Low molecular weight arabinoxylans with branched oligosaccharides

Abstract

The present invention relates to a composition comprising Low Molecular Weight-Arabinoxylan (LMW-AX) with branched oligosaccharides, preferably at least one branched oligosaccharides is positioned at or adjacent to a reducing end of the LMW-AX backbone. The present invention also relates to the production and use thereof. The present invention further relates to a composition comprising Low Molecular Weight-Arabinoxylan (LMW-AX) with oligosaccharides, where a fraction of the Araf units have been removed to improve the yield of oligosaccharides.

Claims

1. A process for the production of at least one of water-soluble Arabinoxylan, branched Low Molecular Weight-Arabinoxylan or Low Molecular Weight-Arabinoxylan comprising the steps of: A. Isolating starting material comprising a fiber fraction from an ethanol process; B. Increasing the concentration of Arabinoxylan in the fiber fraction of step A, by a mechanical process, obtaining a fiber fraction with increased content of Arabinoxylan; C. Treating and extracting the fiber fraction with increased Arabinoxylan of step B with an alkaline or alkaline hydrogen peroxide solution, obtaining a solution comprising cellulosic solids and water-soluble Arabinoxylan; D. Separating the cellulosic solids of step C from extraction liquid containing water-soluble Arabinoxylan to obtain a step D extraction liquid followed by neutralizing, washing and drying the cellulosic solids; E. Optionally repeating extraction of additional fiber fraction re-using the step D extraction liquid, to obtain step E extraction liquid, and optionally repeating step E using the last step E extraction liquid; F Reducing the pH, with CO.sub.2, of the step D extraction liquid or, if applicable, step E extraction liquid; G. Treating the step D extraction liquid or, if applicable, step E extraction liquid with a calcium salt to precipitate impurities and removing precipitate; H. Using precipitation or filtration to recover water-soluble Arabinoxylan; I. Treating the water-soluble Arabinoxylan with one or more enzymes comprising Arabinoxylan specific endoxylanase to make or Low Molecular Weight-Arabinoxylan or branched Low Molecular Weight-Arabinoxylan; and J. Concentrating or drying the Low Molecular Weight-Arabinoxylan products from H and/or I, wherein the Arabinoxylan specific endoxylanases specifically hydrolyze substituted regions on an Arabinoxylan molecule to produce (1.fwdarw.3) linked disaccharides and optionally branched poly- or oligo-saccharides as end products, wherein the (1.fwdarw.3) linkages of the disaccharides comprise linkages between one arabinose and a xylose unit and optionally between two xylose units.

2. The process according to claim 1, wherein the starting material is a fiber isolated from at least one of a fiber containing kernel, fiber containing wet process streams or dry Dried Distillers Grains with Solubles (DDGS).

3. The process according to claim 1, wherein the starting material is obtained from a cereal ethanol plant.

4. The process according to claim 1, wherein a portion of arabinoxylan having lower substitution is precipitated by acidification of an alkali extraction.

5. The process according to claim 1, wherein the water-soluble Arabinoxylan is obtained using a pre-treatment comprising at least one of HCl, H.sub.2SO.sub.4, NaOH, Ca(OH).sub.2, NH.sub.4, and alkaline hydrogen peroxide.

6. The process according to claim 5, wherein any cellulosic solids remaining after step D or step E of the process is reintroduced into the starting material.

7. The process according to claim 6, wherein the process is for production of feed products.

8. The process according to claim 1, comprising a fiber processing unit including a reaction unit adapted for alkali extraction and neutralization, or autohydrolysis, or acid hydrolysis and neutralization, or cavitation, or steam explosion, or milling; and a separation unit adapted for at least one solid/liquid separation.

9. The process according to claim 6, wherein the pre-treatment is repeated at least two times.

10. The process according to claim 1, wherein step D extraction liquid or, if applicable, step E extraction liquid containing water-soluble Arabinoxylan is purified by precipitation.

11. The process according to claim 2, wherein the starting material comprises cereal comprising substituted Arabinoxylan.

12. The process according to claim 1, wherein the Arabinoxylan substrate for the Arabinoxylan specific endoxylanase is a water-soluble Arabinoxylan obtained using a pre-treatment of the fiber with alkali, alkaline hydrogen peroxide, acid, autohydrolysis, steam explosion, milling, cavitation or enzymes.

13. The process according to claim 12, wherein the pre-treatment is repeated three or more times.

14. The process according to claim 10, wherein step D extraction liquid or, if applicable, step E extraction liquid containing water-soluble Arabinoxylan is purified by precipitation with calcium salts.

15. The process according to claim 14, wherein the calcium salts comprise calcium hydroxide and/or calcium chloride.

16. The process according to claim 14, wherein the purity of water-soluble Arabinoxylan after calcium salt precipitation and filtration or precipitation is at least 50%.

17. The process according to claim 14, wherein the purity of water-soluble Arabinoxylan after calcium salt precipitation and filtration or precipitation is at least 80%.

18. The process according to claim 14, wherein the purity of water-soluble Arabinoxylan after calcium salt precipitation and filtration or precipitation is at least 90%.

19. The process according to claim 14, wherein the purity of water-soluble Arabinoxylan after calcium salt precipitation and filtration or precipitation is at least 95%.

20. The process according to claim 14, wherein the purity of water-soluble Arabinoxylan after calcium salt precipitation and filtration or precipitation is at least 99%.

Description

SHORT DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Molecular structures of molecules derived by hydrolysis of arabinoxylans A) Arabinoxylan-oligosaccharides which is a mixture between xylo-oligosaccharides and arabinoxylo-oligosaccharides obtained by commercial xylanases from family 10 or 11, B) disaccharides that are (1.fwdarw.3) linked xylose-xylose or arabinose-xylose generated by an arabinoxylan specific endoxylanase from corn fiber arabinoxylan, C) branched oligosaccharide from corn fiber arabinoxylan generated by an arabinoxylan specific endoxylanase and D) branched oligosaccharide from wheat bran arabinoxylan generated by an arabinoxylan specific endoxylanase.

(2) FIG. 2. Flow chart describing the separation of fibers from kernels, fiber bypass or fiber separation by wet fractionation or from dry fractionated fiber from DDGS. These fibers are then fed to a fiber-processing unit where LMW-AX with branched oligosaccharides is produced as the main product. The cellulosic solid residue after fiber processing is optionally returned to a stream for feed while a fraction of LMW-AX with branched oligosaccharides can be added to introduce prebiotic properties in novel feed product.

(3) FIG. 3. Flow chart describing the fiber-processing operations in connection to an ethanol process to obtain LMW-AX with branched oligosaccharides.

(4) FIG. 4. A) Picture of corn stillage sample, B) picture of fractionated fiber from corn stillage sample, C) freeze dried LMW-AX with branched oligosaccharides from corn AX and D) freeze dried cellulosic residue after alkaline hydrogen peroxide extraction of corn fiber.

(5) FIG. 5. HPAED-PAD chromatogram showing oligosaccharides present in samples A) corn arabinoxylan hydrolyzed with 3 different xylanases and B) branched oligosaccharides obtained from corn arabinoxylan by an arabinoxylan specific endoxylanase CtXyl5A before and after incubation with arabinofuranosidases. Standards are A: arabinose, X: xylose, X.sub.2: xylobiose, X.sub.3: xylotriose, X.sub.4: xylotetraose, X.sub.5: xylopentaose, X.sub.6: xylohexaose, A.sup.2XX: α-(1.fwdarw.2)-arabinofuranosyl-O-(1.fwdarw.4)-xylopyranosyl-β-(1.fwdarw.4)-xylopyranosyl-β-(1.fwdarw.4)-xylopyranosyl, A.sup.3X: α-(1.fwdarw.3)-arabinofuranosyl-β-(1.fwdarw.4)-xylopyranosyl-β-(1.fwdarw.4)-xylopyranosyl.

(6) FIG. 6. HPAED-PAD chromatogram showing oligosaccharides present in the different SEC fractions obtained from wheat bran LMW-AX with branched oligosaccharides that was produced by an arabinoxylan specific endoxylanase CtXyl5A. Standards are A: arabinose, X: xylose, X.sub.2: xylobiose, X.sub.3: xylotriose, X.sub.4: xylotetraose, X.sub.5: xylopentaose, X.sub.6: xylohexaose, A.sup.2XX: α-(1.fwdarw.2)-arabinofuranosyl-β-(1.fwdarw.4)-xylopyranosyl-β-(1.fwdarw.4)-xylopyranosyl-β-(1.fwdarw.4)-xylopyranosyl, A.sup.3X: α-(1.fwdarw.3)-arabinofuranosyl-β-(1.fwdarw.4)-xylopyranosyl-β-(1.fwdarw.4)-xylopyranosyl.

(7) FIG. 7. Arabinoxylan-oligosaccharides derived by hydrolysis of arabinoxylans which is a mixture between xylo-oligosaccharides and arabinoxylo-oligosaccharides.

(8) FIG. 8. Flow chart describing the separation of fibers from kernels, fiber bypass or fiber separation by wet fractionation or from dry fractionated fiber from DDGS. These fibers are then fed to a fiber-processing unit where LMW-AX with oligosaccharides is produced as the main product. The cellulosic solid residue after fiber processing is optionally returned to a stream for feed while a fraction of LMW-AX with oligosaccharides can be added to introduce prebiotic properties in novel feed product.

(9) FIG. 9. Flow chart describing the fiber-processing operations in connection to an ethanol process to obtain LMW-AX with oligosaccharides.

(10) FIG. 10. A) Picture of corn stillage sample and B) picture of fractionated fiber from corn stillage sample.

(11) FIG. 11. HPAEC-PAD chromatogram showing oligosaccharides present in samples of enzymatically hydrolyzed corn arabinoxylan. Different combinations of endoxylanase and arabinoxylan arabinofuranohydrolases have been used. “+” sign indicate that the enzymes are used at the same time in an enzyme cocktail while the “.fwdarw.” sign indicate that the enzyme treatments have been sequential with an enzyme inactivation step between.

(12) FIG. 12. Picture of foam produced by washed fiber (left) and de-starched corn bran (right) with alkaline hydrogen peroxide treatment.

DETAILED DESCRIPTION OF THE INVENTION

(13) LMW-AX with branched oligosaccharides was prepared from a cereal fiber containing AX by alkaline hydrogen peroxide extraction followed by incubation with an AX specific endoxylanase.

(14) In the first example a fiber is isolated from a sample of corn stillage (FIG. 4A) by fractionation and washing of the fiber (FIG. 4B). The fiber is processed by alkaline hydrogen peroxide to yield water-soluble AX with a high yield 76-88% based on recovered AX or on remaining AX in the solid fraction respectively. The soluble phase is then separated and dialyzed to remove colored components and salts. Finally the water-soluble AX after dialysis is incubated with an AX specific endoxylanase to yield LMW-AX with branched oligosaccharides (FIG. 4C) and a cellulosic solid residue (FIG. 4D). The sugar composition of corn stillage, washed fractionated fiber and LMW-AX with branched oligosaccharides is shown in Table 1. The purity of water-soluble AX can be further improved by an optional protease treatment before the dialysis. The purity of LMW-AX with branched oligosaccharides can be further improved by ultrafiltration and nanofiltration to recover the added xylanase and to remove the added CaCl.sub.2 respectively.

(15) A comparison with endoxylanases from family 10 and 11, the most commonly used xylanase families to produce LMW-AX, showed that the AX specific endoxylanase was much more efficient in producing branched oligosaccharides from corn AX (FIG. 5A). These oligosaccharides were confirmed to be branched by incubating the sample with two arabinofuranosidases that selectively removed Araf units attached to the oligosaccharides (FIG. 5B). The LMW-AX with branched oligosaccharides obtained by the AX specific endoxylanase contained no detectable β-(1.fwdarw.4) linked XOS or xylose which means that all the products produced by this type of enzymes are truly branched or (1.fwdarw.3) linked disaccharides as opposed to hydrolysis products obtained with a family 10 or 11 xylanase (FIG. 5A).

(16) Isolated water-soluble AX from corn fiber had an average molecular weight of 55 kDa and cotton white in color after dialysis. After incubation with the AX specific endoxylanase the average molecular weight decreased to 2.5 kDa corresponding to 18-19 sugar units. This experiment proved that an AX specific endoxylanse is excellent in producing branched oligosaccharides even from complex AX substrates such as isolated from corn. While state of the art processes that use either family 10 or 11 xylanases are less efficient in hydrolyzing complex AX and cannot produce sufficient amounts of oligosaccharides (FIG. 5A). Analysis of the Mw obtained by the family 10 xylanase revealed that this xylanase family can only reduce the molecular weight to 24 kDa while family 11 did not reduce the molecular weight at all (55 kDa). This explains why very few oligosaccharides are obtained using the family 10 xylanase and why family 11 xylanases are unable to hydrolyse corn AX (FIG. 5A). This motivates using a chemical pre-treatment such as alkaline extraction in combination with an AX specific endoxylanase to obtain LMW-AX with branched oligosaccharides.

(17) The cellulosic residue obtained after corn fiber alkali hydrogen peroxide extraction had a cellulose content of 36% and an AX content of 14% (A/X=0.38) based on dry mass. This residue can be added back to a process stream introducing an easily digestible cellulosic fiber for ruminant animals. Alternatively this residue could be used as a cellulosic ethanol feedstock or as a food additive.

(18) Detailed Description of LMW-AX with Branched Oligosaccharides from Corn Fiber

(19) Average molecular weight: 2.5 kDa

(20) A/X ratio: 0.56

(21) Contain branched oligosaccharides with at least one (1.fwdarw.3) linked Araf or (1.fwdarw.3) linked Xylp unit on the reducing end xylose of the backbone. Can also contain two different disaccharides consisting of (1.fwdarw.3) linked xylose-xylose (D-xylopyranose-β-(1.fwdarw.3)-xylose) or arabinose-xylose (L-arabinofuranose-α-(1.fwdarw.3)-xylose).

(22) TABLE-US-00001 TABLE 1 Total sugar composition (g/g), arabinoxylan content (% dry mass) and arabinose to xylose ratio (A/X) of corn stillage, isolated corn fiber, water-soluble AX from corn fiber and LMW-AX with branched oligosaccharides from corn AX. Table 1 also shows the total sugar composition (g/g), arabinoxylan content (% dry mass) and arabinose to xylose ratio (A/X) of front separated fiber (corn bran) and de-starched front separated fiber (corn bran). Arabinoxylan content Sample Arabinose Galactose Glucose Xylose (% dry mass) A/X Corn stillage 0.06 0.02 0.15 0.09 13 0.58 Isolated corn 0.15 0.04 0.14 0.31 40 0.48 fiber Water-soluble AX 0.24 0.06 0.01 0.43 59 0.55 from corn fiber LMW-AX with 0.22 0.06 0.01 0.40 54 0.56 branched oligosaccharides from corn AX Front 0.09 0.03 0.35 0.19 25 0.47 separated fiber (corn bran) De-starched 0.13 0.04 0.09 0.29 37 0.45 front separated fiber (corn bran) Note: AX content is calculated as (arabinose + xylose) × 0.88. The relatively lower arabinoxylan content in LMW-AX with branched oligosaccharides from corn AX relative to Water-soluble AX from corn fiber is due to the added CaCl.sub.2 and enzyme to the reaction mix. A/X: arabinose to xylose ratio.

(23) In the second example a highly branched AX fraction (A/X=0.88 and average molecular weight of 87 kDa) is isolated by alkaline hydrogen peroxide from wheat bran (A/X=0.55 and 26% AX on dry mass basis) and treated with an AX specific endoxylanase. A low branched AX fraction A/X=0.34 was removed by lowering the pH of the extract prior to enzyme treatment. Alternatively not lowering the pH and keeping both AX fractions in the same fraction would result in a substrate with an average A/X of 0.81.

(24) LMW-AX with branched oligosaccharides was obtained by hydrolysis of the highly branched AX fraction (A/X of 0.88) with an arabinoxylan specific endoxylanase. The resulting LMW-AX was fractionated using SEC and the fractions were freeze dried, weighted and analyzed for their arabinose to xylose ratios (Table 2).

(25) TABLE-US-00002 TABLE 2 Fractions 1-5, percent of mass, molecular weight range and A/X ratio of LMW-AX with branched oligosaccharides produced from highly branched wheat bran arabinoxylan. Fraction Percent of total mass Mw range (kDa) A/X I 12.2% 88.3-24.5 0.94 II 6.4% 24.5-6.8  0.99 III 32.6% 6.8-1.9 0.85 IV 43.6%  1.9-0.53 0.85 V 5.2% 0.53-0.15 0.85

(26) An AX specific endoxylanase is highly efficient in hydrolyzing very complex AX from wheat bran. The majority of the hydrolysis products (76.2%) are in the range 6.8-0.5 kDa in size corresponding to 52-5 sugar units and 43.6% are in the range 1.9-0.5 kDa in size corresponding 14-5 sugar units which means that there is a large portion of branched oligosaccharides present in the LMW-AX sample. This is also confirmed by the results obtained from HPAEC-PAD oligosaccharide analysis (FIG. 6), where fraction III and IV contains most of the branched oligosaccharides.

(27) Detailed Description of LMW-AX with Branched Oligosaccharides from Wheat Bran

(28) Average molecular weight: 3.6 kDa

(29) A/X ratio: 0.88 and 0.85 for the fraction that is less than 5 kDa in size.

(30) Contain branched oligosaccharides with at least one (1.fwdarw.3) linked Araf or (1.fwdarw.3) linked Xylp unit on the reducing end xylose of the backbone. Can also contain two different disaccharides consisting of (1.fwdarw.3) linked xylose-xylose (D-xylopyranose-β-(1.fwdarw.3)-xylose) or arabinose-xylose (L-arabinofuranose-α-(1.fwdarw.3)-xylose).

(31) One aspect of the present invention relates to a process for production of water-soluble arabinoxylan or arabinoxylan derived oligosaccharides by isolation of fiber from a cereal ethanol plant. One embodiment relates to a process wherein the fibers are isolated from at least one of the group consisting of the kernel, wet process streams or dry DDGS. Another embodiment relates to a process wherein the cereal ethanol plant is a dry mill ethanol plant. Another embodiment relates to a process wherein the water-soluble arabinoxylan or arabinoxylan derived oligosaccharides are obtained using treatment of wet fractionated fiber with alkali, acid, steam or enzymes. The wet fractionated fiber may be from dry mill ethanol plants, but not exclusively. Another embodiment relates to a process wherein an obtained fraction of the arabinoxylan or arabinoxylan derived oligosaccharides is introduced into a stream for production a prebiotic product. Also other additives are thinkable to add into the stream for the purpose to enhance the end product. Another embodiment relates to a process wherein an obtained fraction of the arabinoxylan or arabinoxylan derived oligosaccharides is introduced into a stream for production of feed to obtain a prebiotic feed product. Another embodiment relates to a process wherein an obtained fraction of the arabinoxylan or arabinoxylan derived oligosaccharides is introduced into a stream of said cereal ethanol plant. Another embodiment relates to a process wherein spent solids remaining after production of water-soluble arabinoxylan or arabinoxylan derived oligosaccharides are introduced into a stream for production of feed products. The spent solids may be introduced as a modified fiber that aid in digestion in ruminant animals. Further, the present invention may comprise a fiber processing unit comprising a reaction unit adapted for an extraction or hydrolysis, and neutralization; a separation unit adapted for at least one solid/liquid separation.

(32) Another aspect of the present invention relates to a product comprising water-soluble arabinoxylan or arabinoxylan derived oligosaccharides obtainable by the process according to the present invention. Another aspect of the present invention relates to the use of the process according to the present invention, for the production of water-soluble arabinoxylan or arabinoxylan derived oligosaccharides. Yet another aspect of the present invention relates to a food, feed, beverage ingredient, or nutritional supplement comprising the water-soluble arabinoxylan or arabinoxylan derived oligosaccharides obtainable by the process according to the present invention.

(33) In another example alkaline hydrogen peroxide extraction was repeated once to increase the concentration of water-soluble AX in the extraction liquid. The results showed that repeating the extraction a second time did not significantly reduce the yield of extracted AX, 79% and 78% for extraction 1 and 2 respectively. Instead repeating the extraction by re-using the extraction liquid increased the concentration of water-soluble AX by 98%, from 15.5 g/L to 30.7 g/L in the first extraction to the second respectively. In one embodiment the concentration of water-soluble AX is 15 g/L to 35 g/L. This exemplifies that re-using the extraction liquid for one time or multiple times is useful to increase the water-soluble AX content, thereby reducing the need for chemicals and making down-stream processing more efficient. The quality of the cellulosic solids were similar for each extraction, demonstrating that re-using the extraction liquid does not change the characteristics of the cellulosic solids significantly.

(34) In another example water-soluble AX from alkaline hydrogen peroxide extraction was purified by calcium chloride precipitation. The calcium chloride was added after the solution was neutralized to pH 7.5 by carbon dioxide. Upon adding the calcium chloride solution a white precipitate formed instantly. The precipitate consisted of calcium carbonate which co-precipitates contaminants from the solution increasing the purity of the water-soluble AX by 31%, from 59% to 77% by selectively removing non-sugar molecules from the solution. In one embodiment the increase in concentration of water-soluble AX is from 59% to 77%. The loss of AX was not significant showing the potential of purifying water-soluble arabinoxylan in a cost efficient way. The only requirement necessary is to de-salt the water-soluble arabinoxylan by for example ultra-filtration to reach a purity exceeding 70% on dry mass basis.

(35) TABLE-US-00003 TABLE 3 Total arabinoxylan content (% dry mass) after dialysis of water-soluble arabinoxylan samples Arabinoxylan content Sample (% dry mass) Water-soluble AX from corn fiber 59 Calcium salt treated water-soluble AX 77 from corn fiber

(36) In another example two different fiber sources where compared with regard to foaming during alkaline hydrogen peroxide extraction. The results as shown in FIG. 12 exemplify the importance to use a fiber source that is washed to remove contaminants. Having a fiber source with more than 40% AX on dry matter basis and reduced amount of protein significantly reduced foam during alkaline extraction. By using a washed fiber source with 41% AX on dry mass basis compared to de-starch front separated fiber (corn bran) reduced the foam fivefold from 50 ml down to 10 ml using a total extraction volume of 20 ml. This means that choosing a fiber with more than 40% AX seems crucial to reduce foaming problems during extraction. This example shows that it is crucial to use a fiber with a high AX content to reduce problems with foaming during alkaline hydrogen peroxide extraction.

(37) LMW-AX with oligosaccharides was prepared from a cereal fiber containing AX by alkaline hydrogen peroxide extraction followed by incubation with arabinofuranosidases and an endoxylanase.

(38) A fraction of the easily removable Araf units was removed in order to open up holes in the AX structure for xylanase hydrolysis, thereby facilitating the production of oligosaccharides from a complex AX substrate.

(39) In the first example a fiber is isolated from a sample of corn stillage (FIG. 4A) by fractionation and washing of the fiber (FIG. 4B). The fiber is processed by alkaline hydrogen peroxide to yield water-soluble AX with a high yield 76-88% based on recovered AX or on remaining AX in the solid fraction respectively. The soluble phase is then separated and dialyzed to remove colored components and salts. Finally the water-soluble AX after dialysis is incubated with a combination of different arabinoxylan arabinofuranohydrolases (AXHs) and a representative endoxylanase from family 10. The resulting oligosaccharides were analyzed on HPAEC-PAD to evaluate which combination of enzymes yields the highest amount of oligosaccharides (FIG. 11). The sugar composition of corn stillage washed fractionated fiber and LMW-AX with oligosaccharides is shown in Table 1. The purity of water-soluble AX can be further improved by an optional protease treatment before the dialysis. The purity of LMW-AX with oligosaccharides can be further improved by ultrafiltration and nanofiltration to recover the added enzymes and to remove the monosaccharides respectively.

(40) A comparison using only an endoxylanase, the most commonly used xylanase activity to produce oligosaccharides, showed that the endoxylanase alone was not efficient in hydrolyzing the AX substrate into oligosaccharides. Instead the additions of arabinofuranosidases, that selectively removed Araf units, were necessary to have a more efficient production of oligosaccharides from corn AX (FIG. 11).

(41) Isolated water-soluble AX from corn fiber had an average molecular weight of 55 kDa and cotton white in color after dialysis. Analysis of the Mw obtained after incubation with a xylanase 10 revealed that this xylanase alone can only reduce the molecular weight to more than 24 kDa. This demonstrates that additional enzymes are necessary for generating oligosaccharides. Only after incubation with an enzyme cocktail comprising both arabinofuranosidases and xylanases there was a reduction in the Mw. Two fractions were obtained, one with a higher Mw of 10-20 kDa and a low Mw of 0.4-10 kDa. This motivates using arabinofuranosidases together with xylanases to obtain LMW-AX with oligosaccharides from complex or densely substituted AX. The experiment also showed that there are more oligosaccharides formed by simultaneous enzymatic treatment with arabinofuranosidases and xylanases than if the treatment is performed in a sequence. Therefore, an enzyme cocktail comprising arabinofuranosidases and xylanases is the preferred method for generating oligosaccharides.

(42) The cellulosic residue obtained after corn fiber alkali hydrogen peroxide extraction had a cellulose content of 36% and an AX content of 14% (A/X=0.38) based on dry mass. This residue can be added back to a process stream introducing an easily digestible cellulosic fiber for ruminant animals. Alternatively this residue could be used as a cellulosic ethanol feedstock or as a food additive.

(43) Detailed Description of LMW-AX with Oligosaccharides from Corn Fiber

(44) Molecular weight: 0.4-20 kDa

(45) A/X ratio: 0.4-0.5

(46) TABLE-US-00004 TABLE 4 Total sugar composition (g/g), arabinoxylan content (% dry mass) and arabinose to xylose ratio (A/X) of corn stillage, isolated corn fiber, water-soluble AX from corn fiber and LMW-AX with oligosaccharides from corn AX Arabinoxylan content Sample Arabinose Galactose Glucose Xylose (% dry mass) A/X Corn stillage 0.06 0.02 0.15 0.09 13 0.58 Isolated corn 0.15 0.04 0.14 0.31 40 0.48 fiber Water-soluble AX 0.24 0.06 0.01 0.43 59 0.55 from corn fiber LMW-AX with 0.17 0.08 0.02 0.38 48 0.44 oligosaccharides from corn AX obtained using a cocktail of GH10 + AXH-m2,3 + AXH-d3 Note: AX content is calculated as (arabinose + xylose) × 0.88. The relatively lower arabinoxylan content in LMW-AX with oligosaccharides from corn AX relative to Water-soluble AX from corn fiber is due to the enzyme added to the reaction mix. A/X: arabinose to xylose ratio.

(47) In one embodiment the present invention relates to a composition comprising Low Molecular Weight-Arabinoxylan (LMW-AX) with oligosaccharides, where a fraction of the Araf units have been removed to improve the yield of oligosaccharides. In another embodiment the Low Molecular Weight-Arabinoxylan (LMW-AX) with oligosaccharides comprise Arabinose and Xylose units, preferably with an Arabinose/Xylose ratio of 0.4-1.0, preferably a ratio of 0.4-0.9, preferably a ratio of 0.4-0.8, preferably a ratio of 0.4-0.7, preferably a ratio of 0.4-0.6, preferably a ratio of 0.4-0.5. In another embodiment the average molecular weight is less than 10 kDa, preferably less than 7.5 kDa, preferably less than 5 kDa, preferably less than 2.5 kDa, preferably less than 2 kDa. In another embodiment of the present invention a yield of oligosaccharides is improved by combining a xylanase with arabinofuranosidases, preferentially ones able to remove α-(1.fwdarw.3)-linked Arafat double substituted Xylp units (dXyl) or able to remove single α-(1.fwdarw.2)-linked and α-(1.fwdarw.3)-linked Arafat single substituted Xylp units (mXyl). Preferably using a combination of the two different activities of arabinofuranosidases to obtain a high yield of oligosaccharides; In another embodiment the arabinofuranosidases are selected from the group Arabinoxylan arabinofuranohydrolases (AXHs), preferably selected from the group consisting of arabinoxylan arabinofuranohydrolase-d3 (AXH-d3) or arabinoxylan arabinofuranohydrolase-m2,3 (AXH-m2,3). In another embodiment, the arabinofuranosidase treatment is replaced by a treatment with a weak acid solution before the xylanase treatment, such as, but not limited to, inorganic acids, preferably hydrochloric acid, preferably sulfuric acid, preferably phosphoric acid, preferably nitric acid or preferably acetic acid. In another embodiment the arabinofuranosidase treatment is replaced by a treatment with steam or hot pressurized water before the xylanase treatment, such as, steam explosion or autohydrolysis. In another embodiment the xylanase is an endoxylanase, preferably from family 8, 10 or 11, more preferably from family 10 or 11, most preferably from family 10. In another embodiment the present invention is a composition wherein the substrate is a fiber isolated from at least one of a fiber containing kernel, fiber containing wet process streams or dry Dried Distillers Grains with Solubles (DDGS), preferably from cereals comprising fibers, preferably cereals comprising arabinoxylan.

(48) Another aspect of the present invention relates to a process wherein the starting material is a fiber isolated from at least one of a fiber containing kernel, fiber containing wet process streams or dry Dried Distillers Grains with Solubles (DDGS), preferably from cereals comprising fibers, preferably cereals comprising arabinoxylan, preferably a highly or densely substituted arabinoxylan fraction. In another embodiment the starting material is obtained from a cereal ethanol plant, preferably a dry mill ethanol plant. In another embodiment the arabinoxylan is a water-soluble arabinoxylan, preferably obtained using a pre-treatment of the fiber with alkali, acid, autohydrolysis, steam explosion, milling, cavitation or enzymes. In another embodiment low substituted AX is precipitated by acidification of an alkali extraction solution, and preferably is removed. In another embodiment the water-soluble arabinoxylan is obtained using a pre-treatment comprising at least one of HCl, H.sub.2SO.sub.4, NaOH, Ca(OH).sub.2, NH.sub.4, or alkaline hydrogen peroxide. In another embodiment, any cellulosic solids remaining after the pre-treatment is reintroduced into a stream from a cereal ethanol plant, preferably a dry mill ethanol plant. In another embodiment the process is for production of feed products. In another embodiment the present invention comprises a fiber processing unit including a reaction unit adapted for alkali extraction and neutralization, or autohydrolysis, or acid hydrolysis and neutralization, or cavitation, or steam explosion, or milling; and a separation unit adapted for at least one solid/liquid separation.

(49) Another aspect of the present invention relates to use of a composition according to the present invention or an obtained fraction of LMW-AX with oligosaccharides from the process according to the present invention, preferably for introduction into a stream of a cereal ethanol plant, preferably a dry mill ethanol plant. Another embodiment relates to use of the present invention for production of a prebiotic product, preferably food, feed, beverage ingredient or nutritional supplements.

(50) Another aspect of the present invention relates to a food, feed, beverage ingredient, or nutritional supplement or a personal care product ingredient, comprising the LMW-AX with oligosaccharides according to the present invention or obtainable by the process according to the present invention.

EXAMPLES

Example 1: Preparation of LMW-AX with Branched Oligosaccharides from Fractionated Corn Fiber

(51) Materials and Methods

(52) Enzymes

(53) Arabinoxylan specific endoxylanase (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 α-L-arabinofuranosidases (E-ABFCJ and E-AFAM2) were purchased from Megazyme (Wicklow Ireland).

(54) Substrate

(55) A sample of dry corn stillage was received from an U.S. corn dry mill ethanol producer. A sample of 27 g was sieved through 1 mm and the large fiber particles separated out by hand and washed with water and then freeze dried.

(56) Process Steps

(57) Freeze-dried fiber (150 mg) was extracted with a 2% (w/w) H.sub.2O.sub.2 solution (Sigma) adjusted to pH 11.5 by 50% NaOH (Merck). Equal volumes of freshly prepared alkaline H.sub.2O.sub.2 solution was added at time intervals 0, 20 and 40 min to a final dry weight content of 5%. Antifoam TRITON X-100 was added to reduce foaming. The extraction temperature was 90° C. and the total extraction time was 90 min. The reaction was performed in a glass tube with continuously stirring using a magnetic bar.

(58) After the extraction the soluble and insoluble phases where separated by centrifugation at 3900 g for 10 min. The pellet was washed once with 5 volumes of deionized water and centrifuged again to recover remaining solubles trapped in the pellet. Carbon dioxide in the form of dry ice was added to neutralize the soluble and insoluble phase. The soluble fraction was dialyzed against deionized water in a 3.5 kDa dialysis bag (Spectra/por, Spectrum labs, USA) for 24 h. After dialysis the sample was recovered as water-soluble corn arabinoxylan and a fraction was taken out and freeze dried. The solid cellulosic fraction was freeze-dried and a mass corresponding to 34% of the initial fiber was recovered.

(59) Xylanase incubations were all performed using 1.25% of enzyme on arabinoxylan dry basis at pH 7 and 50° C. for 24 h. The reactions were incubated in a water bath. LMW-AX with branched oligosaccharides described in the present invention was produced from 40 mg of dry weight water-soluble corn arabinoxylan by the arabinoxylan specific endoxylanase CtXyl5A with 2 mM CaCl.sub.2 added to the reaction mix. After the reaction all samples were freeze-dried.

(60) Characterization of branched oligosaccharides in the sample was performed by incubating the sample with 1.0 U/mg of two different arabinofuranosidases removing (1.fwdarw.3) double linked arabinofuranosyl units (E-AFAM2) or single linked (1.fwdarw.2) or (1.fwdarw.3) arabinofuranosyl units (E-ABFCJ) respectively. The reaction was performed at pH 5.8 using a 20 mM sodium phosphate buffer at 50° C. for 24 h.

(61) Characterization of Total Sugars

(62) Total sugar composition of the water-soluble arabinoxylan and LMW-AX with branched oligosaccharides fractions were hydrolyzed with 2 M TFA for 60 min at 110° C. Neutralization of TFA was done by evaporation. Total sugar composition in corn stillage, isolated corn fiber and the cellulosic residue after alkaline extraction were pre-hydrolyzed with 72% H.sub.2SO.sub.4 for 1 h at 30° C. after which the samples were diluted to 4% H2SO4 and hydrolyzed at 100° C. for 3 h. Neutralization of H2SO4 was done by adding Ca(OH)2. Analysis of the recovered neutral monosaccharides from TFA and H2SO4 hydrolysis were done by HPAEC-PAD using a CarboPac PA20 column (250 mm×3 mm, 5.5 μm) and a mobile phase of 20 mM NaOH at 0.5 mL/min. Monosaccharide standards (SIGMA) were as follows: arabinose, galactose, glucose and xylose. Total arabinoxylan content in the samples was calculated as 0.88 times (% arabinose+% xylose) after subtracting any free arabinose. Total cellulose content in the cellulose sample was calculated as 0.90 times % glucose after subtracting any free glucose.

(63) Characterization of Oligosaccharides

(64) Analysis of the obtained oligosaccharides was done by High-Performance Anion-Exchange Chromatography Coupled with Pulsed Electrochemical Detection (HPAEC-PAD) using (ICS-5000) using a CarboPac PA200 column (250 mm×3 mm, 5.5 μm) 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, xylo-oligosaccharide and arabinoxylo-oligosaccharide standards used were as follows: arabinose and xylose (Sigma), xylobiose, xylotriose, xylotetraose, xylopentaose, xylohexaose, arabinoxylobiose and arabinoxylotriose (Megazyme). All samples were filtered through a 0.22 μm filter before analysis. Determination of branched oligosaccharides was done by incubating the branched low molecular weight arabinoxylan sample with two different arabinofuranosidases as described above, releasing double and single Araf units, and analyzing the sample on HPAEC-PAD.

(65) Characterization of Molecular Weight

(66) Analysis of molecular weight was determined using by high-performance liquid chromatography (HPLC; Dionex Ultimate 3000) with an IR detector (RI-101, Shodex, Japan) using a column for polysaccharide analysis (Shodex, Japan) SB-806HQ and a mobile phase (0.5 mL min-1) of constant 25 mM sodium acetate buffer pH 5.0. Injection volume was 20 μL. Standards were used containing pullulan standards (Shodex, Japan) with molecular weights P-400 (36.6×10.sup.4), P-200 (20.0×10.sup.4), P-100 (11.3×10.sup.4), P-50 (4.88×10.sup.4), P-20 (2.17×10.sup.4), P-10 (1.00×10.sup.4) and P-5 (0.62×10.sup.4) and xylose based standards xylohexaose (MW=810.7 Da) (Megazyme, Ireland) and xylose (MW=150.13 Da) (Sigma).

Example 2: Preparation of LMW-AX with Branched Oligosaccharides from Wheat Bran

(67) Enzymes

(68) Arabinoxylan specific endoxylanase (Arabinoxylanase) from Clostridium thermocellum (CtXyl5A) was purchased from Nzytech (Lisboa, Portugal). Thermostable α-amylase 0.12 U/g (Thermamyl) and protease (Neutralse 0.8 L) were purchased from Sigma.

(69) Substrate

(70) Commercial wheat bran (Lantmânnen Mill Malmö, Sweden) was used as starting material. A fraction of the bran was grinded in a knife mill (FOSS) through a 1.5 mm mesh size for analysis of total sugars.

(71) Process

(72) A suspension (1:9 w/v) of 250 g wheat bran (94% dry mass) in 2.5 L DI water was adjusted to pH 6.0 with HCl 8 M and treated with a thermostable α-amylase 0.12 U/g 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 the bran with a protease 0.035 U/g for 4 h at 50° C. Thereafter the bran was rinsed with hot tap water, then with DI water and then vacuum dried.

(73) De-starched and de-proteinized wheat bran (50 g dry weight) was extracted with 1 L of a dilute alkaline solution of sodium hydroxide containing 2% hydrogen peroxide at pH 11.5 for 4 h at 60° C. with 200 rpm stirring. 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 containing AX (A/X=0.81) was centrifuged again at 6000 g for 20 minutes to remove precipitate.

(74) The recovered supernatant was then adjusted to pH 4.0 and left for 3 h to precipitate low branched AX (A/X=0.34). The highly branched fraction of AX (A/X=0.88) was recovered by centrifugation at 6000 g for 20 minutes and the low branched AX discarded. The supernatant after centrifugation was neutralized with NaOH to pH 7. A protease 0.00125 U/mL was added and the sample was incubated at 50° C. overnight. AX was recovered from the solution by ethanol precipitation to a final concentration of 80% and the sample was left stirring for 1 h. The precipitated AX was recovered by centrifugation 3000 g for 3 min. The pellet was washed once with 80% ethanol and recovered by centrifugation. The pellet was then left to air dry and then dissolved in MQ water at 50° C. and the pH adjusted to 7 with NaOH.

(75) Xylanase incubation of 30 mL of highly substituted water-soluble wheat bran arabinoxylan was performed at 50° C. for 24 h using a water bath. Arabinoxylan specific endoxylanase CtXyl5A (0.5 mg) was used to hydrolyze the arabinoxylan. In the reaction mix was also 2 mM CaCl.sub.2 in order to stabilize the enzyme. After the reaction the sample was freeze dried and referred to as LMW-AX with branched oligosaccharides from wheat bran.

(76) Characterization of Total Sugars

(77) Total sugar composition of the water-soluble arabinoxylan and LMW-AX with branched oligosaccharides and isolated fractions from SEC were hydrolyzed with 2 M TFA for 60 min at 110° C. Neutralization of TFA was done by evaporation. Total sugar composition in milled wheat bran was determined by pre-hydrolyzed with 72% H.sub.2SO.sub.4 for 1 h at 30° C. after which the sample was diluted to 4% H2SO4 and hydrolyzed at 100° C. for 3 h. Neutralization of H2SO4 was done by adding Ca(OH)2. Analysis of the recovered neutral monosaccharides from TFA and H2SO4 hydrolysis were done by HPAEC-PAD using a CarboPac PA20 column (250 mm×3 mm, 5.5 μm) and a mobile phase of 20 mM NaOH at 0.5 m L/min. Monosaccharide standards (SIGMA) were as follows: arabinose, galactose, glucose and xylose. Total arabinoxylan content in the samples was calculated as 0.88 times (% arabinose+% xylose) after subtracting any free arabinose.

(78) Characterization of Oligosaccharides

(79) Analysis of oligosaccharides was done by High-Performance Anion-Exchange Chromatography Coupled with Pulsed Electrochemical Detection (HPAEC-PAD) using (ICS-5000) using a CarboPac PA200 column (250 mm×3 mm, 5.5 μm) 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, xylo-oligosaccharide and arabinoxylo-oligosaccharide standards used were as follows: arabinose and xylose (Sigma), xylobiose, xylotriose, xylotetraose, xylopentaose, xylohexaose, arabinoxylobiose and arabinoxylotriose (Megazyme).

(80) Characterization of Molecular Weight and Fraction Collection

(81) Analysis of molecular weights and fraction collection was done using a Sephacryl S-200 HR column (600×16 mm) and an IR detector and a mobile phase (0.3 mL min-1) of constant degassed filtered water. A 2.5 mL sample with 10 g/L of LMW-AX with branched oligosaccharides obtained by arabinoxylan hydrolysis with the arabinoxylan specific endoxylanase CtXyl5A was filtered through a 0.45 μm and injected. Fractions of 3 mL were collected. Fractions were pooled together for 5 different time interval (60 min each) and freeze dried. Standards were used to calculate the molecular weight range of each fraction using pullulan standards (Shodex, Japan) with molecular weights P-400 (36.6×10.sup.4), P-200 (20.0×10.sup.4), P-100 (11.3×10.sup.4), P-50 (4.88×10.sup.4), P-20 (2.17×10.sup.4), P-10 (1.00×10.sup.4) and P-5 (0.62×10.sup.4). Also xylose based standards xylohexaose (MW=810.7 Da) (Megazyme, Ireland) and xylose (MW=150.13 Da) (Sigma) were used.

Example 3: Preparation of LMW-AX with Oligosaccharides from Fractionated Corn Fiber

(82) Materials and Methods

(83) Enzymes

(84) A family 10 xylanase from Rhodothermus marinus (RmXyn10A) was prepared as described in Falck et al. (2013). High purity recombinant α-L-arabinofuranosidases were purchased from Megazyme (Wicklow Ireland): Cellvibrio japonicus (E-ABFCJ) removing Araf from (1.fwdarw.2) or (1.fwdarw.3) single substituted Xylp units, mXyl2 and mXyl3 respectively, referred to as AXH-m2,3 in the text and FIG. 11. Bifidobacterium adolescentis (E-AFAM2) removing (1.fwdarw.3) Araf from double (1.fwdarw.2) and (1.fwdarw.3) substituted Xylp units, dXyl, referred to as AXH-d3 in the text and in FIG. 11.

(85) Substrate

(86) A sample of dry corn stillage was received from an U.S. corn dry mill ethanol producer. A sample of 27 g was sieved through 1 mm and the large fiber particles separated out by hand and washed with water and then freeze dried.

(87) Process Steps

(88) Freeze-dried fiber (150 mg) was extracted with a 2% (w/w) H.sub.2O.sub.2 solution (Sigma) adjusted to pH 11.5 by 50% NaOH (Merck). Equal volumes of freshly prepared alkaline H.sub.2O.sub.2 solution was added at time intervals 0, 20 and 40 min to a final dry weight content of 5%. Antifoam TRITON X-100 was added to reduce foaming. The extraction temperature was 90° C. and the total extraction time was 90 min. The reaction was performed in a glass tube with continuously stirring using a magnetic bar.

(89) After the extraction the soluble and insoluble phases where separated by centrifugation at 3900 g for 10 min. The pellet was washed once with 5 volumes of deionized water and centrifuged again to recover remaining solubles trapped in the pellet. Carbon dioxide in the form of dry ice was added to neutralize the soluble and insoluble phase. The soluble fraction was dialyzed against deionized water in a 3.5 kDa dialysis bag (Spectra/por, Spectrumlabs, USA) for 24 h. After dialysis the sample was recovered as water-soluble corn arabinoxylan and a fraction was taken out and freeze dried. The solid cellulosic fraction was freeze-dried and a mass corresponding to 34% of the initial fiber was recovered.

(90) LMW-AX with oligosaccharides described in the present invention was produced from water-soluble corn arabinoxylan (10 g/L) by incubating the sample with different combinations of 1.0 U/mg arabinofuranosidases (AXH-m2,3 and/or AXH-d3) and 1% (wt/wt) xylanase (Xyn10). Where the “+” sign indicate that the enzymes are used at the same time in an enzyme cocktail while the “.fwdarw.” sign indicate that the enzyme treatments have been sequential with an enzyme inactivation step by boiling for 15 min have between used between the treatments. All reactions were performed at pH 7 at 50° C. for 24 h. The reactions were incubated in a heat block.

(91) Cocktail Combinations

(92) Xyn10 only Xyn10+AXH-m2,3 Xyn10+AXH-d3 Xyn10+AXH-m2,3+AXH-d3 (AXH-m2,3+AXH-d3).fwdarw.Xyn10
Characterization of Total Sugars

(93) Total sugar composition of the water-soluble arabinoxylan and LMW-AX with oligosaccharides fractions were hydrolyzed with 2 M TFA for 60 min at 110° C. Neutralization of TFA was done by evaporation. Total sugar composition in corn stillage, isolated corn fiber and the cellulosic residue after alkaline extraction were pre-hydrolyzed with 72% H.sub.2SO.sub.4 for 1 h at 30° C. after which the samples were diluted to 4% H2SO4 and hydrolyzed at 100° C. for 3 h. Neutralization of H2SO4 was done by adding Ca(OH)2. Analysis of the recovered neutral monosaccharides from TFA and H2SO4 hydrolysis were done by HPAEC-PAD using a CarboPac PA20 column (250 mm×3 mm, 5.5 μm) and a mobile phase of 20 mM NaOH at 0.5 mL/min. Monosaccharide standards (SIGMA) were as follows: arabinose, galactose, glucose and xylose. Total arabinoxylan content in the samples was calculated as 0.88 times (% arabinose+% xylose) after subtracting any free arabinose. Total cellulose content in the cellulose sample was calculated as 0.90 times % glucose after subtracting any free glucose.

(94) Characterization of Oligosaccharides

(95) Analysis of the obtained oligosaccharides was done by High-Performance Anion-Exchange Chromatography Coupled with Pulsed Electrochemical Detection (HPAEC-PAD) using (ICS-5000) using a CarboPac PA200 column (250 mm×3 mm, 5.5 μm) 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, xylooligosaccharide and arabinoxylooligosaccharide standards used were as follows: arabinose and xylose (Sigma), xylobiose, xylotriose, xylotetraose, xylopentaose, xylohexaose, arabinoxylobiose and arabinoxylotriose (Megazyme). All samples were filtered through a 0.22 μm filter before analysis on HPAEC-PAD.

(96) Characterization of Molecular Weight

(97) Analysis of molecular weight was determined using by high-performance liquid chromatography (HPLC; Dionex Ultimate 3000) with an IR detector (RI-101, Shodex, Japan) using a column for polysaccharide analysis (Shodex, Japan) SB-806HQ and a mobile phase (0.5 mL min-1) of constant 25 mM sodium acetate buffer pH 5.0. Injection volume was 20 μL. Standards were used containing pullulan standards (Shodex, Japan) with molecular weights P-400 (36.6×10.sup.4), P-200 (20.0×10.sup.4), P-100 (11.3×10.sup.4), P-50 (4.88×10.sup.4), P-20 (2.17×10.sup.4), P-10 (1.00×10.sup.4) and P-5 (0.62×10.sup.4) and xylose based standards xylohexaose (MW=810.7 Da) (Megazyme, Ireland) and xylose (MW=150.13 Da) (Sigma).

Example 4 Reuse of Extraction Liquid from Pre-Treatment

(98) In order to increase the concentration of water-soluble arabinoxylan in the extraction liquid without increasing the fiber to extraction liquid ratio, the extraction liquid was re-used in another subsequent extraction.

(99) Substrate

(100) A sample of a separated fiber fraction after fermentation was received from an U.S. corn dry mill ethanol producer. The fiber wash washed with water and filtered through 1 mm filter to recover a fraction with an AX content more than 40% on dry mass basis (41%). The AX content was determined with 72% H2SO4 for 1 h at 30° C. after which the samples were diluted to 4% H2SO4 and hydrolyzed at 100° C. for 3 h. Neutralization of H2SO4 was done by adding Ba(OH)2.

(101) Process for Repeated Extraction

(102) Washed fiber (1.44 g dry weight) was extracted with a 2% (w/w) H.sub.2O.sub.2 solution (Sigma) adjusted to pH 11.5 by 50% NaOH (Merck). Equal volumes of freshly prepared alkaline H.sub.2O.sub.2 solution was added at time intervals 0, 20 and 40 min to a final dry weight content of 5%. Antifoam TRITON X-100 was added to reduce foaming. The extraction temperature was 90° C. and the total extraction time was 90 min. The reaction was performed in a glass tube with continuously stirring using a magnetic bar.

(103) After the extraction the soluble and insoluble phases where separated by centrifugation at 3900 g for 10 min. The pellet was washed once with 10 ml of deionized water and centrifuged again to recover remaining solubles trapped in the pellet. The wash water was added back with the supernatant to a final volume of 30 ml and sample was taken for sugar analysis. The pH was re-adjusted to 11.5 with NaOH and then the solution was used to extract 1.44 g of washed fiber using the same process as described for the first extraction. After the second extraction the soluble and insoluble phases where separated by centrifugation at 3900 g for 10 min. The pellet was washed once with 10 ml of deionized water and centrifuged again to recover remaining solubles trapped in the pellet. The wash water was added back with the supernatant to a final volume of 30 ml and sample was taken for sugar analysis. Deionized water was added to the two pellets and then carbon dioxide in the form of dry ice was added to neutralize the insoluble phase containing the cellulosic fiber. The pellet was washed another time using 40 ml deionized water to remove remaining salts and then freeze dried.

(104) Characterization of Total Sugars

(105) Total sugar composition of the water-soluble arabinoxylan fractions were hydrolyzed with 2 M TFA for 60 min at 110° C. Neutralization of TFA was done by evaporation. Total sugar composition in washed fiber and the cellulosic residue after alkaline extraction were pre-hydrolyzed with 72% H.sub.2SO.sub.4 for 1 h at 30° C. after which the samples were diluted to 4% H2SO4 and hydrolyzed at 100° C. for 3 h. Neutralization of H2SO4 was done by adding Ba(OH)2. Analysis of the recovered neutral monosaccharides from TFA and H2SO4 hydrolysis were done by HPAEC-PAD using a CarboPac PA20 column (250 mm×3 mm, 5.5 μm) and a mobile phase of 20 mM NaOH at 0.5 m L/m in. Monosaccharide standards (SIGMA) were as follows: arabinose, galactose, glucose and xylose. Total arabinoxylan content in the samples was calculated as 0.88 times (% arabinose+% xylose) after subtracting any free arabinose. Total cellulose content in the cellulose sample was calculated as 0.90 times % glucose after subtracting any free glucose.

Example 5: Process for Purification of Water-Soluble Arabinoxylan by Precipitation with Calcium Chloride

(106) A supernatant recovered after a second extraction from a process described in example 4 was neutralized to pH 7.5 by adding carbon dioxide in the form of dry ice. A 10 ml sample was dialyzed against deionized water in a 3.5 kDa dialysis bag (Spectra/por, Spectrum labs, USA) for 24 h. After dialysis the sample was recovered as water-soluble corn arabinoxylan and a fraction was taken out and freeze dried. Another 10 ml sample of the supernatant mixed with 3 ml 1 M CaCl2 to precipitate impurities together with CaCO3. The precipitate was removed by centrifugation at 6000 g for 20 min and 10 ml of supernatant was dialyzed as described above and freeze died.

(107) Characterization of Total Sugars

(108) Total sugar composition of the water-soluble arabinoxylan fractions were hydrolyzed with 2 M TFA for 60 min at 110° C. Neutralization of TFA was done by evaporation. Analysis of the recovered neutral monosaccharides from TFA hydrolysis were done by HPAEC-PAD using a CarboPac PA20 column (250 mm×3 mm, 5.5 μm) and a mobile phase of 20 mM NaOH at 0.5 mL/min. Monosaccharide standards (SIGMA) were as follows: arabinose, galactose, glucose and xylose. Total arabinoxylan content in the samples was calculated as 0.88 times (% arabinose+% xylose) after subtracting any free arabinose. Total cellulose content in the cellulose sample was calculated as 0.90 times % glucose after subtracting any free glucose.

Example 6: Comparison of Fiber Sources

(109) In order to compare different fiber sources as substrates for making cellulose, corn fiber gum, and AXOS from two different fiber sources where compared with regard to foam production during extraction. A sample of front separated fiber also known as corn bran was received from a European corn dry mill ethanol producer.

(110) The front separated fiber was de-starched in a suspension (1:9 w/v) of 35.7 g dry mass of corn bran in 0.6 L DI water was adjusted to pH 6.0 with HCl 8 M and treated with a thermostable α-amylase 0.12 U/g 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. The bran was then freeze dried.

(111) Washed fiber (1.0 g dry weight) and de-starched front separated fiber (corn bran) were extracted with a 2% (w/w) H.sub.2O.sub.2 solution (Sigma) adjusted to pH 11.5 by 50% NaOH (Merck). The foam level was recorded once it was stable.

(112) Total sugar composition in front separated fiber (corn bran) and de-starched front separated fiber (corn bran) were pre-hydrolyzed with 72% H.sub.2SO.sub.4 for 1 h at 30° C. after which the samples were diluted to 4% H2SO4 and hydrolyzed at 100° C. for 3 h. Neutralization of H2SO4 was done by adding Ba(OH)2. Analysis of the recovered neutral monosaccharides from H2SO4 hydrolysis were done by HPAEC-PAD using a CarboPac PA20 column (250 mm×3 mm, 5.5 μm) and a mobile phase of 20 mM NaOH at 0.5 mL/min. Monosaccharide standards (SIGMA) were as follows: arabinose, galactose, glucose and xylose. Total arabinoxylan content in the samples was calculated as 0.88 times (% arabinose+% xylose) after subtracting any free arabinose. Total cellulose content in the cellulose sample was calculated as 0.90 times % glucose after subtracting any free glucose.

REFERENCES

(113) FALCK, P., PRECHA-ATSAWANAN, S., GREY, C., IMMERZEEL, P., STÅLBRAND, H., ADLERCREUTZ, P., NORDBERG KARLSSON, E. 2013. 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.