PREPARATION COMPRISING ARABINOXYLO-OLIGOSACCHARIDES

Abstract

The present invention related to an arabinoxylo-oligosaccharide composition comprising at least one arabinose unit linked to one of the xylose units of the backbone, per molecule, wherein the at least one arabinose unit is an -L-arabinofuranosyl, wherein said composition has an xylo-oligosaccharide backbone with a degree of polymerization of 1-10.

Claims

1-23. (canceled)

24. Process for producing an arabinoxylo-oligosaccharide composition from flour or bran comprising the steps of: if flour, (i) extracting and isolating an endosperm arabinoxylan fraction from flour, (ii) optionally removing starch and proteins and recovering a solid phase, and (iii) optionally treat of the endosperm arabinoxylan or solid phase with arabinofuranosidases, preferably one able to remove -(1.fwdarw.3)-linked L-arabinofuranosyl at double substituted -(1.fwdarw.4)-linked D-xylopyranosyl units (dXyl); if bran, (i) removing starch and proteins and recovering a solid phase, (ii) treating the solid phase with alkaline solution, alkaline and peroxide solution or treating the solid phase with heat to provide a soluble phase, (iii) neutralizing the soluble phase which comprises arabinoxylan and recovery of said soluble phase comprising arabinoxylan, (iv) removing arabinose from the arabinoxylan containing soluble phase in step D using arabinofuranosidases or a weak acid solution to obtain a molar ratio of arabinose to xylose of 0.2-0.7, and (v) separating to recover arabinoxylan; adding an arabinoxylanase to the obtained product of step A or step B; and drying the obtained material of step D.

25. Process for producing an arabinoxylo-oligosaccharide composition according to claim 24, wherein the arabinoxylo-oligosaccharide composition is produced using an arabinoxylan specific endoxylanase.

26. Process for producing an arabinoxylo-oligosaccharide composition according to claim 24, wherein arabinoxylo-oligosaccharide composition is produced using an arabinoxylan specific endoxylanase and the arabinoxylan specific endoxylanase is arabinoxylanase.

27. Process for producing an arabinoxylo-oligosaccharide composition according to claim 24, wherein step (B)(ii) includes an optional treatment with arabinofuranosidases to increase the yield of arabinoxylo-oligosaccharides.

28. Process for producing an arabinoxylo-oligosaccharide composition according to claim 24, wherein the step (B)(I) includes removal of starch and proteins with amylases and proteases respectively.

29. Process for producing an arabinoxylo-oligosaccharide composition according to claim 24, wherein the step (B)(ii) includes extraction with alkali and peroxide,

30. Process for producing an arabinoxylo-oligosaccharide composition according to claim 29, wherein the step (B)(ii) includes steam treatment to increase the water soluble arabinoxylan content.

31. Process for producing an arabinoxylo-oligosaccharide composition according to claim 24, wherein the step (B)(iv) includes an optional treatment with arabinofuranosidases or a weak acid to increase the yield of arabinoxylo-oligosaccharides.

32. Process for producing an arabinoxylo-oligosaccharide composition according to claim 24, wherein the separating of step (B)(iv) is by precipitation or membrane separation.

33. Process for producing an arabinoxylo-oligosaccharide composition according to claim 24, wherein the obtained molar ratio of arabinose to xylose in step (B)(iv) is 0.35-0.5.

34. Process for producing an arabinoxylo-oligosaccharide composition according to claim 24, wherein the obtained molar ratio of arabinose to xylose in step (B)(iv) is 0.38-0.45.

35. Process for producing an arabinoxylo-oligosaccharide composition according to claim 24, wherein the obtained molar ratio of arabinose to xylose in step (B)(iv) is about 0.4.

Description

SHORT DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1. AXOS generated by an arabinoxylanase are different from the XOS and AXOS mixtures obtained by family 10 and 11 xylanases indicated by the different products obtained as seen by the different retention times (Table 1).

[0042] FIG. 2. Lower chromatogram: Major AXOS generated from wheat endosperm AX (peaks 1-13) and upper chromatogram: arabinofuranosidase treated sample to expose the xylose and XOS backbone. X: xylose, X.sub.2: xylobiose, X.sub.3: xylotriose, X.sub.4: xylotetraose, X.sub.5: xylopentaose, X.sub.6: xylohexaose, X.sub.7: xyloheptaose and X.sub.8: xylooctaose.

[0043] FIG. 3. Lower chromatogram: Major AXOS generated from rye endosperm AX (peaks 1-13) and upper chromatogram: HCl treated sample to expose the xylose and XOS backbone. X: xylose, X.sub.2: xylobiose, X.sub.3: xylotriose, X.sub.4: xylotetraose, X.sub.5: xylopentaose, X.sub.6: xylohexaose, X.sub.7: xyloheptaose, X.sub.8: xylooctaose, X.sub.9: xyloenneaose and X.sub.10: xylodecaose.

[0044] FIG. 4. Optimal yield of arabinoxylanase AXOS are in the range of 0.35-0.61 as indicated by the highest yield at 0.43.

[0045] FIG. 5. Upper chromatogram: Improved generation of AXOS (white arrows) in XOS and AXOS containing mixtures with addition of an arabinoxylanase as indicated as a shift in retention times towards shorter oligosaccharides and disappearance of AXOS peaks (black arrows) in original composition (lower chromatogram).

[0046] FIG. 6. Bifidobacterium adolescentis utilization of arabinoxylanase derived AXOS from rye endosperm AX as indicated by the disappearing peaks 1-6.

[0047] FIG. 7. Lactobacillus brevis does not utilize arabinoxylanase derived AXOS from rye endosperm AX as indicated by the remaining peaks 1-6.

DETAILED DESCRIPTION OF THE INVENTION

[0048] Arabinoxylanases are unique in their specificity for AX since they do not attack unsubstituted xylans. The oligosaccharides generated by these enzymes contain at least one (1.fwdarw.3) Araf group linked to a reducing end Xylp unit. This group of enzymes have not previously been used or considered in the production of prebiotic AXOS from AX or AX containing materials. In the present invention an arabinoxylanase is used to produce AXOS from AX containing materials. These AXOS preparations obtained by the arabinoxylanase are unique prebiotics in their AXOS composition and lack of xylose and XOS. Comparison with state of the art xylanases used to make prebiotics from AX clearly show the difference in hydrolysis products obtained (FIG. 1 and Table 1). Production of AXOS is further optimized by choosing an A/X ratio in the interval of 0.2-0.7 preferably 0.28-0.65, preferably 0.35-0.5, preferably 0.38-0.45, preferably 0.4. The AXOS preparations are particularly useful as selective prebiotics for certain groups of intestinal bacteria belonging to the group of bifidobacteria adapted to ferment AXOS or the arabinose substituents attached to the AXOS molecule(s).

TABLE-US-00001 TABLE 1 Peak retention times for FIG. 1 Retention time (min) Peak RmXyn10A - Arabinoxylanase - Pentopan - number GH10 GH5 GH11 1 2.659 (Xylose) 3.033 2.667 (Xylose) 2 3.684 (Xylobiose) 3.317 3.700 (Xylobiose) 3 4.284 4.683 5.609 (Xylotriose) 4 5.617 6.867 11.059 5 11.342 8.325 12.759 6 12.109 9.675 13.325 7 12.309 10.95 14.075 8 13.117 11.442 14.592 9 18.509 11.942 19.284 10 20.242 12.8 20.342 11 20.892 13.475 21.475 12 21.409 14.075 22.325 13 21.909 14.817 23.209 Note: Xylo-oligosaccharide strandards retention time: Xylose: 2.667; Xylobiose: 3.700; Xylotriose: 5.600; Xylotetraose: 7.825; Xylopentaose: 9.359 and Xylohexaose: 10.384.

[0049] In the first example AXOS are generated from endosperm (flour) AX from but not limited to wheat and rye. The endosperm AX is optionally enzymatically treated with arabinofuranosidases to remove a fraction of the Araf groups in order to improve the yield of AXOS. Pure AXOS generated from endosperm AX is shown in FIG. 2 for wheat and in FIG. 3 for rye. This data shows that there are no xylose or XOS formed in the preparations (less than 0.1% on dry weight basis). Only after removing all Araf groups attached to the obtained AXOS with an arabinofuranosidase or hydrochloric acid are the xylose and XOS backbones exposed. This confirms that all the obtained oligosaccharides are AXOS with no or very little formation of xylose and XOS. From the analysis of the XOS backbone after removing all Araf groups it is determined that the backbone degree of polymerisation (DP) of the AXOS is 1-8 for wheat endosperm AX (FIG. 2) and 1-10 for rye endosperm AX (FIG. 3). The majority of AXOS from wheat and from rye endosperm AX had an average backbone of 3 (Table 2).

TABLE-US-00002 TABLE 2 Xylose and XOS content in arabinofuranosidase or HCl treated AXOS samples from wheat and rye respectively presented as molar percentage Wheat endosperm AX Rye endosperm AX XOS backbone (A/X 0.43) (A/X 0.64) Xylose 16% 20% Xylobiose 10% 15% Xylotriose 29% 17% Xylotetraose 26% 23% Xylopentaose 13% 16% Xylohexaose 6% 10%

[0050] Further was the impact of the arabinose content in the AX substrate determined for the generation of AXOS by an arabinoxylanase. The highest yield of AXOS obtained from AX was achieved using an A/X of 0.43 (FIG. 4) demonstrating that the optimal generation of AXOS is in the interval of A/X=0.61-0.35 with 0.43 closest to the optimal. Significance of this is in the optimized production of AXOS by partially removing Araf groups from the AX substrate prior or during the arabinoxylanase treatment.

[0051] Another application of the technology is demonstrated in improved generation of AXOS in mixtures containing both XOS and AXOS. By adding an arabinoxylanse to an (A)XOS mixture from a family 11 xylanase new AXOS are formed by degrading poly- and oligosaccharides not hydrolysed by the family 11 xylanase (FIG. 5). These XOS and AXOS mixtures can be obtained using state of the art xylanase treatments and the addition of the arabinoxylanase is a way to improve the generation AXOS in such preparations. Preparations similar to the ones mentioned in EP 2 265 127 B1 to improve the prebiotic properties of such products containing XOS and AXOS.

[0052] In the second example wheat bran is used as a substrate to make different fractions of AX suitable for making AXOS by an arabinoxylanase. The fractions are isolated from bran material by first removing starch and proteins followed by an extraction of the AX components from the bran material. The AX is then subsequently treated enzymatically with an arabinofuranosidase, or acid treated to obtain fractions with different A/X ratios (Table 3) that could be used to make different AXOS compositions using an arabinoxylanase.

[0053] In the third example it is demonstrated that the obtained AXOS can be used to selectively stimulate the growth of bifidobacteria over other groups of intestinal bacteria that normally can use xylose or XOS (FIGS. 6 and 7). The reason is that the AXOS obtained do not contain neither xylose nor XOS that normally could be utilized by e.g. Lactobacillus brevis. There is also a difference in the carbohydrate preferences between different bifidobacteria strains which means that the AXOS can be used to stimulate certain strains of bifidobacteria. Strains from either cluster II, III, IV and V could be selectively stimulated with preparations containing only AXOS.

[0054] Representative strains from cluster II-V are but not limited to the following strains of bifidobacteria: [0055] Bifidobacterium longum subsp. longum DSMZ 20219 (Cluster 2) [0056] Bifidobacterium adolescentis DSMZ 20083 (Cluster 3) [0057] Bifidobacterium longum subsp. longum CCUG 15137 (Cluster 4) [0058] Bifidobacterium catenulatum DSMZ 16992 (Cluster 5)

[0059] Especially strains belonging to cluster 4 and 5, are able to efficiently utilize the entire AXOS and are of special interest to combine with the obtained AXOS. However, all bifidobacteria, able to cleave the arabinose substituents present on AXOS or utilize the entire AXOS, are possible to stimulate.

EXAMPLES

Example 1: Preparation of Arabinoxylanase AXOS

Materials and Methods

[0060] Arabinoxylanase from Clostridium thermocellum (CtXyl5A) was purchased from Nzytech (Lisboa, Portugal). A family 10 xylanase from Rhodothermus marinus (RmXyn10A) was prepared as described in Falck et al. (2013). Pentopan mono bg, a commercial family 11 xylanase was obtained from Novozymes (Bagsvaerd, Denmark). High purity recombinant -L-arabinofuranosidase (E-ABFCJ) from Cellvibrio japonicus was purchased from Megazyme (Wicklow Ireland). Endosperm AX extracted by alkali from wheat (P-WAXYM, P-EDWAX30, P-ADWAX26, P-ADWAX22) and rye (P-RAXY) were purchase from Megazyme. AX substrates were dissolved 10 g/L according to manufactures instructions in 50 mL MQ water and the pH was adjusted to 7 with 8M HCl. Arabinoxylanase from family 5 and xylanases from family 10 and 11 were added at an enzyme to substrate ratio of 1:1000 on a mass basis. In the arabinoxylanase reactions 2 mM CaCl.sub.2 was used to stabilize the enzyme. All reactions were performed at 50 C. for 24 h using either a thermoblock or water bath. Enzymes were inactivated by incubating the sample at 95 C. for 30 minutes.

[0061] The comparison between the arabinoxylanase and family 10 and 11 xylanases (FIG. 1) was performed using wheat endosperm AX (P-EDWAX30) with 30% arabinose content equal to an A/X of 0.43. The AX had been treated with an arabinofuranosidase from Bacteroides ovatus to remove all -(1.fwdarw.3)-linked Araf at double substituted Xylp units (dXyl). Characterization of the AXOS backbones of xylose and XOS in the arabinoxylanase sample (FIG. 2) was performed with 0.5 U/(mg AXOS) using an arabinofuranosidase (E-ABFCJ) removing Araf from (1.fwdarw.2) or (1.fwdarw.3) single substituted Xylp units, mXyl2 and mXyl3 respectively. The reaction was performed at pH 5.8 using a 20 mM sodium phosphate buffer at 50 C. for 24 h. The treatment resulted in a complete removal of Araf from the AXOS (FIG. 2). Rye endosperm AX was treated in the same way as wheat with the exception that the arabinoxylanase reaction was performed for 48 hours. To remove all single and double substituted Araf groups from the obtained AXOS a weak acid treatment was used. The pH was set to 2.8 with a diluted HCl solution and the sample (5 mL) was incubated at 90 C. for 24 h resulting in an almost complete removal of all Araf groups from the AXOS (FIG. 3).

[0062] The relation between arabinose content and the yield of arabinoxylanase generated AXOS was determined using wheat endosperm with different arabinose content. P-WAXYM, P-EDWAX30, P-ADWAX26 and P-ADWAX22 with an arabinose content of 38%, 30%, 26% and 22% percent respectively or based on A/X 0.61, 0.43, 0.35 and 0.28 respectively (FIG. 4). The reactions were performed in glass vials using 2 mL reaction volumes and a substrate concentration of 0.2 g/L substrate concentration. Arabinoxylanase was used to treat pentopan generated (A)XOS to generate more and shorter AXOS in the XOS and AXOS mixtures (FIG. 5) using the same reaction conditions as described previously for the xylanase reactions.

Characterization of AXOS and XOS

[0063] Analysis of the obtained AXOS fractions and XOS backbones was done by High-Performance Anion-Exchange Chromatography Coupled with Pulsed Electrochemical Detection (HPAEC-PAD) using (ICS-5000) using a CarboPac PA200 column (250 mm3 mm, 5.5 m) and a guard column (50 mm3 mm) of the same material and a mobile phase of 100 mM NaOH at 0.5 mL/min and a linear gradient (0-30 min) of 0-120 mM of sodium acetate (Sigma). Monosaccharide and xylooligosaccharide standards used were as follows: arabinose and xylose (Sigma), xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose (Megazyme). All samples were filtered through a 0.22 m filter and diluted to a final concentration of 0.2 g/L before analysis.

Example 2: Preparation of AX Substrates with Different A/X Ratios from Bran

Materials and Methods

[0064] Commercial wheat bran (Lantmnnen Mill Malm, Sweden) was used as starting material for the preparation of AX with different arabinose content defined as A/X. A suspension (1:9 w/v) of 250 g wheat bran in 2.5 L DI water was adjusted to pH 6.0 with HCl 8 M and treated with a thermostable -amylase 0.12 U/g (Thermamyl, SIGMA-ALDRICH) for 90 min at 90 C. to hydrolyse the starch. The bran was then rinsed with hot tap water to remove solubles until a clear permeate was obtained. A new suspension in water (1:9 w/v) was prepared to remove proteins by incubating with a protease 0.035 U/g (Neutralse 0.8 L, SIGMA-ALDRICH) for 4 h at 50 C. Thereafter the bran was rinsed with hot tap water, then with DI water and then vacuum dried. Destarched and deproteinised wheat bran was extracted with a dilute alkaline solution (NaOH) of hydrogen peroxide containing 2% hydrogen peroxide at pH 11.5 for 4 h at 60 C. under 200 rpm stirring to obtain soluble AX. Antifoam TRITON X-100 was added to reduce foaming. After the extraction solids were removed by filtration and the solution was centrifuged (SIGMA) 6000 g for 20 min. The supernatant was neutralized with 8 M HCl and horseradish peroxidase was added to remove remaining hydrogen peroxide. The extract was centrifuged again at 6000 g for 20 minutes. The supernatant was divided and 50 mL was adjusted to pH 6 with 8M HCl and treated with 5 U of an arabinofuranosidase from Bifidobacterium adolescentis (Megazyme, E-AFAM2) by incubating the sample at 37 C. for 24 h. Supernatant was also acid debranched by a weak HCl acid at pH 2.5 at 90 C. on a magnetic plate stirrer at 200 rpm. Samples (50 mL) were removed and neutralized with 1M NaOH after 3.4, 5.1, 6.8, and 8.6 h. All fractions were desalted by dialysis bags (SpectrumLab, USA) using a 3500 Da Mw cut off. Dialysis was performed in 5 L DI water twice and then all samples were freeze dried.

Characterization of the Isolated Preparations

[0065] The monosaccharide composition of the AX fractions were analysed by HPAEC-PAD after hydrolysing the samples with 2 M TFA for 60 min at 110 C. Total arabinoxylan content in the samples were calculated as 0.88 times (% arabinose+% xylose) after subtracting any free arabinose.

[0066] Analysis of the obtained monosaccharides was done by HPAEC-PAD using a CarboPac PA20 column (250 mm3 mm, 5.5 m) and a guard column (30 mm3 mm) of the same material and a mobile phase of 0.75 mM NaOH at 0.5 m L/min with a post column addition of base of 100 mM at 0.15 m L/min. Monosaccharide (SIGMA) were as follows: arabinose, galactose, glucose and xylose. The resulting A/X fractions obtained are listed in Table 3.

TABLE-US-00003 TABLE 3 Carbohydrate composition (w/w of total carbohydrates) and A/X of AX fractions obtained from wheat bran Fraction Arabinose Glucose Xylose A/X Supernatant 0.42 0.02 0.57 0.73 Abf. 0.38 0.02 0.60 0.63 HCl 3.4 h 0.37 0.02 0.61 0.60 HCl 5.1 h 0.33 0.02 0.64 0.52 HCl 6.8 h 0.31 0.02 0.67 0.47 HCl 8.6 h 0.29 0.02 0.69 0.41 Note: Abfarabinofuranosidase treated sample

Example 3: Selective Growth of Intestinal Bacteria on Arabinoxylanase AXOS

Materials and Methods

[0067] The bacterial strains used to test the fermentability of the obtained AXOS from rye endosperm AX were Bifidobacteria adolescentis (B. adolescentis) ATCC 15703 and Lactobacillus brevis (L. brevis) DSMZ 1269. B. adolescentis, L. brevis, were all pre-cultivated twice using 5 g/L glucose as carbon source. B. adolescentis was inoculated in Bifidobacterium medium at 37 C. and pH 6.8. The medium contained 12.5 g of casein peptone, tryptic digest, 6.25 g of yeast extract, 6.25 g of meat extract, 6.25 g of bacto soytone, 2.5 g of K.sub.2PO.sub.4, 0.25 g of MgSO.sub.4.7H.sub.2O, 0.0625 g of MnSO.sub.4.H.sub.2O, 6.25 g of NaCl, and 1.25 mL of Tween 80 per litre, respectively. To this solution was added 5 mL of solution with resazurin (25 mg/100 mL) together with 50 mL of salt solution containing 0.25 g of CaCl.sub.2.H.sub.2O, 0.5 g of MgSO.sub.4.7 H.sub.2O, 1 g of K.sub.2HPO.sub.4, 1 g of KH.sub.2PO.sub.4, 10 g of NaHCO.sub.3, and 2 g of NaCl per litre, respectively. The medium was subsequently boiled followed by cooling under N.sub.2 gas. Cysteine was added to a concentration of 0.625 g/L and adjusted to pH 6.8 using NaOH. L. brevis was grown anaerobically in MRS broth at pH 6.5 under anaerobic condition at 37 C. All media for the cultivation experiments, broth as well as agar, were autoclaved at 121 C. for 15 min. All cultivation media used for anaerobic growth were deaerated by replacing the oxygen in the anaerobic tubes with nitrogen gas. Then, all tubes were closed with metal caps and autoclaved at 121 C. for 15 min. The respective carbon sources glucose and AXOS were filter sterilized through a 0.45 m filter and added to the media at a final concentration of 5 g/L and a total volume of 5 mL. The fermentation experiment started from the second pre-culture using 2% vol./vol. inoculum and samples were withdrawn after 24 and 48 h. Optical density and pH was measured after 0, 24 and 48 h, while consumption of oligosaccharides was analysed after 48 hours using HPAEC-PAD with the same conditions as described for the oligosaccharide analysis. B. adolescentis could grow on the arabinoxylanase AXOS produced from rye endosperm AX while L. brevis could not due to the fact that the preparation does not contain any xylose or XOS molecules (FIGS. 6 and 7 respectively).

TABLE-US-00004 TABLE 4 Net change in optical density and pH after 48 h B. adolescentis L. brevis OD: 0.2 OD: 0.0 pH: 0.56 pH: 0.0

REFERENCES

[0068] FALCK, P., PRECHA-ATSAWANAN, S., GREY, C., IMMERZEEL, P., STALBRAND, H., ADLERCREUTZ, P., NORDBERG KARLSSON, E. 2013. [0069] Xylooligosaccharides from hardwood and cereal xylans produced by a thermostable xylanase as carbon sources for Lactobacillus brevis and Bifidobacterium adolescentis. Journal of Agricultural and Food Chemistry 61, 30, 7333-7340. [0070] SWENNEN, K., COURTIN, C. M., LINDEMANS, G. C., & DELCOUR, J. A. 2006. Large scale production and characterisation of wheat bran arabinoxylooligosaccharides. Journal of the Science of Food and Agriculture, 86, 1722-1731.