Method for preparing xyloglucan-oligosaccharides

Abstract

The present invention relates to a method for preparing oligosaccharides which can be used among others as food additives to reduce calorie content, to sweeten food products, to increase the fiber content of food products, to improve the texture of food products and to stimulate the gut microbiome bacteria. Furthermore they can be applied in the fields of animal feed, or other applications. More particularly, this invention is directed to a high temperature hydrolysis of xyloglucan polysaccharide to defined xyloglucan oligosaccharides. The invention further relates to oligosaccharide hydrolysates produced with the method of the invention and to the use of said oligosaccharide hydrolysates in human and/or animal nutrition, as prebiotic or other uses. Further provided are novel endoglucanases for use in the method of the invention as well as in other applications.

Claims

1. A method for producing xyloglucan oligosaccharides (XGOS) from a xyloglucan source, said method comprising: contacting, at a temperature in the range of 50.5° C. to 80° C., a xyloglucan polysaccharide with an enzyme that is a polypeptide selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, wherein the enzyme exhibits xyloglucanase activity at a temperature higher than 50° C., and exhibits an end product inhibition constant (Ki) of at least 5 mM.

2. The method of claim 1, wherein the xyloglucan source is selected from the group consisting of peppergrass, rapeseed, apple, bilberry, blueberry, olives, tamarind or fractions thereof comprising tamarind kernel powder, defatted tamarind kernel powder and tamarind seeds.

3. The method according to claim 1, wherein said enzyme is recombinantly produced in a host cell selected from the group consisting of E. coli, Bacillus subtilis and Bacillus licheniformis.

4. The method according to claim 1, wherein the xyloglucan polysaccharide is hydrolyzed in an aqueous solution.

5. The method according to claim 4, further comprising one or more steps of: removing solids from the solution; removing proteins and salts from the solution; removing coloring agents from the solution; and recovering the xyloglucan source polysaccharide hydrolysate from the solution.

6. The method according to claim 1, wherein said enzyme is present in an amount of 0.005 to 0.03% (w/w) of the xyloglucan source.

7. The method according to claim 1, wherein a xyloglucan hydrolysate is produced that comprises a mixture of DP7 to DP9 XGOS.

8. The method according to claim 1, wherein the amount of the xyloglucan source used is 100 g/l to 750 g/l.

Description

(1) The present invention is further described by the following figures and examples that should not be construed as limiting the scope of the invention.

(2) Herein,

(3) FIG. 1 shows the hydrolysis of tamarind xyloglucan to oligosaccharides DP7 to DP9. Possible sites for hydrolysis by an endoglucanase are indicated by a scissors symbol. In XXXG, XXLG, XLXG and XLLG describes the structure of the oligosaccharides, with G designating an undecorated backbone glucose residue, X a backbone glucose residue decorated with xylose, and L a backbone glucose residue decorated with xylose and galactose; letter code naming convention from Fry et al, 1993.

(4) FIG. 2 shows the typical steps of the method of the invention to produce a DP7-DP9 XGOS containing hydrolysate from tamarind kernel powder.

(5) FIG. 3 shows a photograph of purified DP7-DP9 XGOS mixture in powder form, which was produced with the method of the invention.

(6) FIG. 4 shows the activity profile of the employed endoglucanase SEQ ID NO 3 in dependence of pH and temperature. Relative activity was determined as generation of reduced sugars from barley-beta-glucan determined by DNSA-assay.

(7) FIG. 5 shows a SDS-PAGE of recombinantly produced SEQ ID NOs 1, 2, 3 and 4 respectively. PageRuler Prestained Protein Ladder 10 to 18 kDa (#26616, ThermoFisher Scientific) was used as protein standard.

(8) FIG. 6 shows the relative enzyme activity of SEQ ID NOs 1 and 3 respectively. The enzyme activity of SEQ ID NO 1 was set to 100%. Surprisingly, the SEQ ID NO 3 enzyme activity increases >4 fold in comparison to SEQ ID NO 1 enzyme activity.

(9) Enzyme activity: the release of micromol reducing sugars from arabinoxylan per mg of enzyme was determined with the DNSA assay.

(10) FIG. 7 shows the relative enzyme activity of SEQ ID NOs 2 and 4 respectively. The enzyme activity of SEQ ID NO 2 was set to 100%. Surprisingly, the SEQ ID NO 4 enzyme activity increases >7 fold in comparison to SEQ ID NO 1 enzyme activity.

(11) Enzyme activity: the release of micromol reducing sugars from arabinoxylan per mg of enzyme was determined with the DNSA assay.

(12) FIG. 8 shows the XGOS release from 20% (wt/vol) dTKP by 0.006% (wt/wt) SEQ ID NO 3 and 0.03% (wt/wt dTKP) enzymes SEQ ID NO 2. The enzyme reactions were performed at 60° C. Complete hydrolysis was achieved after 24 h for SEQ ID NO 2 and over 80% after 24 h for SEQ ID NO 3.

(13) FIG. 9 shows the XGOS release from dTKP by 0.65% (wt/wt dTKP) SEQ ID NO 3 and 20% TKP substrate. The enzyme reaction was performed at 60° C. The dTKP contains 65% (wt/wt) xyloglucan which is completely degraded to XGOS DP7-9 by SEQ ID NO 3 within 1 h.

(14) FIG. 10 shows the hydrolysis stability of the XGOS mixture produced with the method of the invention: TKP 20% (wt/vol) hydrolysis, 0-24 h at 60° C. with 3% (wt/wt dTPK) SEQ ID NO 3 and subsequent HPAEC-PAD analytic. The HPAEC results were subsequently converted to oligosaccharide ratios (DP7-DP9) over time.

(15) FIG. 11 shows TKP hydrolysis of high substrate concentration (up to 700 g/L) with the method of the invention: 0-72 h with 0.05% SEQ ID NO 3. Degree of hydrolysis was confirmed by HPAEC-PAD analytic.

EXAMPLES

Example 1: Cloning of SEQ ID NOs 11-14 Encoding Endoglucanases and Expression of SEQ ID NOs 1-4

(16) All Chemicals used as buffers and substrates were commercial products of at least reagent grade.

(17) Cloning of SEQ ID NO 2: A new cellulose degrading bacterial strain named Herbivorax saccincola DSM101079 was isolated from a 20 I fermenter operated with cow manure and fed with maize silage at 55° C. Herbivorax saccincola was classified as a new family in the Ruminococcaceae (Koeck et al. 2016). For genome sequencing of the strain Herbivorax saccincola DSM101079, a total of 4 μg genomic DNA was used to construct an 8-k mate-pair sequencing library (Nextera Mate Pair Sample Preparation Kit, Illumina Inc.), which was sequenced applying the paired-end protocol on an Illumina MiSeq system. Analysis and interpretation of the Herbivorax saccincola DSM101079 genome sequence within GenDB and by means of the Carbohydrate-active-enzyme database dbCAN (Yin et al., 2012) revealed more than 100 genes predicted to encode enzymes that mainly belong to different families of Glycoside Hydrolases (GH) and Carbohydrate-Binding Modules (CBM). SEQ ID NO 2 was identified as coding sequence for a glycoside hydrolase family 5 member.

(18) The genes SEQ ID NOs 11 and 12 encoding endoglucanases were produced by recombinant E. coli strains. DNA was amplified using PCR with primers SEQ ID NOs 5-10 and cloned into NdeI/XhoI-linearized pET24c(+) Vector (Novagen, MerckMillipore) under control of the inducible T7-promoter using Gibson Assembly (NEB, Cat. Nr. E2611S). For SEQ ID NO 11 oligonucleotide primers (SEQ ID NO 5) and (SEQ ID NO 7) were used. Variant SEQ ID NO 13, a shortened variant of SEQ ID NO 11, was amplified using oligonucleotide primers (SEQ ID NO 5) and (SEQ ID NO 6). For SEQ ID NO 12 DNA amplification the oligonucleotide primers (SEQ ID NO 8) and (SEQ ID NO 9) were used. Variant SEQ ID NO 14, a shortened variant of SEQ ID NO 12, was amplified using the oligonucleotide primers (SEQ ID NO 8) and (SEQ ID NO 10). Chemically competent E. coli DH10B (Invitrogen, Fisher Scientific, Schwerte, Germany) were transformed. Positive clones were selected by colony PCR using primer combination as described above. For SEQ ID NOs 1-4 protein expression chemically competent E. coli BL21 Star (DE3) (Invitrogen, Fisher Scientific, Schwerte, Germany) were transformed with the respective expression vector.

(19) Growth of Cells

(20) Fed-batch fermentations of recombinant E. coli strains harbouring the endoglucanase genes from C. thermocellum ATCC27405/DSM1237 of SEQ ID NOs 1 and 3, and Herbivorax saccincola DSM101079 gene SEQ ID NO 2 and 4 were carried out in a 10 L Uni-Vessel controlled and equipped with a Biostat B Twin DCU (Sartorius A G, Gottingen, Germany). Temperature, pH, foam, turbidity, weight and dissolved oxygen were monitored online during fermentation. The dissolved oxygen (DO %) was set to 25% (vol/vol) and maintained with increasing agitation and constant air flow. The formation of foam was controlled by the addition of Antifoam 206 (Sigma Aldrich, St. Louis, Mo., USA). A pH of 6.9 was maintained by addition of a 25% (vol/vol) ammonium hydroxide solution and 25% (vol/vol) HPO.sub.4 solution. E. coli strains were cultivated in Riesenberg medium (Korz et al., 1995) at the 10 L scale, the feeding solution consists of 1021 g/L glycerol, 20 g/L MgSO.sub.4.7H.sub.2O, 13 mg/L EDTA, 4 mg/L CoCl.sub.2.6H.sub.2O, 23.5 mg/L MnCl.sub.2.4H.sub.2O, 2.5 mg/L CuCl.sub.2.2H.sub.2O, 5 mg/L H.sub.3BO.sub.3, 4 mg/L Na.sub.2MoO.sub.4×2 H.sub.2O, 16 mg/L Zn(CH.sub.3C00).sub.2.2H.sub.2O, 40 mg/L Fe(III)citrate (Korz et al., 1995).

(21) After the consumption of the initial carbohydrate substrate, growth rate was controlled according to EQUATION 1, whereby ms, is the mass flow of substrate (g h.sup.−1), μ.sub.set the desired specific growth rate (h.sup.−1), Y.sub.X/S the biomass/substrate yield coefficient (g g.sup.−1), m the specific maintenance coefficient (g g.sup.−1 h.sup.−1), V the cultivation volume (L), and X the biomass concentration (g L.sup.−1):

(22) $\begin{array}{cc}{m}_S=\left(\frac{{\mu }_{\mathrm{set}}}{Y_{X\text{/}S}}+m\right).Math.V\left(t\right).Math.X\left(t\right).Math.e^{{\mu }_{\mathrm{set}}\left(t-t_F\right)}& \mathrm{EQUATION}1\end{array}$

(23) The inoculation procedure was the following: Based on a cryo-stock, a fresh agar plate containing adequate antibiotics was prepared. With one colony an Erlenmeyer flask containing 30 mL Lysogeny Broth (Sambrook et al. 1989) was inoculated and incubated for 12 to 15 h at 30° C. 30 mL of this first preculture was used to inoculate 500 mL of the fermentation medium in a 5 L Erlenmeyer flask and incubated for further 14 h. The 10 L fermenter was filled with 6 L fermentation medium and inoculated with 500 mL of the second preculture. Kanamycin was added at 50 μg/mL. Protein production was induced by changing the glycerol feed to lactose feed. Cells were harvested after 48 h by centrifugation for 1 h at 9000 rpm and 22° C. Portions of 300 g cells were solved in 3 L lysis buffer (50 mM MOPS pH 7.3, 0.1 M NaCl, 20 mM imidazol). Cell lysis was achieved by ultrasonic treatment in a ultrasonic flow through chamber. Cell debris was separated by centrifugation (9000 rpm, 22° C.). Supernatant was clarified from residual cells or debris by tangential filtration applying a 0.2 μm filter cassette and three volumes washing with lysis buffer. The enzyme solution was concentrated employing tangential filtration with a 30 kDa filter cassette followed by dialysis with three volumes lysis buffer. GH10 xylanases were purified by immobilized metal ion affinity chromatography (IMAC). Pure enzymes were eluted with elution buffer containing 50 mM MOPS, pH 7.3, 0.25 M imidazole, 0.1 M NaCl, and 20 mM CaCl.sub.2).

(24) Protein expression was monitored by SDS-PAGE using 12.5% gels (Bio-Rad Laboratories GmbH, Muenchen) and Coomassie blue staining. Proteins were resuspended in denaturating buffer and heated for 15 min at 95° C. The PageRuler Prestained Protein Ladder 10 to 180 kDa (#26616, ThermoFisher Scientific) was used as molecular weight standard. The proteins were stained with Coomassie brilliant blue R-250 (Weber and Osbourne 1969). Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis was performed according to Laemmli (1970). The protein amount was determined by using Pierce™ BCA Protein Assay Kit (#23225, ThermoFisher Scientific).

(25) Another possibility producing the endoglucanases SEQ ID NOs 1-4 of the invention is to transform a competent B. subtilis strain with an appropriate vector containing the DNA and cultivate the recombinant strain according to Park et al. 1991.

Example 2: Characterization of SEQ ID NOs 1-4

(26) Specific enzyme activity was determined by using a model substrate such as barley-beta-glucan (Megazyme). Endoglucanase activity is defined as the generation of reducing sugars from the model substrate. One unit of enzyme activity is defined as the amount of reducing sugars in micromol generated per mg of enzyme in one minute at 60° C. To define the enzyme profile and temperature and pH optimum the endoglucanases (50 ng/reaction) were incubated for 30 minutes at a temperature range from 50 to 80° C. in presence of 1% (wt/vol) barley-beta-glucan solved in citrate buffer with varying pH (range 4.0-8.0) (solution A: 0.2 M citric acid, 0.1 M NaCl; solution B: 0.4 M Na.sub.2HPO.sub.4, 0.1 M NaCl). Reducing sugars were measured by 3,5-dinitrosalicylic acid (DNSA) method: 50 microL sample were mixed with 75 microL DNSA-solution (10 g/L DNSA, 200 g/L K.sup.+-Na.sup.+-Tartrate, 10 g/L NaOH, 0.5 g/L Na.sub.2SO.sub.4, 2 g/L Phenol) in microtiter plates, incubated for 5 minutes at 95° C., cooled on ice and the absorption determined at 540 nm. For calibration, glucose solutions between 0 and 2 mg/mL were used.

(27) The temperature and pH profile of SEQ ID NOs 1-4 showed optimum temperatures at about 60° C., pH 5.5-7.5. As one representative enzyme the SEQ ID NO 3 enzyme activity profile is shown in FIG. 4 and demonstrates a broad temperature profile above 50° C. The other SEQ ID NOs 1, 2 and 4 show a similar enzyme profile. As described in example 2 the SEQ ID NOs 11 and 12 represents the wildtype genes. SEQ ID NOs 13 and 14 are variants of SEQ ID NOs 11 and 12 respectively, lacking the dockerin modules thereby reducing the overall protein size by approximately 15% (FIG. 5). Surprisingly compared to the wildtype protein SEQ ID NOs 1 and 2, the specific enzyme activities of SEQ ID NOs 3 and 4 are at least 4,5 fold increased (FIGS. 6 and 7). The activity increase cannot be explained by the size reduction only. All enzymes SEQ ID NOs: 1-4 are characterized by an enzyme activity suitable to hydrolyze TKP at temperatures above 50° C. equally well as shown for SEQ ID NOs 2 and 3 in FIG. 8.

(28) To evaluate the suitability of SEQ ID NOs 1-4 for TKP hydrolysis, the Ki value of SEQ ID NO 3 was determined. Enzymatic activity was measured on Azo-Xyloglucan (Megazyme) according to the manufacturer's instruction. Substrate concentrations of 1-20 g/I were chosen and enzymatic activity was measured in the presence of increasing concentrations of xyloglucan oligosaccharides. Mode of Inhibition was assessed using a Lineweaver-Burke plot. Nonlinear regression fit of experimental data for SEQ ID NO 3 with GraphPad Prism6 using non-competitive inhibition model gave a K.sub.i value of 16.75 mM (±1.35).

Example 3: Hydrolysis of TKP

(29) The tamarind kernel powder (TKP) and deoiled tamarind kernel powder (dTKP) were purchased from Tamarind Magic, Hyderabad, Phase-IV, 3rd Gate, IDA, Cherlapally, Hyderabad—500051, Telangana, India. Both, TKP and dTKP consist of approximately 65% (wt/wt) xyloglucan (https://www.altrafine.com/tamarind-kernel-powder//.

(30) For XGOS release 20% (wt/vol) 8 g of TKP or dTKP was dissolved in 40 ml demineralized water at 60° C. The enzyme was added at the desired concentrations, 3%, 0.65%, 0.05%, 0.03% or 0.006% (wt/wt TKP) referred to the substrate load. Incubation of the reaction mixture was carried out on a rotary shaker at 125 rpm and 60° C. The hydrolysis process was monitored by taking samples at different time points. The samples were centrifuged and the supernatant boiled for 5 min at 95° C. to denature residual proteins. For analysis, samples of the supernatant were withdrawn, tenfold diluted with double deionized water and measured by HPAEC-PAD.

Example 4: Hydrolysis of up to 700 g TKP/I

(31) 25 g/L TKP was dissolved in demineralized water at 60° C. The enzyme was added at the desired concentration of 0.05% related to the final substrate load of 700 g/L. The hydrolysis process was carried out in a 2 L stirred tank reactor (Sartorius AG, Gottingen, Germany) equipped with a three segment impeller agitated between 250 and 500 rpm at 60° C. for 72 h. The substrate was fed with a rate of 25 g/Uh until the final substrate load 700 g/I was achieved. For process monitoring, samples were taken from the reactor at different time points. The samples were centrifuged and the supernatant boiled for 5 min at 95° C. to denature residual proteins. XGOS were isolated in 5 mL samples from the supernatant by alcoholic precipitation as described in example 5 and weighted. For analysis, samples of the supernatant were withdrawn diluted with double deionized water and measured by HPAEC-PAD as described in example 7. A hydrolysis degree over 75% was reached after 24 h and was even further enhanced to over 80% by prolonging the reaction time to 72 h.

Example 5: XGOS Purification Including Ethanol Precipitation

(32) To obtain pure XGOS powder (FIG. 3), hydrolysates from example 3 and 4 were separated by centrifugation to separate the insoluble fraction of TKP/dTKP (9,000 rpm, 20 min, RT). One example to further use the insoluble fraction might be as protein enriched, high fat matrix for animal feed. The supernatant is clarified first by microfiltration (tangential filtration, 0.2 micrometer filter cassettes) and second by ultrafiltration (tangential filtration, 10 kDa filter cassettes). Residual proteins, peptides, fatty acids or pigments were removed by ethanol precipitation with ethanol concentrations up to 90% (vol/vol) and incubation on ice for one hour. The precipitate is removed by centrifugation and the supernatant is concentrated and ethanol recycled by rotary evaporation. The precipitate is dried by lyophilisation, desiccator or spray drying.

Example 6: XGOS Purification without Ethanol Precipitation

(33) To obtain pure XGOS powder (FIG. 3), hydrolysates from example 3 were separated by centrifugation to separate the insoluble fraction of TKP/dTKP (9,000 rpm, 20 min, RT). The supernatant is clarified first by microfiltration (tangential filtration, 0.2 microm filter cassettes) and second by ultrafiltration (tangential filtration, 10 kDa filter cassettes). Concentration of DP7-DP9 was achieved by using (sequential) simulated moving bed chromatography or nanofiltration for larger scale. The filtrates or extracts were dried by lyophilisation, desiccator or spray drying.

Example 7: Oligosaccharide Analytics

(34) For the analysis by HPAEC-PAD an ICS 3000 Dionex chromatography system from Thermo Fisher Scientific (Waltham, USA) equipped with a CarboPac™ PA1 column (4×250 mm) and a PA1-precolumn (4×50 mm) was used. The system was set up using PEEK tubing (0.25 mm i.d.), a GM-4 gradient mixer (2 mm), an ED amperometry cell with 0.25 microL channel volume, a pH-Ag/AgCl reference electrode, 0.002″ gasket and disposable gold electrodes. Runs were performed at a column temperature of 30° C. with an injection volume of 25 microL and a flow rate of 1 mL/min. The eluent gradient used for the analyte separation started at 0 min with 100 mM NaOH and 7.5 mM sodium acetate (NaOAc). The latter was linearly increased to 100 mM at 67.5 min while 100 mM NaOH was maintained. To wash the column, the concentration of NaOAc was increased to 650 mM with 100 mM NaOH for 4 min and subsequently re-equilibrated with 100 mM NaOH for 16.3 min after each run. The carbohydrate detection with the PAD was based on the waveform “standard carbohydrate quad” (Waveform B) which was set to 2 Hz.

(35) Prior to the analysis of the polysaccharide hydrolysates, the samples were diluted with double-distilled water to a final XGOS concentration between 10 and 200 mg/I. The degradation products were identified by comparison to oligosaccharide standards (10-200 mg L.sup.−1 each) as listed above.

(36) Analysis of the hydrolysates by TLC was used for quick analysis of multiple samples in parallel. TLC was performed using silica gel 60 plates from Merck Millipore GmbH (Düsseldorf, Germany) as stationary and acetonitrile-water 8:2 v/v as mobile phase. For staining, the plates were sprayed with 5 mL staining solution (100 mL acetone, 1 g diphenylamine, 1 mL anilin) with 0.5 mL 85% freshly added phosphoric acid. Spraying was done using the DESAGA ChromaJet DS 20 from Sarstedt (Nuermbrecht, Germany). Subsequently, the plates were developed for 20 min at 120° C. The applied volume for each hydrolysate and negative control was 4.5 μL and 1 microL of a 1 microg/μL stock solution for each analyte.

(37) Quantification was performed using a XGOS standard mixture from Megazyme with 0.4 g/L, 0.2 g/L, 0.1 g/L, and 0.05 g/L.

(38) Results: In contrast to the prior art (WO1991011112) enzymatic TKP (20% wt/vol) hydrolysis to the maximum DP7-DP9 amounts is achieved by 0.65% (wt/wt TKP) in less than 1 hour using SEQ ID NO 3 (FIG. 9). Another results was obtained by using SEQ ID NO 2 enzyme concentration below 0.03% (wt/wt TKP) which is sufficient to hydrolyze TKP (20% wt/vol) to DP7-DP9 oligosaccharides completely within 24 h (FIG. 8). Further reduction of SEQ ID NO 3 enzyme concentration to 0.006% (wt/wt TKP) leads to a sufficient hydrolysis over 80% within 24 hours (FIG. 8).

Example 8: Peak Area Determination

(39) 20% (wt/wt) TKP was hydrolyzed for 24 hours with 3% (wt/wt TKP) SEQ ID NO 3 in accordance with example 3. Samples were taken after 15 min, 30 min, 45 min, 1 h, 2 h, 3 h, 4 h, 8 h, and 24 h and analyzed using HPAEC-PAD described in example 7. Peak areas (nC*min) for the produced oligosaccharides were determined for DP7, DP8 and DP9 oligosaccharides separately and the percentage of each oligosaccharide was determined.

(40) Result: Once the TKP is hydrolyzed completely the ratio between the DP7, DP8 and DP9 oligosaccharides is is stable and does not change with prolonged reaction times. In contrast to the prior art (WO1991011112) we do not observe any further oligosaccharide degradation causing unwanted monosaccharides or loss of XGOS yield (FIG. 10). Even using a very high enzyme concentration (3%) and complete TKP hydrolysis reached after less than an hour the ratio between DP 7, 8 and 9 oligosaccharides is stable over time (FIG. 10).

Example 9: Detection of Monosaccharides

(41) The polysaccharide xyloglucan has a backbone of beta1.fwdarw.4-linked glucose residues, most of which are substituted with 1,6 linked xylose sidechains. The xylose residues are in many cases decorated with galactose. To analyze possible further xyloglucan hydrolysis to monosaccharides we specifically analyzed the content of monomeric glucose and galactose after enzymatic hydrolysis.

(42) Residual galactose in XGOS powder was determined using a Lactose/D-Galactose detection kit as described by the manufacturer (Megazyme, Ireland). Measurement was performed with a Perkin Elmer Lambda 35 UV/Vis spectrophotometer at 340 nm. A 20% (wt/vol) purified XGOS solution, whereby the XGOS was produced as described in example 3 and purified as described in example 6 with 0.05% SEQ ID NO 3, was used to determine the galactose concentration. The galactose concentration in the XGOS solution was determined by inferring the galactose concentration from a standard curve prepared with galactose. The apparent galactose content in the preparation was 72 ppm.

(43) Residual glucose in the XGOS preparation was determined using a coupled enzymatic assay, with Aspergillus niger glucose oxidase (Type VII, Sigma Aldrich) and horseradish peroxidase (type VI, Sigma Aldrich) as described by (Kovacevic et al. 2014). The assay was performed in 0.1 M sodium acetate buffer pH 5.5 containing 2 mM ABTS, 1.3 U/mL glucose oxidase and 0.5 U/mL horseradish peroxidase using a 20% (wt/vol) purified XGOS solution as sample. Change of absorbance at 410 nm was followed in a Tecan Sunrise microplate reader. No glucose was detected in the 20% (wt/vol) purified XGOS solution. Control experiment: Through addition of glucose to the same TKP solution the minimal detectable glucose concentration in the 20% (wt/vol) purified XGOS solution was determined to be 54 ppm. Hence residual glucose content in the XGOS preparation is below 54 ppm.

(44) In contrast to the prior art (WO1991011112) the described hydrolysis using enzymes SEQ ID NOs 1-4 process does not produce monosaccharides.

Example 10: Metabolic Stability XGOS

(45) Metabolic stability of the oligosaccharide was assessed using the integrated total dietary fiber assay kit (Megazyme, Ireland). XGOS samples were digested with enzymes as described in AOAC Method 2011.25 and oligosaccharide composition was analyzed after enzymatic treatment as described in example 7 using HPAEC-PAD. Xyloglucan oligosaccharides were resistant to hydrolysis by porcine pancreatic amylase and amyloglucosidase, qualifying XGOS as soluble dietary fiber.

Example 11: Water Content aW-Value

(46) The water activity (aw value) was determined using a AW device from Sprint from Novasina (Lachen, Switzerland) at 25° C. The water content of oligosaccharide purified without ethanol precipitation was 0.311. The aw XGOS value prepared including ethanol precipitation was 0.327.

Example 12: XGOS Stability Against Acid Treatment

(47) For food and feed products, acid stability is necessary. To test the stability of DP7-DP9 XGOS toward acids, 10% (wt/vol) XGOS were solved in water (pH 7.0), incubated for 2 h at 37° C. and compared to the same amount of XGOS treated with 10 mM HCl (pH 2.0) and incubated at the same conditions. The analysis was performed by HPAEC-PAD. There were no differences in oligosaccharide composition between the pH 7.0 and pH 2.0 samples observed.

Example 13: XGOS Stability Against Heat

(48) For pelleting, extruding or pasteurization processes, heat stability is necessary. To test the stability of DP7-DP9 XGOS toward heat 10% (wt/vol) XGOS were solved in water (pH 7.0), incubated for 10 min at 97° C. and compared to the same amount of untreated XGOS. The analysis was performed by HPAEC-PAD. After heat treatment a light precipitation was observed, which didn't affect XGOS solubility. Heat treatment didn't alter the composition of DP7-DP9 XGOS and equal amounts of XGOS were detected in heat treated and non-treated samples. No increase of mono- or disaccharides could be detected. The 10% (wt/vol) XGOS solution was incubated for 20 min at 121° C. and 2 bar and more than 80% XGOS were left in the supernatant. The XGOS composition was not altered. Furthermore, the monomer fraction did not increase.

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