AU4990199A - Extraction of hemicellulosic materials - Google Patents

Description

WO 00/04053 PCT/US99/15811 TITLE EXTRACTION OF HEMICELLULOSIC MATERIALS FIELD OF THE INVENTION The present invention relates to processes for producing hemicellulose gels which 5 have a wide variety of uses in industry, including the food and medical industries and in agriculture. BACKGROUND TO THE INVENTION Plant tissue is made up of several different components, including cellulose, hemicellulose, P-glucan, starch, protein, phenolic acids, lignin, waxes, cutin and suberin. 10 The present invention is particularly concerned with the extraction and processing of hemicelluloses. The term "hemicellulose" is a term of art used to embrace non-cellulosic, non-starch plant polysaccharides. The term therefore embraces inter alia pentosans, pectins and gums. Some hemicelluloses (including arabinoxylan and pectin) are suitable as substrates for 15 oxidative gelation ("gelling hemicelluloses"): such hemicelluloses often have substituents with phenolic groups which are cross-linkable with certain oxidizing agents. Arabinoxylan and pectin constitute two particularly important classes of hemicellulose. Arabinoxylans consist predominantly of the pentoses arabinose and xylose, and are therefore often classified as pentosans. However, in many cases hexoses and 20 hexuronic acid are present as minor constituents, and therefore they may also be referred to descriptively as heteroxylans. The arabinoxylan molecule consists of a linear backbone of (1-4)-P3-xylopyranosyl units, to which substituents are attached through C2 and C3 atoms of the xylosyl residues. The major substituents are single c-L-arabinofuranosyl residues. Single a-D-glucoronopyranosyl residues and their 4-O-methyl ethers are also common 25 substituents. Arabinoxylan preparations are usually heterogeneous with respect to the ratio of xylose to arabinose (i.e. the degree of substitution) and in the pattern of substitution of the arabinosyl units along the (1-4)-P3-xylan backbone. Phenolic acid (including ferulic acid) and acetyl substituents occur at intervals along the arabinoxylan chains. These substituents to some extent determine the solubility of the 30 arabinoxylan. Arabinoxylan preparations bearing phenolic (e.g. ferulic acid substituents) are referred to herein as "AXF", while those bearing acetyl substituents are designated "AXA". Similarly, preparation bearing both phenolic (e.g. ferulic acid) and acetyl substituents are hereinafter abbreviated to the designation "AXFA". Arabinoxylan preparations having few phenolic (e.g. ferulic acid) substituents are designated "AX": when the degree of 35 substitution falls below that required for oxidative gelation, the arabinoxylan is designated a "non-gelling arabinoxylan" (a term which therefore embraces AX and AXA). Pectins constitute another important class of hemicelluloses. As used herein and unless otherwise indicated, the term "pectin" is used sensu lato to define hemicellulose WO 00/04053 PCT/US99/15811 polymers rich in D-galacturonic acid. Many (but not all) are cell wall components. The term "pectin" is also used herein sensu stricto to define the so-called "true pectins", which are characterized by the presence of an O-(u.-D-galacturonopyranosyl)-(1-2)-L-rhamnopyranosyl linkage within the molecule. The pectins may be subcategorized on the basis of their 5 structural complexity. At one extreme are "simple pectins", which are galacturonans. At the other extreme are "complex pectins" exemplified by rhamnogalacturonan II, which contains at least 10 different monosaccharide components in the main chain or as a components of branches. Pectins of intermediate complexity (herein referred to as "mesocomplex pectins" contain alternate rhamnose and galacturonic acid units, while others have branches of 10 glucoronic acid linked to galacturonic acid. Complex and mesocomplex pectins are made up of "smooth" regions (based on linear homogalacturonan) and "hairy" regions corresponding to the rhamnogalacturonan backbone with side-branches of varying length. Certain pectins (for example, pectins obtainable from representatives of the plant family Chenopodiaceae, which include beets (e.g. sugar beet), spinach and mangelwurzels) are substituted to some 15 extent with substituents derived from carboxylic acids (usually substituted cinnamic acids) containing phenolic groups. Such pectins may be oxidatively cross-linked to produce viscous solutions or gels via their phenolic substituents. This can be achieved by powerful oxidants (e.g. persulfate - see J.-F. Thibault et alia, in The Chemistry and Technolovgy of Pectin, Academic Press 1991, Chapter 7, pages 119-133) or a combination of peroxidase and 20 hydrogen peroxide (see Thibault et alia, ibidem). FR 2 545 101 Al also describes the gelling of beet pectins using an oxidant (e.g. hydrogen peroxide) and an enzyme (peroxidase). Such pectins are referred to herein as "gelling pectins". Sugar beet pectin is especially rich in arabinan. Arabinan contains 3-1,5-linked arabinose in the backbone with a-(1->3) or a-(1->2)-linked arabinose residues, whereas 25 arabinogalactan contains P3-1,4-linked galactose in the backbone, with a-(1->3) or a-(1->2) linked arabinose residues. Ferulyl substituents are linked to the arabinose and/or the galactose in the arabinan and arabinogalactan side-branches of the rhamnogalacturonan part. The "ferulic acid" content varies according to the extraction method, but is often about 0.6%. 30 Beet pectins obtained by processes which partially remove arabinose residues may exhibit improved gelling properties. Thus, procedures involving mild acid treatment and/or treatment with an a-arabinofuranosidase will improve the gelling properties of the pectin (see F. Guillon and J.-F. Thibault, ibidem). Such pectins are hereinafter referred to as "treated pectins". 35 Oxidative gelation, gelling hemicelluloses and hemicellulose gels Aqueous extracts of several different types of hemicelluloses are known to form gels (or viscous liquids) when treated with certain oxidizing agents. For example, it has long 2 WO 00/04053 PCT/US99/15811 been known that certain flour extracts (e.g. wheat and rye flour extracts) can form gels in the presence of certain oxidants (e.g. upon the addition of hydrogen peroxide). The phenomenon is known in the art as "oxidative gelation", and an extensive literature exists on the subject of oxidative gelation of wheat flour extracts. The term 5 "oxidative gelation" is used herein in a broad sense to include the case where viscous solutions are produced rather than true gels, and the term "gel" is therefore to be interpreted loosely to cover viscous liquids. This reflects the fact that oxidative gelation is a progressive phenomenon which may be controlled to vary the degree of gelation to the extent that hard, brittle gels are formed at one extreme and slurries or viscous liquids at the other. 10 The biochemical basis of the gelling process is not completely or consistently described in the prior art. According to one model, the gels arise as high molecular weight arabinoxylan and protein molecules become inter- and/or intra-linked (via inter alia phenolic substituents, for example ferulic acid-derived diferulate bridges): see e.g. Hoseney and Faubion (1981), Cereal Chem., 58:421. 15 In another model, gel formation and/or viscosity increases arise (at least in part) from cross-linking within and/or between macromolecular components of the hemicellulose mediated by ferulic acid residues (for example, involving diferulate generated by oxidative coupling of the aromatic nucleus of ferulic acid). It should be noted that, as used herein (and as is usual in the art), the terms "ferulic 20 acid" and "ferulate" are used sensu lato encompass ferulyl (often denoted feruloyl) groups (i.e. 4-hydroxy-3-methoxy-cinnamyl groups) and derivatives (particularly oxidized derivatives) thereof. Only a few oxidizing agents are known to have the ability to induce gelation, and these include hydrogen peroxide (usually in conjunction with a peroxidase), ammonium 25 persulphate and formamidine disulphide. Most of the work in the area of oxidative gelation has focused on water soluble pentosans from wheat flour. In these studies, wheat flour is extracted with water (usually at room temperature) to yield gelling arabinoxylans. However, water-insoluble wheat pentosans extracted from wheat flours with various concentrations of cold sodium hydroxide 30 have also been shown to form gels (Michniewicz et alia, Cereal Chemistry 67(5):434-439 (1990), and oxidative gelation of beet pectins has also been described: see J.-F. Thibault et alia, in The Chemistry and Technolovgy of Pectin, Academic Press 1991, Chapter 7, pages 119-133) and FR 2 545 101 Al, discussed earlier. WO 93/10158 describes the preparation of hemicellulosic material from various 35 brans and the oxidative gelation of maize-derived hemicelluloses using an oxidizing system comprising a peroxide (such as hydrogen peroxide) and an oxygenase (such as a peroxidase). The hemicellulosic material for use as a gelling agent is prepared by hot water or mild alkali extraction. 3 WO 00/04053 PCT/US99/15811 Extraction of hemicelluloses There are many known methods for fractionating plant material (such as testaceous or cell wall material) to produce gelling hemicelluloses. Such methods usually involve alkali and/or water extraction to yield insoluble cellulose and soluble hemicellulose fractions, 5 followed by separation. The soluble extract is then often neutralized (or acidified) to precipitate hemicelluloses. Organic solvents are also commonly used instead of (or in addition to) acidification to precipitate further hemicellulose fractions. In the past, gelling hemicelluloses such as arabinoxylan ferulate have been isolated from plants or hemicellulosic starting material by extracting into water or alkaline solutions. 10 Extensive hydrolysis (by e.g. harsh alkaline treatments) is known to strip the ferulic acid residues from the bulk pentosans, and so hemicelluloses for use as starting materials in the production of gels or viscous solutions are usually extracted by water (particularly hot water) or mild alkali extraction. However, water extraction can be used only with a relatively small class of gelling 15 hemicelluloses (and so such extractions are not generally applicable). Moreover, both water and mild alkaline extraction procedures may result in coloured products (usually brown, yellow or tan). There is therefore a need for alternative processes for isolating gellable hemicellulosic compositions from hemicellulosic starting materials. 20 It is also known that substantially colourless hemicellulose products can be extracted from plant sources using alkaline peroxide extraction (Gould (1984), Biotechnol. Bioeng. 26:46-52; Gould (1985) Biotechnol. Bioeng. 27:893-896; Gould (1985) Biotechnol. Bioeng. 27:225-231; U.S. 4,806,475; Doner and Hicks (1997), Cereal Chem. 74(2):176-181). SUMMARY OF THE INVENTION 25 It has now surprisingly been discovered that the hemicellulose extracted by alkaline peroxide treatment is gellable: contrary to all expectations, the hydrolytic activity of the alkali together with the oxidative activity of the peroxide does not strip off the ferulate residues to render the product non-gelling. The present inventors have also found that several important advantages are attendant 30 on the use of alkaline hydrogen extraction, including the bleached/light colour of the end product, high yield and the fact that the entire process can be (but is not necessarily) run at cold temperatures, facilitating enzymic co-processing. It has also been found that hydrogen peroxide is just one of a number of different oxidants that can be used to extract gelling hemicelluloses under alkaline conditions. Thus, 35 the oxidising species which may be used in the method of the invention include the hydrogen peroxide anion, the hydrogen peroxide radical, the hydroxyl radical, the superoxide radical and the oxide radical. Any suitable source of these radicals may therefore be used, including hydrogen peroxide, sodium peroxide, ozone and oxygen. Other oxidising species that have 4 WO 00/04053 PCT/US99/15811 been found useful include the chloronium ion and protonated hypochlorous acid. These latter species may be generated under alkaline conditions by sodium hyperchlorite, chlorine or chlorine dioxide. Thus, according to the present invention there is provided a process for producing a 5 hemicellulose gel comprising the steps of providing a hemicellulosic material; subjecting the hemicellulosic material to alkaline oxidant extraction; isolating the extracted hemicellulose and oxidatively gelling the isolated hemicellulose to produce a cross-linked hemicellulose gel. In another aspect, the invention provides a process for producing a gelling 10 hemicellulose comprising the steps of providing a hemicellulosic material, subjecting the hemicellulosic material to alkaline oxide extraction, isolating the extracted hemicellulose and supplementing the isolated hemicellulose with an oxidase (e.g. glucose oxidase) supplement and optionally: (i) a peroxidase (e.g. horse radish peroxidase) supplement and/or (ii) an oxidase substrate (e.g. glucose) supplement. 15 The process is preferably an industrial process. As used herein, the term "industrial" is used in contradistinction to laboratory scale extractions undertaken in the course of academic and commercial research. The term therefore implies the involvement of large scale apparatus (plant) for producing large (commercial) quantities of products over relatively long periods of time (months or years). 20 The hemicellulosic material may be derived from any suitable source, for example from cereal flour, husk or bran (e.g. from maize, wheat, barley, rice, oats or malt) or from legumes or from the other sources described below. The hemicellulose extracted in step (b) may be any gelling hemicellulose. Preferred are pentosans, e.g. a water soluble or alkali soluble pentosans. The pentosan may comprise 25 arabinoxylan, for example arabinoxylan ferulate. In preferred embodiments the hemicellulose consists (e.g. consists essentially of) arabinoxylan ferulate, so that the gels are cross-linked arabinoxylan ferulate gels. The conditions under which the alkaline oxide extraction is conducted are readily optimized by routine trial and error in respect of the particular hemicellulosic material 30 selected as starting material. In preferred embodiments, the extraction comprises holding the hemicellulosic material at an alkaline pH in an aqueous solution of H 2 0 2 . The holding time may vary over a wide range, and may for example be anywhere between 0.5-50 hours (for example, up to 1, 2, 4, 5, 6, 7 or 10 hours). The pH is preferably at least 11 (for example, 11-12, e.g. about 11.5), while the 35 aqueous solution of H 2 0 2 may be a 0.1-10.0% (for example, 0.5-5%, e.g. about 1-2%) solution. Any suitable alkalifying agent may be used, but particularly convenient is sodium hydroxide, potassium hydroxide or ammonium hydroxide. 5 WO 00/04053 PCT/US99/15811 The hemicellulosic material may be present at any suitable concentration, but extraction efficiencies may decline if the material is present at very high levels. Preferred are levels of between 0.1-50% w/v (for example, 0.5-10 w/v, e.g. 1-5% w/v). In many applications, the process is preferably conducted in the absence of heating 5 ("cold" extraction) while for other applications hot alkaline oxidant can be used to advantage, particularly where high yields are critical and/or enzymic modification is to be carried out. Thus, cold extractions may be conducted at a temperature of between 10 and 40oC (for example, 20-30 0 C, e.g. about 25 0 C), while hot extractions may be conducted at 10 temperatures in excess of 40 0 C (e.g. in excess of 50, 60, 70, 80, 90 or 100oC). The alkaline oxide extraction preferably at least partially decolourizes the hemicellulosic extract (i.e. has a bleaching effect on the product). This has many important advantages, and extends the range of applications of the end product. The alkaline oxide extraction may be preceded by a preliminary alkaline (e.g. mild 15 alkaline) extraction (e.g. at elevated temperature), and in some circumstances higher yields may be obtained. Such two stage extraction process preferably further comprise an enzymic modification step. Where employed, enzymic modification is preferably effected by incorporating one or more enzymes into the aqueous solution of H 2 0 2 . The enzymic treatment may adjust the 20 degree of acetyl ester substitution in the hemicellulose extract (and e.g. comprises treating the hemicellulose with an acetyl esterase). Alternatively, or in addition, the treatment may comprise acetyl esterase treatment under condensing conditions (e.g. low water activity) to form acetyl hemicellulose esters and/or under hydrolytic conditions (e.g. high water activity) to at least partially de-acetylate the hemicellulose. 25 Alternatively, or in addition, the enzyme treatment may adjust the degree of phenolic ester substitution in the hemicellulose extract. It may comprise treating the hemicellulose with a ferulic acid esterase, the treatment conveniently being sequential or simultaneous with respect to an acetyl esterase treatment described earlier. The enzyme treatment may also comprise ferulic acid esterase treatment under condensing conditions (e.g. low water 30 activity) to form ferulic acid hemicellulose esters and/or hydrolytic conditions (e.g. high water activity) to at least partially de-feruloylate the hemicellulose. The esterase treatment may modify the solubility of the hemicellulose. For example, the acetyl esterase treatment may be carried out under condensing conditions (e.g. low water activity) to form acetyl hemicellulose esters and/or hydrolytic conditions (e.g. high water 35 activity) to at least partially de-acetylate the hemicellulose. Treatment with the acetyl esterase under condensing conditions (e.g. low water activity) to form acetyl hemicellulose esters effects a decrease in solubility of the hemicellulose, while treatment under hydrolytic 6 WO 00/04053 PCT/US99/15811 conditions (e.g. high water activity) to at least partially de-acetylate the hemicellulosic starting material effects an increase in solubility of the hemicellulose. The modification of the solubility of the hemicellulose has great significance for the fractionation of various kinds of plant material, and in particular facilitates the extraction of 5 gelling hemicelluloses therefrom. This follows from the fact that the solubility of the hemicellulose can be increased to the extent that highly efficient (in some circumstances essentially quantitative) extraction into water (or buffered aqueous solutions at or around neutral pH, e.g. between pH 6 and 8) can be achieved under mild conditions without: (a) hydrolysing crosslinkable phenolic substituents that may be present on the hemicellulose; 10 and (b) co-extracting undesirable contaminants. The residue remaining forms a particularly useful source of co-products present in a substantially unhydrolysed state, including proteins, starches, p-glucans, celluloses, lignins, phenolic extracts etc. Adjusting the degree of phenolic ester substitution in the hemicellulose via treatment with a ferulic acid esterase (the treatment being either sequential or simultaneous with 15 respect to the acetyl esterase treatment) may modify the cross-linking potential of the hemicellulose. For example, the ferulic acid esterase treatment may be carried out under condensing conditions (e.g. low water activity) to form ferulic acid hemicellulose esters and/or hydrolytic conditions (e.g. high water activity) to at least partially de-feruloylate the hemicellulose. 20 In such embodiments, treatment with ferulic acid esterase under condensing conditions (e.g. low water activity) to form ferulic acid hemicellulose esters may effect an increase in crosslinking potential (and ultimate gel strength), while treatment under hydrolytic conditions (e.g. high water activity) to at least partially de-feruloylate the hemicellulosic starting material may effect a decrease in crosslinking potential (and a 25 decrease in ultimate gel strength). Treatment with both acetyl and ferulic acid esterases may be carried out, and here the treatment may be conducted simultaneously or sequentially. When conducted sequentially, the hemicellulose or starting material may be first treated with either the acetyl esterase or the ferulic acid esterase. However, in many circumstances it is desirable to first treat with 30 acetyl esterase to facilitate extraction and then treat the extracted hemicellulose with ferulic acid esterase. The invention also contemplates a gel or gelling hemicellulose obtainable by the process of any one of the preceding claims. The gel may be in dehydrated form. Also contemplated are rehydrated dehydrated gels obtainable by the processes of the invention. 35 The invention also contemplates an industrial installation specifically adapted for conducting the process of the invention. 7 WO 00/04053 PCT/US99/15811 DETAILED DESCRIPTION Starting materials for use in the invention Suitable starting materials containing hemicellulose (hemicellulosic materials) for use in the processes of the invention (either as starting materials in the fractionation 5 processes or as sources of hemicellulose per se) typically include plant material of various kinds and any part or component thereof. Plant materials useful as a starting material in the invention include the leaves and stalks of woody and nonwoody plants (particularly monocotyledonous plants), and grassy species of the family Gramineae. Particularly preferred are gramineous agricultural residues, 10 i.e. the portions of grain-bearing grassy plants which remain after harvesting the seed. Such residues include straws (e.g. wheat, oat, rice, barley, rye, buckwheat and flax straws), corn stalks, corn cobs and corn husks. Other suitable starting materials include grasses, such as prairie grasses, gamagrass and foxtail. Other suitable sources include dicotyledonous plants such as woody dicots (e.g. 15 trees and shrubs) as well as leguminous plants. Another preferred source are fruits, roots and tubers (used herein in the botanical sense). The term "fruit" includes the ripened plant ovary (or group thereof) containing the seeds, together with any adjacent parts that may be fused with it at maturity. The term "fruit" also embraces simple dry fruits (follicles, legumes, capsules, achenes, grains, samaras 20 and nuts (including chestnuts, water chestnuts, horsechestnuts etc.)), simple fleshy fruits (berries, drupes, false berries and pomes), aggregate fruits and multiple fruits. The term "fruit" is also intended to embrace any residual or modified leaf and flower parts which contain or are attached to the fruit (such as a bract). Encompassed within this meaning of fruit are cereal grains and other seeds. Also contemplated for use as starting materials are 25 fruit components, including bran, seed hulls and culms, including malt culms. "Bran" is a component of cereals and is defined as a fraction obtained during the processing of cereal grain seeds and comprises the lignocellulosic seed coat as separate from the flour or meal. Other suitable component parts suitable as starting materials include flours and meals (particularly cereal flours and meals, and including nonwoody seed hulls, such as the bracts 30 of oats and rice). The term "root" is intended to define the usually underground portion of a plant body that functions as an organ of absorption, aeration and/or food storage or as a means of anchorage or support. It differs from the stem in lacking nodes, buds and leaves. The term "tuber" is defined as a much enlarged portion of subterranean stem (stolon) provided with 35 buds on the sides and tips. Preferred lignocellulosic starting materials include waste stream components from commercial processing of crop materials such as various beets and pulps thereof (including sugar beet pulp), citrus fruit pulp, wood pulp, fruit rinds, nonwoody seed hulls and cereal 8 WO 00/04053 PCT/US99/15811 bran. Suitable cereal sources include maize, barley, wheat, oats, rice, other sources include pulses (e.g. soya), legumes and fruit. Other suitable starting materials include pollen, bark, wood shavings, aquatic plants, marine plants (including algae), exudates, cultured tissue, synthetic gums, pectins and 5 mucilages. Particularly preferred as a starting material is testaceous plant material, for example waste testaceous plant material (preferably containing at least about 20% of arabinoxylan and/or glucoronoarabinoxylan). The starting material may be treated directly in its field-harvested state or (more 10 usually) subject to some form of pre-processing. Typical pre-processing steps include chopping, grinding, cleaning, washing, screening, sieving etc. Preferably, the starting material is in a substantially ground form having a particle size of not more than about 100 microns. It may be air classified or sieved (for example to reduce the level of starch). Alternatively, or in addition, the starting material may be treated 15 with enzymes to remove starch (e.g. alpha- and/or beta-amylase). The starting material may also be pre-digested with a carbohydrase enzyme to remove P-glucan. Suitable washing treatments include washing with hot water or acid (e.g. at a pH of 3-6, e.g. about 5). This at least partially separates protein. Other pre-treatments include protease treatment. 20 Hemicelluloses for use in the invention The hemicelluloses extracted in the processes of the invention may be any hemicellulose which is suitable as a substrate for oxidative gelation (i.e. a "gelling hemicellulose"). Such hemicelluloses often have substituents with phenolic groups which are cross-linkable with certain oxidizing agents. The hemicellulose may be an arabinoxylan, 25 heteroxylan or pectin. Alternatively, the hemicellulose may be a synthetic hemicellulose (i.e. a structural analogue of a naturally-occurring hemicellulose synthesised in vitro by any chemical/enzymic synthesis or modification). Thus, a wide variety of non-cellulosic, non starch plant polysaccharides may be extracted in the processes of the invention, including pentosans, pectins and gums. Preferred are arabinoxylans, heteroxylans and pectins. Of the 30 arabinoxylans, particularly preferred are AXFA and AXF. Also suitable are pectins, including the true pectins, simple pectins, complex pectins, mesocomplex pectins and gelling pectins (e.g. those obtainable from representatives of the plant family Chenopodiaceae, which include beets (e.g. sugar beet), spinach and mangelwurzels). Particularly preferred is sugar beet pectin (for example in the form of sugar 35 beet pulp). Also useful in the invention are treated pectins (as hereinbefore defined). 9 WO 00/04053 PCT/US99/15811 Post-extraction processing/isolation Once extracted and prior to oxidative gelation, the hemicelluloses may be further processed to concentrate, purify or simply isolate the hemicellulose from the unextracted residue. 5 Other post-extraction treatments include supplementing the extracted hemicellulose with an oxidase (e.g. glucose oxidase) supplement, optionally together with a peroxidase (e.g. horse radish peroxidase) and/or an oxidase substrate (e.g. glucose) supplement. This supplementing step is carried out when gelation is to be carried out subsequently by in situ generation of hydrogen oxide by redox enzymes (as described infra). 10 Particularly preferred are post-extraction processes which avoid the use of alcohol precipitation, so avoiding the costs associated with this step. Preferred processing steps include any of centrifugation, filtration (e.g. ultrafiltration or filtration of vega clay), precipitation (e.g. isoelectric precipitation), chromatography (e.g. silica hydrogel and/or ion exchange chromatography). Particularly preferred is ultrafiltration 15 or concentration by spray-, drum- or freeze-drying, vacuum rotary drying or ammonium sulphate precipitation. Other treatments include desalting treatments, for example dialysis or tangential flow ultrafiltration. Although not preferred, alcohol (e.g. IMS, methanol, ethanol or iso-propanol) precipitation, for example with up to 30% v/v alcohol, may be employed. However, 20 particularly preferred is direct spray or freeze drying followed by drying, in the absence of an alcohol precipitation step. Any of the aforementioned processes may be applied directly to the extracted hemicellulose. The extracted hemicellulose may be dried, either before or after oxidative gelation. Dried preparations may be supplemented with carriers or dispersants, such as 25 glucose. Oxidative Relation Any of a variety of known oxidative gelation process can be used to gel the extracted hemicelluloses of the invention. Only a few oxidizing agents are known to have the ability to induce gelation, and these include hydrogen peroxide (usually in conjunction with a 30 peroxidase), ammonium persulphate and formamidine disulphide. The oxidative gelation may also be accomplished enzymically, for example as described in WO 96/03440 in which an oxidase (preferably a laccase) is used to promote oxidative gelation of inter alia arabinoxylans. Other enzymic approaches include promoting the generation of hydrogen peroxide in 35 situ by redox enzymes. The redox enzymes preferably comprise an oxidase (e.g. glucose oxidase) and a peroxidase (e.g. horse radish peroxidase), which are preferably present as supplements in the hemicellulosic material. 10 WO 00/04053 PCT/US99/15811 Alternatively, gelation may be achieved as described in WO 93/10158, which describes an oxidizing system comprising a peroxide (such as hydrogen peroxide) and an oxygenase (such as a peroxidase). Applications 5 The hemicellulose products (i.e. the gels, dehydrated gels, rehydrated dehydrated gels and viscous liquids of the invention) find a variety of applications various therapeutic, surgical, prophylactic, diagnostic and cosmetic (e.g. skin care) applications. For example, the aforementioned materials may be formulated as a pharmaceutical or cosmetic preparation or medical device, for example selected from: a wound plug, wound 10 dressing, wound debriding system, controlled release device, an encapsulated medicament or drug, a lotion, cream (e.g. face cream), suppository, pessary, spray, artificial skin, protective membrane, a nutraceutical, prosthetic, orthopaedic, ocular insert, injectant, lubricant or cell implant matrix. The gelling and gelled hemicelluloses (e.g. AXF and gelled AXF) are particularly useful as agents which maintain the integrity of the gut wall lining, and as agents 15 for coating the luminal wall of the gastrointestinal tract. They may therefore find particular application in animal feeds and in the treatment of gastrointestinal disorders. In such embodiments the material, gel or viscous medium of the invention may further comprise an antibiotic, electrolyte, cell, tissue, cell extract, pigment, dye, radioisotope, label, imaging agent, enzyme, co-factor, hormone, cytokine, vaccine, growth 20 factor, protein (e.g. a therapeutic protein), allergen, hapten or antigen (for e.g. sensitivity testing), antibody, oil, analgesic and/or antiinflammatory agent (e.g. NSAID). Thus, the above-listed materials find application in therapy, surgery, prophylaxis or diagnosis, for example in the treatment of surface (e.g. skin or membrane lesions, e.g. burns, abrasions or ulcers). In a particularly preferred embodiment, the invention contemplates a 25 wound dressing comprising the above listed materials of the invention, for example in the form of a spray. Such wound dressings are particularly useful for the treatment of burns, where their great moisture retaining properties help to prevent the wound drying out. Particularly preferred for such application is a self-gelling liquid comprising gelling hemicellulose supplemented with glucose and peroxidase and/or oxidase enzymes which gels 30 on contact with oxygen in the air. Such compositions can be provided in the form of oxygen-free liquids in airtight containers which can be sprayed onto the skin, whereupon the liquid gels after exposure to the air. Such composition may advantageously be formulated so as to produce a slight excess of hydrogen peroxide on exposure to oxygen, so that a sterilizing, antibacterial, bacteriostatic and/or cleansing effect is obtained which helps 35 promote healing. The invention also contemplates water absorbent nappies, diapers, incontinence pads, sanitary towels, tampons and panty liners comprising the above-listed materials, as well as 11 WO 00/04053 PCT/US99/15811 domestic and industrial cleaning or liquid (e.g. water) recovery operations (e.g. in the oil industry). Alternatively, the gels of the invention can be provided in the form of hydrated or dehydrated sheets or pellicles for application to various internal or external surfaces of the 5 body, for example during abdominal surgery to prevent adhesions. Other applications include enzyme immobilizing systems, brewing adjuncts and bread improvers. The materials listed above also find application as a foodstuff, dietary fibre source, food ingredient, additive, lubricant, supplement or food dressing. Such products are 10 preferably selected from crumb, alginate replacer, cottage cheeses, aerosol toppings, frozen yoghurts, milk shakes, ice cream, low calorie products such as dressings and jellies, batters, cake mixes, frozen chips, binders, gravies, pastas, noodles, doughs, pizza toppings, sauces, mayonnaise, jam, preserve, pickles, relish, fruit drinks, a clouding agent in drinks, syrups, toppings and confectionary (e.g. soft centres), petfood (wherein the gel e.g. acts as a binder), 15 a flavour delivery agent, a canning gel, fat replacer (e.g. comprising macerated gel), a coating, a glaze, a bait, a binder in meat and meat analogue products (for example vegetarian products), an edible adhesive, a gelatin replacer or dairy product or ingredient (e.g. a yoghurt supplement). When used as a fat replacer the gel of the invention is preferably macerated to 20 optimize its mouthfeel and fat mimetic properties. The invention will now be further illustrated by way of specific Examples, which are purely illustrative and not intended to limit the scope of the invention in any way. EXAMPLE 1 Six extraction vessels containing 400 ml of 0.025 M sodium acetate buffer (pH 5.0) 25 with 1% v/v hydrogen peroxide were agitated (200 rpm) at 25 0 C. To each vessel, 8 g of maize bran (2% w/v) was added and dispersed over 15 minutes. Potassium hydroxide was then added to raise the pH to 11.5 (approximately 4.2 g per vessel). Extracts were sacrificed at 1, 2.5, 5, 7, 18 and 24 hour intervals. Sacrificed extracts were adjusted to pH 7.0 with acetic acid, filtered to remove the 30 "bran" and chilled overnight at 4'C. The glucan precipitate formed overnight was removed by centrifugation. The pH was then adjusted to pH 5.5 with glacial acetic acid and 1.5 volumes of 99% IMS added. The pH was then re-adjusted to pH 5.0. The precipitate was agitated for 30 minutes and then allowed to stand for 1 hour at RTP. The supernatant was removed and the precipitate washed (triturated) 3 times with IMS and dried rapidly in a 35 rotary evaporator under vacuum at 50 0 C. The clarity of the recovered AXF/AX was found to improve with increasing extraction time up to 7 hours, but at extractions beyond 7 hours no further improvement was 12 WO 00/04053 PCT/US99/15811 noted. Gel strength of gelled extracts decreased with increasing extraction time, but gels were formed even after 7 hour extractions. EXAMPLE 2 Extracts were prepared as described in Example 1, except that extracts were 5 sacrificed at 2, 3, 4, 5, 6 and 7 hours. The yields increased with extraction time, from about 4% w.r.t the bran (at 2 hours) to about 11% (at 7 hours). The extracts were oxidatively gelled. The extract obtained after 2 hours produced a very brittle gel, while the extract from the 6 and 7 hour extractions thickened (but did not gel). 10 13

Claims (22)

1. A process for producing a hemicellulose gel comprising the steps of: (a) providing a hemicellulosic material; (b) subjecting the hemicellulosic material to alkaline oxide (e.g. alkaline 5 peroxide) extraction; (c) isolating hemicellulose extracted in step (b); and (d) oxidatively gelling the hemicellulose isolated in step (c) to produce a cross-linked hemicellulose gel.

2. A process for producing a gelling hemicellulose comprising the steps of: 10 (a) providing a hemicellulosic material; (b) subjecting the hemicellulosic material to alkaline oxide (e.g. alkaline peroxide) extraction; (c) isolating hemicellulose extracted in step (b); and (d) supplementing the hemicellulose isolated in step (c) with an oxidase 15 (e.g. glucose oxidase) supplement and optionally: (i) a peroxidase (e.g. horse radish peroxidase) supplement and/or (ii) an oxidase substrate (e.g. glucose) supplement.

3. The process of Claim 1 or Claim 2 wherein the hemicellulosic material is derived from cereal flour, husk or bran (e.g. from maize, wheat, barley, rice, oats or malt) or from 20 legumes.

4. The process of any one of Claims 1 to 3 wherein the hemicellulose extracted in step (b) comprises a pentosan, e.g. a water soluble or alkali soluble pentosan fraction.

5. The process of Claim 4 wherein the pentosan or extracted hemicellulose consists, consists essentially of, or comprises arabinoxylan (for example arabinoxylan ferulate). 25

6. The process of any one of the preceding claims wherein the oxide is selected from: (a) the hydrogen peroxide anion; (b) the hydrogen peroxide radical; (c) the hydroxyl radical; 30 (d) the superoxide radical; (e) the oxide radical, the oxidising species (a)-(e) for example being generated by hydrogen peroxide, sodium peroxide, ozone or oxygen; (f) the chloronium ion; 35 (g) protonated hypochlorous acid, the oxidising species (f) and (g) for example being generated under alkaline conditions by sodium hyperchlorite, chlorine or chlorine dioxide. 14 WO 00/04053 PCT/US99/15811

7. The process of any one of the preceding claims wherein the alkaline oxide extraction comprises holding the hemicellulosic material at an alkaline pH in an aqueous solution of H 2 0 2 .

8. The process of Claim 7 wherein the hemicellulose is held for 0.5-50 hours (for 5 example, up to 1, 2, 4, 5, 6, 7 or 10 hours).

9. The process of Claim 7 or Claim 8 wherein the alkaline pH is at least 11 (for example, 11-12, e.g. about 11.5).

10. The process of any one of Claims 7-9 wherein the aqueous solution of H 2 0 2 is a 0.1-10.0% (for example, 0.5-5%, e.g. about 1-2%) solution. 10

11. The process of any one of Claims 7-10 wherein the alkalifying agent is sodium hydroxide, potassium hydroxide or ammonium hydroxide.

12. The process of any one of Claims 7-11 wherein the hemicellulosic material is present at 0.1-50% w/v (for example, 0.5-10 w/v, e.g. 1-5% w/v).

13. The process of any one of Claims 7-12 wherein the extraction is conducted: 15 (a) at a temperature in excess of 40oC (e.g. in excess of 50, 60, 70, 80, 90 or 100 0 C); or (b) at a temperature of between 10 and 40 0 C (for example, 20-30 0 C, e.g. about 25 0 C).

14. The process of any one of Claims 7-13 wherein the alkaline oxide extraction at 20 least partially decolourizes the hemicellulosic extract.

15. The process of any one of Claims 7-14 wherein the alkaline oxide extraction is preceded by a preliminary alkaline (e.g. mild alkaline) extraction (e.g. at elevated temperature).

16. The process of any one of Claims 7-15 further comprising an enzymic 25 modification step.

17. The process of Claim 16 wherein the enzymic modification is effected by incorporating one or more enzymes into the aqueous solution of H 2 0 2 .

18. The process of Claim 16 or 17 wherein the enzyme treatment: (a) adjusts the degree of acetyl ester substitution in the hemicellulose 30 extract (and e.g. comprises treating the hemicellulose with an acetyl esterase); and/or (b) comprises acetyl esterase treatment under condensing conditions (e.g. low water activity) to form acetyl hemicellulose esters and/or under hydrolytic conditions (e.g. high water activity) to at least partially de 35 acetylate the hemicellulose; and/or (c) adjusts the degree of phenolic ester substitution in the hemicellulose extract (and e.g. comprises treating the hemicellulose with a ferulic acid esterase, the treatment e.g. being sequential or simultaneous 15 WO 00/04053 PCT/US99/15811 with respect to an acetyl esterase treatment as defined in (a) or (b), above); and/or (d) comprises ferulic acid esterase treatment under condensing conditions (e.g. low water activity) to form ferulic acid hemicellulose esters 5 and/or hydrolytic conditions (e.g. high water activity) to at least partially de-feruloylate the hemicellulose.

19. A gel or gelling hemicellulose obtainable by the process of any one of the preceding claims.

20. The gel of Claim 19 in dehydrated form. 10

21. A rehydrated gel as defined in Claim 20.

22. An industrial installation specifically adapted for conducting the process of any one of Claims 1 to 18. 16