub.W 70-110 kDa M.sub.W >70 kDa M.sub.W 40-70 kDa M.sub.W <40 kDa code Treatment [wt %] [wt %] [wt %] [wt %] [wt %] P-W1 water 100 C., 30 min 9 14 23 17 60 P-W2 water 100 C., 60 min 15 13 28 19 53 P-W3 water 100 C., 90 min 12 16 28 21 51 P-W4 water 70 C., 30 min 23 19.5 42.5 17 40.5 P-W5 water 70 C., 60 min 11 15 26 11 63 P-W6 water 70 C., 90 min 9 16 25 21 54 P-A1 HCl 70 C., 30 min n.a.* n.a. n.a. n.a. n.a. P-A2 HCl 70 C., 60 min n.a. n.a. n.a. n.a. n.a. P-A3 HCl 70 C., 90 min n.a. n.a. n.a. n.a. n.a. P-B1 NaHCO.sub.3 100 C., 30 min 16 17.5 33.5 28 38.5 P-B2 NaHCO.sub.3 100 C., 60 min 13 13.5 26.5 23 50.5 P-B3 NaHCO.sub.3 100 C., 90 min 18 13 31 27 42 *indicates that the extract was not run in GPC due to gel character.

(33) TABLE-US-00004 TABLE 3 Composition analysis of extracts of bell pepper using water, acid, or bicarbonate. Rha subst.sup.(1) Gal/Rha.sup.(2) Ara/Rha.sup.(3) GalA OCH.sub.3.sup.(4) acetylation.sup.(5) GalA/Rha.sup.(6) code [%] [mol/mol] [mol/mol] [%] [%] [mol/mol] P-W1 25 0 4.4 50 15 8.5 P-W2 30 0 10.5 50 20 6.8 P-W3 30 0 4.4 50 20 6.9 P-W4 20 0 16.2 50 35 4.8 P-W5 15 0 3.3 50 15 10.0 P-W6 20 0 6.5 50 20 9.2 P-A1 35 0 3.1 0 10 8.1 P-A2 40 0 5.0 0 10 7.3 P-A3 35 0 5.1 0 10 7.1 P-B1 30 0 0 0 5 13.9 P-B2 30 0 0 0 0 12.1 P-B3 45 0 0 0 0 5.3 Legend: .sup.(1)Rha subst.: molar fraction of the Rha moieties in the RG-I core which is substituted at the C-4 position with a side chain; as measured from the Rha CH.sub.3 signal shift; .sup.(2)Gal/Rha: molar ratio of beta(1,4)-linked Gal as compared to Rha (mol/mol); relates to the length of side chains containing beta(1,4)-linked galactan residues; .sup.(3)Ara/Rha: molar ratio of alpha(1,5)-linked arabinan as compared to Rha (mol/mol); relates to the length of side chains containing alpha(1,5)-linked arabinosyl residues; .sup.(4)GalA OCH.sub.3: degree of methylation of galacturonic acid residues (mol %); .sup.(5)acetylation: degree of acetylation of galacturonic acid residues (mol %), 1 acetylgroup per galacturonic acid residue is set as 100%; .sup.(6)GalA/Rha: alpha-1,4-galacturonyl acid residues vs. rhamnosyl residues (mol/mol); i.e. this number indicates the molar ratio between GalA residues in the alpha(1,4)-linked polygalacturonic acid or alpha(1,4)-linked oligogalacturonic acid cores in the polysaccharide and Rha residues in the RG-I core of the polysaccharide;

(34) TABLE-US-00005 TABLE 4 Yield of extraction of carrot using water 3% (w/v), acid, or bicarbonate. AIR starting extraction yield after material volume extraction yield code Treatment [g] [mL] [g] [%] C-W1 water 100 C., 30 min 5.01 50 0.43 8.6 C-W2 water 100 C., 60 min 5.01 50 0.25 5.0 C-W3 water 100 C., 90 min 5.00 50 0.26 5.2 C-W4 water 100 C., 1.00 30 0.14 14.0 3x vol., 30 min C-W5 water 100 C., 1.00 30 0.16 16.0 3x vol., 60 min C-W6 water 100 C., 1.07 30 0.18 16.4 3x vol., 90 min C-W7 water 70 C., 30 min 5.00 50 0.37 7.5 C-W8 water 70 C., 60 min 5.04 50 0.42 8.3 C-W9 water 70 C., 90 min 5.02 50 0.42 8.3 C-A1 HCl 70 C., 30 min 5.06 100 0.65 12.9 C-A2 HCl 70 C., 60 min 5.09 100 0.67 13.2 C-A3 HCl 70 C., 90 min 5.04 100 0.56 11.2 C-B1 NaHCO.sub.3 100 C., 5.04 66 0.86 17.1 30 min C-B2 NaHCO.sub.3 100 C., 5.04 66 0.92 18.2 60 min C-B3 NaHCO.sub.3 100 C., 5.04 66 1.03 20.4 90 min

(35) TABLE-US-00006 TABLE 5 Molecular weight distribution of carrot extracts. M.sub.W >110 kDa M.sub.W 70-110 kDa M.sub.W >70 kDa M.sub.W 40-70 kDa M.sub.W <40 kDa code treatment [wt %] [wt %] [wt %] [wt %] [wt %] C-W1 water 100 C., 30 min 9 16 25 22 53 C-W2 water 100 C., 60 min 13 17.5 30.5 25.5 44 C-W3 water 100 C., 90 min 13 18 31 25.5 43.5 C-W4 water 100 C., 3x vol., 30 min 25 19.5 44.5 23 32.5 C-W5 water 100 C., 3x vol., 60 min 26 22 48 29 23 C-W6 water 100 C., 3x vol., 90 min 28 22 50 27 23 C-W7 water 70 C., 30 min 15 24 39 24 37 C-W8 water 70 C., 60 min 21 24 45 17 38 C-W9 water 70 C., 90 min 17 23 40 24.5 35.5 C-A1 HCl 70 C., 30 min n.a.* n.a. n.a. n.a. n.a. C-A2 HCl 70 C., 60 min n.a. n.a. n.a. n.a. n.a. C-A3 HCl 70 C., 90 min n.a. n.a. n.a. n.a. n.a. C-B1 NaHCO.sub.3 100 C., 30 min 27 19.5 46.5 13 40.5 C-B2 NaHCO.sub.3 100 C., 60 min 34 25 59 14 27 C-B3 NaHCO.sub.3 100 C., 90 min 33 23 56 17 27 *indicates that the extract was not run in GPC due to gel character.

(36) TABLE-US-00007 TABLE 6 Composition analysis of extracts of carrot using water, acid, or bicarbonate. Rha subst.sup.(1) Gal/Rha Ara/Rha GalA OCH.sub.3 acetylation GalA/Rha code [%] [mol/mol] [mol/mol] [%] [%] [mol/mol] C-W1 45 5.0 12.3 60 25 9.4 C-W2 45 5.2 11.7 60 20 11.9 C-W3 50 5.3 11.8 60 20 10.9 C-W4 45 4.0 10.0 60 20 8.5 C-W5 40 4.2 11.1 60 20 8.6 C-W6 45 4.2 9.7 60 20 8.8 C-W7 40 6.9 16.5 55 20 11.2 C-W8 45 4.7 13.0 55 25 8.3 C-W9 45 4.2 11.0 55 25 7.8 C-A1 60 4.2 10.5 0 15 6.7 C-A2 55 5.6 5.4 0 15 7.8 C-A3 55 5.8 10.9 0 10 7.3 C-B1 55 5.9 5.5 0 10 9.0 C-B2 50 5.5 3.2 0 10 5.1 C-B3 55 5.3 0.0 0 0 7.6 .sup.(1)for legend see Table 3

(37) TABLE-US-00008 TABLE 7 Yield of extraction of apple using water 3.3% (w/v), acid, or bicarbonate 6.7% (w/v). AIR starting extraction yield after material volume extraction yield code treatment [g] [mL] [g] [%] A-W1 water 100 C., 30 min 5.01 50 0.19 3.8 A-W2 water 100 C., 60 min 5.00 50 0.18 3.6 A-W3 water 100 C., 90 min 5.00 50 0.15 3.0 A-W4 water 70 C., 30 min 5.02 50 0.20 3.9 A-W5 water 70 C., 60 min 5.01 50 0.20 3.9 A-W6 water 70 C., 90 min 5.07 50 0.14 2.8 A-A1 HCl 70 C., 30 min 5.06 75 0.40 7.9 A-A2 HCl 70 C., 60 min 5.00 75 0.43 8.6 A-A3 HCl 70 C., 90 min 5.02 75 0.47 9.4 A-B1 NaHCO.sub.3 100 C., 5.09 66 0.73 14.4 30 min A-B2 NaHCO.sub.3 100 C., 5.09 66 0.48 9.5 60 min A-B3 NaHCO.sub.3 100 C., 5.09 66 0.40 8.0 90 min A-B4 NaHCO.sub.3 100 C., 1.00 30 0.10 10.1 2.5x vol, 30 min A-B5 NaHCO.sub.3 100 C., 1.00 30 0.097 9.7 2.5x vol, 60 min A-B6 NaHCO.sub.3 100 C., 1.00 30 0.13 12.5 2.5x vol, 90 min A-B7 NaHCO.sub.3 100 C., 0.50 20 0.10 20.4 3x vol, 30 min A-B8 NaHCO.sub.3 100 C., 0.50 20 0.11 22.6 3x vol, 60 min A-B9 NaHCO.sub.3 100 C., 0.52 20 0.11 20.7 3x vol, 90 min

(38) TABLE-US-00009 TABLE 8 Molecular weight distribution of apple extracts. M.sub.W M.sub.W M.sub.W M.sub.W M.sub.W >110 kDa 70-110 kDa >70 kDa 40-70 kDa <40 kDa code treatment [wt %] [wt %] [wt %] [wt %] [wt %] A-W1 water 100 C., 30 min 13 4 17 28.5 54.5 A-W2 water 100 C., 60 min 6 12 18 24 58 A-W3 water 100 C., 90 min 17 24 41 30 29 A-W4 water 70 C., 30 min 8 12 20 14 66 A-W5 water 70 C., 60 min 11.5 11.5 23 20.5 56.5 A-W6 water 70 C., 90 min 11.5 13 24.5 12.5 63 A-A1 HCl 70 C., 30 min n.a.* n.a. n.a. n.a. n.a. A-A2 HCl 70 C., 60 min n.a. n.a. n.a. n.a. n.a. A-A3 HCl 70 C., 90 min n.a. n.a. n.a. n.a. n.a. A-B1 NaHCO.sub.3 100 C., 30 min 26 19.5 45.5 15.5 39 A-B2 NaHCO.sub.3 100 C., 60 min 22.5 12.5 35 11.5 53.5 A-B3 NaHCO.sub.3 100 C., 90 min 32 19 51 13 36 A-B4 NaHCO.sub.3 100 C., 2.5x vol, 30 min 22 9 31 14 55 A-B5 NaHCO.sub.3 100 C., 2.5x vol, 60 min 20 8 28 13.5 58.5 A-B6 NaHCO.sub.3 100 C., 2.5x vol, 90 min 22 12 34 13 53 A-B7 NaHCO.sub.3 100 C., 3x vol, 30 min 17 9 26 13 61 A-B8 NaHCO.sub.3 100 C., 3x vol, 60 min 15 10 25 13 62 A-B9 NaHCO.sub.3 100 C., 3x vol, 90 min 29 10.5 39.5 14 46.5 *indicates that the extract was not run in GPC due to gel character.

(39) TABLE-US-00010 TABLE 9 Composition analysis of extracts of apple using water, acid, or bicarbonate. Rha subst.sup.(1) Gal/Rha Ara/Rha GalA OCH.sub.3 acetylation GalA/Rha code [%] [mol/mol] [mol/mol] [%] [%] [mol/mol] A-W1 35 0.0 16.8 50 10 10.6 A-W2 40 0.0 20.3 50 15 9.2 A-W3 40 1.4 23.2 55 15 8.9 A-W4 30 2.7 19.7 50 15 8.1 A-W5 40 0.0 19.1 50 15 7.8 A-W6 25 0.0 18.6 50 20 6.5 A-A1 50 3.2 33.5 0 10 12.3 A-A2 50 2.2 24.3 0 5 13.7 A-A3 50 2.1 28.5 0 5 13.7 A-B1 55 0.0 7.6 0 5 12.0 A-B2 55 0.0 3.8 0 0 11.9 A-B3 50 0.0 6.9 0 0 7.5 A-B4 45 0.0 15.0 5 5 9.4 A-B5 40 0.0 13.8 0 0 8.7 A-B6 45 0.0 11.7 0 0 7.1 A-B7 40 0.0 11.3 0 0 6.4 A-B8 40 0.0 11.8 0 0 6.4 A-B9 45 0.0 11.7 0 0 6.7 .sup.(1)for legend see Table 3
Extractions (Table 1, Table 4, Table 7)

(40) Bell pepper: highest extraction yields were obtained with the extractions using bicarbonate, as compared to extractions using water or acid. The yields were on average 2 to 3% higher than those obtained with water. The temperature of the water extraction did not have much influence. Also with carrot or apple as vegetable material, the use of bicarbonate yielded most extracts, as compared to extractions using water or acid.

(41) In case of carrot, the extraction yields at 100 C. are lower after 60 and 90 min than at 30 min. This can be explained by the extraction solution that after 60 min was much more viscous than at 30 min, and part of the extracted polysaccharides are not recovered but stay in the gel. This behavior was not observed at 70 C., and neither at the bicarbonate extractions. The formation of gel seems to be concentration dependent, as extractions at 100 C. at three-fold dilution (samples C-W4, C-W5, C-W6) did not show the reduction in yield.

(42) Also in case of apple that were extracted using bicarbonate (samples A-B1, A-B2, A-B3), a decrease in yield was observed with increasing extraction time. Also in this case some gelling occurred. Extractions at lower concentrations of the AIR revealed that the drop in yield with increasing extraction time could be prevented.

(43) Molecular Weight Distribution (Table 2, Table 5, Table 8)

(44) With bell pepper as vegetable material, the molecular weight distribution of the materials extracted using bicarbonate was more to the higher end of the molecular weights. As compared to extraction using water only, the bicarbonate extractions during 60 and 90 minutes (samples P-B2, P-B3) yielded a higher proportion of material with a molecular weight above 70 kDa than the extractions with water (samples P-W1 to P-W6).

(45) Also in case of carrot as source material, the proportion of polysaccharides having a molecular weight above 70 kDa extracted using bicarbonate during at least 60 minutes was higher than the corresponding extractions with water. The >110 kDa material was the major component in the fractions extracted using bicarbonate.

(46) The extractions of apple using bicarbonate also led to a higher proportion of polysaccharides having a molecular weight above 70 kDa as compared to extractions with water. Dilution of the vegetable material during the bicarbonate extractions did not lead to improved yield of the polysaccharides of interest. The >110 kDa fraction is dominating in all the apple samples extracted using bicarbonate. This fraction is expected to give the best immuno-modulating results. By using bicarbonate the extraction yield can be increased, without giving in on immuno-modulation.

(47) The viscosity of extracts obtained using acid was too high to be able to determine the molecular weight distribution. This shows that acid extraction of these vegetable materials was not a suitable method to isolate the polysaccharides of interest.

(48) Compositions of the Extracts (Table 3, Table 6, Table 9)

(49) The extracts obtained contain RG-I fragments which have been released from the polysaccharides in the vegetable materials. If the ratio GalA/Rha is 1, then an extract contains only an RG-I core. If the ratio GalA/Rha is larger than 1, then in addition to the RG-I core, stretches of homogalacturonic acid stretches are attached to the RG-I core. The RG-I core usually contains side chains of mainly arabinan and galactan, which are attached to the rhamnose residues.

(50) Bell pepper: the bicarbonate extracts have a higher share of polygalacturonic acid (GalA/Rha) than water extracts. An explanation for this may be that in the bicarbonate solution de-esterification is more rapid than beta-elimination, which actually decreases the polygalacturonic acid chain cleavage.

(51) Carrot: the extracts prepared using water extraction at 100 C. (samples C-W1, C-W2, C-W3) are practically identical with regard to amount of Gal and Ara in the side chain of the RG-I polysaccharide. Samples obtained at 70 C. (C-W7, C-W8, C-W9) contain somewhat larger side chains, especially the ratio Ara/Rha is larger. The extractions with bicarbonate lead to reduction of the side chains of the RG-I polysaccharides, as the ratio Gal/Rha and Ara/Rha decrease with increasing extraction times.

(52) Apple: the samples extracted using bicarbonate show that galactan in side chains was not detected anymore, while in case of acid and water extractions, some of the extracts still some galactan in the side chains can be detected. Also the amount of arabinan in the side chains decreases due to the extractions with bicarbonate. The extracts obtained from diluted samples (A-B4 to A-B9) show that higher-volume extractions seem to produce, on average, slightly higher arabinan ratio and lower polygalacturonic acid share than the extracts obtained from non-diluted samples (A-B1 to A-B3).

(53) For all three crops studied here, the acidic extractions led to viscous solutions, for which it was not possible to determine molecular weight distribution. This may reflect a high amount of polygalacturonic acid which is in the extract (i.e. a high ratio of GalA/Rha), although NMR data does not prove this assumption. However, as NMR analysis does not tolerate particulate matter in the tube, the routine is to centrifuge the samples, and thus the NMR analysis of these extracts may be biased as gel-type of material was also lost.

Example 2: Immuno-Modulating Activity of the Vegetable Extracts

(54) A whole blood assay has been used to determine the in vitro immunomodulating response of the extracts obtained from the methods as described in example 1. This assay is based on phagocytosis activity.

(55) Phagocytosis activity in whole blood is evaluated using the Phagotest kit of Orpegen Pharma (Heidelberg, Germany) using an adjusted protocol. Fresh blood is obtained from healthy human volunteers in sodium heparin vacutainers (BD Biosciences). 30 microliter of whole blood and 5 microliter of the ingredient are incubated in duplicates for 30 minutes in a polypropylene 96-well plate at 37 C. in a water bath. Control incubations consisted of PBS (=basal phagocytosis activity) or 100 ng/mL E. coli-lipopolysaccharide (LPS) (=positive control sample) in triplicate measurements. After the incubation step, 10 microliter of FITC-labeled E. coli (white blood cell to E. coli ratio of 25:1) is added. This incubation at 37 C. is stopped after 6.5 minutes by adding 50 microliter of ice-cold quencher solution. The cells are washed three times by adding 230 microliter of ice-cold wash-buffer and centrifugation for 3 min at 300 g (4 C.). The erythrocytes are lysed using 290 microliter of lysis buffer. After incubation in the dark for 20 minutes at room temperature, the cells are centrifuged for 5 min at 300 g (4 C.). Cells are resuspended in 150 microliter of wash-buffer and stained with propidium iodide. Analysis is performed by flow cytometry (Coulter FC500MPL flow cytometer, Beckman Coulter Nederland BV, Mijdrecht). Within the leucocytes, granulocytes are gated according to the FSC/SSC profile. The percentage of phagocytosing cells in the granulocyte population is determined. The results are normalized to the dynamic range between basal and LPS-stimulated phagocytosis and expressed as a relative percentage phagocytosis activity. A normalized percentage of more than 40% is considered to be positive. This assay gives an estimation of the immunomodulating activity, trends can be observed from this. All samples were passed over a preconditioned C-18 cartridge before application to the immuno measurement.

(56) The following extracts were tested in the assay:

(57) TABLE-US-00011 P-W1 bell pepper water 100 C., 30 min P-W3 bell pepper water 100 C., 90 min P-A1 bell pepper HCl 70 C., 30 min P-A3 bell pepper HCl 70 C., 90 min P-B1 bell pepper NaHCO.sub.3 100 C., 30 min C-W1 carrot water 100 C., 30 min C-W3 carrot water 100 C., 90 min C-A1 carrot HCl 70 C., 30 min C-A3 carrot HCl 70 C., 90 min C-B1 carrot NaHCO.sub.3 100 C., 30 min C-B3 carrot NaHCO.sub.3 100 C., 90 min A-W1 apple water 100 C., 30 min A-W3 apple water 100 C., 90 min A-A1 apple HCl 70 C., 30 min A-A3 apple HCl 70 C., 90 min A-B1 apple NaHCO.sub.3 100 C., 30 min A-B3 apple NaHCO.sub.3 100 C., 90 min

(58) The measured phagocytosis data of these extracts are given in FIG. 1, FIG. 2, and FIG. 3.

(59) The extracts obtained from bell pepper showed already some phagocytosis activity at a concentration as low as 0.003 microgram per mL (FIG. 1). The extract obtained using bicarbonate at 30 min (P-B1) showed similar activity as the extract obtained using water at 100 C. at 30 min (P-W1). Also the activity of the bicarbonate sample as compared to acid extraction had improved. The activity of the 30 min samples was higher than the 90 min samples, especially at the low concentrations of the extract (0.3 microgram per mL and less).

(60) Monosaccharide analysis showed that Ara and Gal could not be detected anymore in the bicarbonate samples (P-B1, P-B3). Hence in these extracts, side chains are not required to stimulate phagocytosis.

(61) With carrot as vegetable material, the extracts obtained using bicarbonate were similarly active as the water extracts at an extract concentration of 30 microgram per mL. The bicarbonate extract obtained at 30 minutes extraction was more active than the 90 minute extract.

(62) When comparing the monosaccharide compositions of the extracts, for the bicarbonate extracts (C-B1, C-B3), the ratio Ara/Rha in the side chains is lower than for the water extracts (A-W1, A-W3), and the ratio GalA/Rha is lower than for the water samples. This shows that the extract obtained from carrot using bicarbonate is richer in RG-I core with a smaller amount of homogalacturonic acid residues attached to the RG-I core than the water extract, while the side chains of the RG-I core contain less Ara.

(63) In case of apple, the activity of the 30 minute bicarbonate sample (A-B1) was higher than the water (A-W1) and acid (A-A1) samples. The 90 minute samples were similar in activity. Also here the ratio of Ara/Rha of the bicarbonate extracts is lower than the water extracts. In both cases the Gal/Rha ratio is very low or zero.

CONCLUSIONS

(64) These results show that the extracts obtained using bicarbonate possess immuno-modulating activity as they are active in the whole blood assay. The ratio of GalA/Rha of the tested extracts ranges from about 5 and 14, and these extracts showed phagocytosis activity. This shows that the RG-I core may contain attached homogalacturonic acid stretches of various lengths. Some of the side chains of the RG-I core are very low in Ara and/or Gal, meaning that these side chains are not specifically required for immuno-stimulating activity.

(65) The combination of relatively high yield of the extracts using bicarbonate as compared to water and acid extractions, combined with the activity of the extracts in the in vitro assays, show that the method of the invention is advantageous to obtain the extract containing polysaccharides that modulate immune response.