US 2005.00896O2A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0089602 A1 Kvist et al. (43) Pub. Date: Apr. 28, 2005

(54) PROCESS FOR THE FRACTIONATION OF Publication Classification BRANS (51) Int. Cl." ...... A23K 1100 (76) Inventors: Sten Kvist, Odakra (SE); Tommie (52) U.S. Cl...... 426/52 Carlsson, Hoganas (SE); John Mark Lawther, Roskilde (DK); Fernando (57) ABSTRACT Bastile DeCastro, Campinas/SP (BR) A process for the fractionation of valuable fractions from Correspondence Address: cereal brans (e.g. , and oat brans, and Matthew E. Connors polish) is described. In particular, this invention describes a Gauthier & Connors LLP two step process, in which the Said is first Subjected to Suite 3300 a combination of enzymatic treatment and wet milling, 225 Franklin Street followed by Sequential centrifugation and ultrafiltration, Boston, MA 02110 (US) which aims at physically Separating the main bran factions, i.e. insoluble phase (pericarp and aleurone layer), germ-rich (21) Appl. No.: 10/643,402 fraction, residual fraction and Soluble . A Second Step consists of fractionating cereal brans Substan (22) Filed: Aug. 19, 2003 tially free of Soluble compounds, hence insoluble phase from the above-mentioned first Step, by enzymatic treatment Related U.S. Application Data with Xylanases and/or beta-glucanase and wet milling, fol (63) Continuation of application No. PCT/SE02/00309, lowed by Sequential centrifugation and ultrafiltration, which aims at physically Separating the main fractions, i.e. filed on Feb. 21, 2002. insoluble phase (remaining cell wall components), (30) Foreign Application Priority Data rich fraction, Soluble hemicellulose and oligosaccharide, and therefore maximizes the extraction rate of valuable cell wall

Feb. 26, 2001 (SE). ... O1OO655-0 components and aleurone cells from previously cleaned Oct. 4, 2001 (SE)...... O103328-1 bran. Patent Application Publication Apr. 28, 2005 Sheet 1 of 2 US 2005/0089602 A1

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Patent Application Publication Apr. 28, 2005 Sheet 2 of 2 US 2005/0089602A1

Bran

Insoluble fraction Soluble fraction

("cleaned bran')

Hemicellulose Olig. Syrup Aleurone rich Insol. diet, fibre Protein (ARP)

Defatted ARP Aleurone oil

Germrich protein Endosperm (GRP) rich protein

Defatted GRP Germ oil

FIG. 2 US 2005/0O896O2A1 Apr. 28, 2005

PROCESS FOR THE FRACTIONATION OF 0007 Patent application WO 99/11672 discloses a pro CEREAL BRANS ceSS that uses Selective enzymes, Such as acetyl Xylan esterase and ferulic acid esterase, to both facilitate the TECHNICAL FIELD removal of hemicellulose from various plant materials and 0001. The present invention relates to process for the alter its degree of phenolic ester Substitution. Despite the extraction of Soluble , non-Starch , and fact that functional hemicellulose with high solubility and optionally oils from commercially available cereal bran. It gelling Strength can be produced yields are rather low. In also allows the production of cell wall-derived materials and fact, the inventors reported a 3 and 6% yield of arabinoxylan less accessible proteins from cereal brans that are Substan ferulate (hemicellulose), when wheat bran was treated with tially free of Soluble compounds, the compounds thus recov acetyl Xylan esterase for 90 and 180 min, respectively. ered as well as their use. Furthermore, the invention does not make any reference to the use of Xylanases, or its combination with wet milling in BACKGROUND OF THE INVENTION order to overcome the low yields reported. 0002 Bran is defined as the coat of cereal grains 0008 U.S. Pat. No. 5,308,618 discloses a process to Such as wheat, barley, rye, triticale, oat or rice. Anatomically, extract soluble dietary fibre hemicelluloses from wheat bran bran comprises the outer layers of the Seed, known as the by applying a heat pre-treatment in aqueous Solution. Fur pericarp-testa and an inner layer known as the aleurone ther processing Such as filtration, Salting out, dialysis, ultra layer, which is often classified as the Outermost layer of the filtration, reverse Osmosis, gel filtration and precipitation in endosperm. However, from the practical point of view cereal order to remove contaminants from the hemicellulose frac bran is herein defined as the remaining material after the tion, follows this. The inventors make no claims with conventional milling or polishing of cereal grains and con regards to the use of enzymes and production of products tains primarily pericarp-testa and aleurone layer compo Streams other than hemicellulose. Furthermore, the inven nents, along with the cereal germ and residual parts of the tion highlights the need of run costly procedures to remove endosperm. The relative amounts of each component will contaminants, which were once present in the original wheat depend upon the type of cereal and milling technique bran. The bran is extracted at high temperatures and pres applied. Sures in water (180-200° C.), producing a glucose rich 0003. Within this definition, bran therefore contains all of dietary fibre component in the water phase. The process the pericarp-testa components, the aleurone layer, the germ Specifically targets the production of dietary fibre and is not components including germ proteins and oils, along with a really/strictly a fractionation procedure in that other prod residual amount of endosperm Starch, and pentosans. ucts is largely ignored. 0004 U.S. Pat. No. 4,361,651 describes a process for 0009 U.S. Pat. No. 3,879,373 discloses a process to making fermentable Sugars and high protein products from extract hemicelluloses from wheat bran by applying alkali grain, mainly . In this method, grain is steeped for treatment to dissolve hemicelluloses and other bran compo 10-30 h, prior to milling and Separation of the germ com nents followed by ethanol eXtraction to Separate the hemi ponent, Saccharification of carbohydrates (mainly starch), celluloses. Alkali (Sodium hydroxide) extraction of hemi and separation of fibre. The yield of starch is maximised for cellulose has also been disclosed in U.S. Pat. No. 5,174,998 fermentation to alcohol. Within the described process there as an intermediate Step to produce controlled-release com is no specific fractionation of the bran component, Separa positions containing the Said alkali-extracted hemicellulose tion of protein types or consideration of the germ compo and an active Substance. Similar alkali-extraction procedure nent. is disclosed in U.S. Pat. No. 4,927,649 to produce hemicel lulose, which is then used in coating compositions contain 0005 U.S. Pat. No. 5,312,636 discloses a process for ing insoluble dietary fibre. fractionating crop into industrial raw material. This is focused on oat grain and incorporates bran fractionation 0010 WO 00/04053 patent application describes a procedures that involve the extraction of more hydrophobic chemical process using alkaline peroxide treatment to pro components Such as lipids in polar organic Solvents prior to duce high yields of light coloured gelling hemicelluloses the alkaline extraction of residual bran to produce beta from products derived from , or bran. Another glucan, protein and degummed fibres. The use of the organic chemical extraction process of hemicellulose from wheat Solvent is a key Step in the proceSS and hydrolysing enzymes bran has been disclosed in WO98/31713 patent application, are not utilised during the fractionation procedure. whereby the inventors combine a washing procedure to remove the starch fraction followed by an alkaline treatment 0006 Two related US patents (U.S. Pat. Nos. 4,171,383 with sodium hydroxide to extract the hemicellulose from the and 4,171,384) disclose dry and wet milling procedures for Starch-free raw material. refining whole wheat grain. U.S. Pat. No. 4,171,383 focuses on wet milling of the whole kernel. The bran produced is 0011. It appears from above-described prior art on alkali mixed with a separated (mainly) endosperm protein fraction extraction of hemicellulose that this is an old, proven and to produce animal feed. U.S. Pat. No. 4,171,384 describes effective way to yield high quantities of Soluble hemicellu dry milling of the whole kernel to produce an endosperm lose with interesting functionalities Such as gelling, dietary fraction, a germ fraction and a bran fraction. The endosperm fibre and as an inert material for controlled-release compo fraction is then Subjected to wet milling and Separation of Sitions. The drawback of Such technology is the associated Starch-rich and protein-rich fractions. The protein rich frac problems of utilising chemicals. Firstly, chemicals eventu tion is added to the bran to produce an animal feed. There is ally become contaminants in various product Streams, and no description of a specific wet fractionation of the bran therefore require additional purification. This normally has itself within either patent. Significant cost implications. Secondly, innovative industrial US 2005/0O896O2A1 Apr. 28, 2005

processes based on chemical extraction are not always SUMMARY OF THE PRESENT INVENTION attractive from the marketing point of View, particularly in food applications. 0018. The main objectives of the present invention are to: 0019 1. Arrive at an efficient and cost effective indus 0012 Production of insoluble dietary fibres from oats is trial wet process to extract and yield germ-, endosperm disclosed in U.S. Pat. No. 5,023,103, which describes a and aleurone-rich fractions, glucose, Soluble hemicel chemical procedure (alkali and bleaching treatment) for the lulose, Soluble oligosaccharides, insoluble fibre, and production of insoluble dietary fibre with high water holding optionally oils from cereal bran. capacity and non-gritty mouth feel. A water holding capacity 0020 2. Combine the use of enzymatic treatment with of 6.9 g watering oat fibre has been reported. wet milling to improve the efficiency of extraction and 0013. Other references have disclosed processes for the Separation in an industrial process. extraction of proteins from cereal brans. U.S. Pat. No. 0021 3. Ensure that in the fractionation process pro 4,746,073 discloses a physical process to Separate aleurone tein fractions of distinct physical properties and there cell particles and pericarp-testa particles from commercial fore functionalities were obtained. wheat bran. The process consists of milling the bran par 0022 4. Ensure that the intermediate fibre raw material ticles to a specific particle size distribution, electroStatically contains the least amount of readily extractable com charge the Said particles and then pass the Said charged ponents, hence Solubles, So that contamination with the particles through a magnetic field, which Separates aleurone Said Solubles in the end products is kept to a minimum. from pericarp-testa particles. The Separation is achieved by 0023 5. The process is carried out in such a way so that hammer-milling the bran and then Subjecting the resultant use of chemical extraction procedures are avoided and that, particles to a physical Separation regime achieve the Sepa preferably food grade and non-genetically modified (non ration. No acqueous wet processing is employed during the GMO), Xylanases and/or beta-glucanases are used in order to fractionation procedure described therein. broaden the market opportunities for the end products. 0.014. This is a rather different concept from the current 0024. In this description, the term cereal bran substan invention, which is based upon the use of enzymes and tially free of soluble compounds or “cleaned bran” refers to aqueous wet milling. any cereal bran, which has been processed, after conven tional milling or polishing, by any means, So as to remove 0.015 Waszczynskyi et al. (1981) have proven that pro Substantial amounts of Soluble components, which are tein extraction rate of alkali-treated full wheat bran can extracted by water or less polar Solvents. The resulting be increased from 30% up to 38.5% when it is preceded by material, hereafter referred to as cleaned bran, should con polysaccharidase treatment. The above-mentioned figures tain rather limited amounts of Soluble Sugars, Starch and are significantly lower than those described in the present gluten (less than 1%), but it may still contain Some proteins invention whereby up to 60% protein extraction rates were and , which are leSS accessible and/or Soluble. The achieved without using alkali treatment. Furthermore, U.S. cleaned bran consists primarily of cell wall components, of Pat. No. 5,622,738 discloses a method to extract soluble which hemicellulose is the most abundant. hemicelluloses, for use as a Source of dietary fibre, from 0025 The invention relates to methods, procedures and various fibrous materials including cereal brans using alkali an industrial process for the wet-fractionation of cereal bran digestion followed by Xylanase treatment. AS in other prior into two protein rich fractions, one of which contains the art, Waszczynskyi et al made use of alkali digestion to germ oils and related components, a fibre fraction, which improve extraction rates. Additionally, the residence time for also retains most of the aleurone proteins, and a Syrup the enzymatic treatment was rather long (3 to 96 h), which fraction. makes the proceSS not very attractive from a production cost point of view. 0026. The invention is centred around the wet-milling of cereal bran in the presence of enzymes: a) starch degrading 0016 WO 01/60180 relates to process for separating oil enzymes of the group amylases, and amyloglucosidases, and from rice bran, whereby bran having a Suitable particle size optionally b) non-Starch degrading enzymes (polysacchari in a slurry is Subjected to an enzymatic treatment, and dases) and optionally a phytase, under appropriate condi Subjecting the enzymatically treated Slurry for a separation tions of temperature, i.e. from 50 to 90° C., more preferably to recover an oil phase for further isolation of Specific lipids. from 50 to 75° C., and pH from 4 to 7.5. This is followed by The process is carried out under alkaline conditions for a the Separation of the above listed components from aqueous considerable time period, normally 15 hours. AS no degra Suspension using mainly centrifugal Separation methods. dation of Starch present, about 15% of the ingoing bran, The pH when using an alpha amylase is normally around 7, takes place, any end product will be heavily contaminated and when using an amyloglucosidase it is around 4.5. The with starch. enzymes are used normally in a cocktail comprising 200 to 0.017. It is clear that the above-mentioned prior art has not 1500 IU/g of substrate, but should contain at least 1 IU/g of Succeeded to arrive at a process of cereal bran fractionation, Substrate. which is both chemical-free and yields different food-grade 0027. This invention also relates to methods, procedures fractions and Simultaneously yield aleurone proteins, oli and an industrial process for the wet-fractionation of cleaned gosaccharides and hemicelluloses, and yet produce insoluble bran into one protein rich fraction, which contains primarily dietary fibre from previously cleaned cereal bran, i.e. Sub proteins from the aleurone cells, a Soluble hemicellulose Stantially free of Soluble components, using Xylanases and/ fraction, a Soluble oligosaccharide fraction and an insoluble or beta-glucanases in combination with wet milling. fibre fraction. US 2005/0O896O2A1 Apr. 28, 2005

0028. This invention further aims at the fractionation of intermittent wet milling, followed by an optional Step of cleaned bran by combining wet-milling and enzymatic enzyme inactivation by wet heat treatment. hydrolysis specifically with food grade Xylanases and/or 0038. In a preferred embodiment the inactivated hydroly beta-glucanase under well-controlled conditions of tempera Sate is then fractionated by centrifugal forces into an ture, such as i.e. from 35 to 80°C., more preferably from 40 insoluble phase containing primarily cellulose, lignin, leSS to 50° C., and pH from 4 to 7, preferably 4.5 to 5.5. accessible hemicellulose, residual aleurone cells and call 0029 Nowhere during the degradation of the bran and wall bound proteins, and an aqueous phase containing bran components pH exceeds 7.5 as alkaline hydrolysis Soluble hemicellulose, oligosaccharides, Sugars and pro Seems to have a detrimental effect on the fractionation and teins, and that the aqueous phase is further Separated by the end products. centrifugal force into protein-rich fraction and a carbohy drate-rich fraction, and that the -rich fraction is 0.030. A further effect of the present invention is that the further Separated by Size exclusion technique into a hemi end products will contain no, or Substantially no Starch, Starch derivates, or Starch fragments due to the primary cellulose-rich fraction (medium molecular size) and an hydrolysis using a Starch hydrolysing enzyme treatment. oligosaccharide-rich fraction (Small molecular size). 0039. In a preferred embodiment cereal bran substan 0031. This step is followed by the separation of insoluble tially free of both in water or less polar solvents soluble fibre and protein fractions from aqueous Suspension using compounds are derived from wheat, rice, barley, oat, rye or centrifugal Separation methods while both hemicellulose triticale. and oligosaccharide fractions are Separated by size exclusion techniques Such as ultrafiltration. 0040. In a preferred embodiment the combination of intermittent wet milling with enzymatic treatment is 0.032 Presently there exist no commercial enzyme-based arranged to increase Substrate accessibility to the cell wall methods for wet-fractionating of cereal bran, which is degrading enzymes thereby improving the Overall hydroly capable of extracting the above-mentioned fractions. sis performance and the Subsequent Separation of the various fractions by density/Solubility and molecular size. DETAILED DESCRIPTION OF THE PRESENT INVENTION 0041. In a preferred embodiment the enzymatic treatment is carried out using at least one non-Starch degradable 0033. It has now surprisingly been shown possible to polysaccharidase in the form of cellulases, hemicellulases solve the problems identified above and meet the objectives mainly Xylanases, beta-glucanases, and pectinases, and by means of the present invention which IS characterized in optionally phytases. that bran is first Subjected to a combination of enzymatic treatment with enzymes of the group Starch- and optionally 0042. In a preferred embodiment the enzymatic treatment phytate-hydrolysing enzymes, and aqueous wet milling, is accomplished by using Xylanases with high beta 1-4- followed by an optional Step of enzyme inactivation by wet Xylanase (pentosanase) and/or beta-glucanase activity. heat treatment, and a Subsequent Step whereby the insoluble 0043. In a preferred embodiment the said fraction con phase containing a cleaned bran consisting of both pericarp tains at least 35% protein and 10% oil on dry matter basis and aleurone fractions are separated by centrifugal forces and exhibits a high emulsifying capacity and an increased into an aqueous phase containing a germ-rich fraction and an Shelf life with regards to resistance to oxidation compared to aqueous phase containing residual endosperm components, the original bran, and that the Said fraction contains less than and that the proteins contained in the endosperm-rich frac 5% fibre. tion are concentrated. 0044) In a preferred embodiment the said fraction con 0034. In a preferred embodiment cereal brans are the tains at least 25% protein and 10% sugar and less than 3% fibrous-residue resulting from a primary grain milling, i.e. oil and 3% fibre, and at least 25% soluble high-molecular after the Separation of the endosperm fraction, of wheat, rice, weight non-Starch polysaccharides of the groups beta-glu barley, oat, rye and triticale, and having variable chemical cans for barley and oat and arabinoxylans for wheat, rice, rye compositions, presence of anti-nutritive factors, and pres and triticale. ence of various anatomical fractions, i.e. pericarp, germ, and residual endosperm. 0045. In a preferred embodiment liquid whey is incorpo rated in to the said fraction at levels varying from 20 to 80% 0035) In a preferred embodiment the enzymatic treatment by weight on dry matter basis, and that the final mixture is is accomplished using a Starch degrading enzyme in the dried. form of a polysaccharidase of amylases and/or amyloglu cosidases. 0046 A further aspect of the invention comprises an insoluble fibre fraction produced wherein the said fraction 0036). In a preferred embodiment a further enzymatic consists of cell wall components of bran (>85%) and aleu treatment is carried out using at least one non-Starch degrad rone proteins (>10%), and Substantially free of gluten and able polysaccharidase in the form of cellulases, hemicellu Starch, and with a high water holding capacity (>6 g water/g lases mainly Xylanases, beta-glucanases, and pectinases, dry product). and/or phytases. 0047 A still further aspect of the invention encompasses 0037. In a preferred embodiment such cleaned bran is a Sugar fraction wherein the Said fraction is originated Subjected to a combination of enzymatic treatment with primarily from the residual endosperm and it contains more Specific enzymes of the group Xylanase and/or beta-gluca than 65%. Sugars (Such as glucose, maltose and maltotriose) nase under Strictly controlled hydrolysis conditions, and on dry matter basis. US 2005/0O896O2A1 Apr. 28, 2005

0.048. A further aspect of the invention encompasses a pH conditions, and the resulting protein hydrolysate has protein fraction derived Substantially from the aleurone enhanced functionalities Such as Solubility, emulsifying and cells, wherein the said fraction contains at least 35% protein foaming capacities. and 10% oil, less than 5% insoluble fibre on dry matter basis, 0060 A further aspect of the invention relates to a use of Substantially free of gluten and Starch and with a high a protein fraction, as described in feed and food applications emulsifying capacity. to replace other protein products from Vegetable and animal 0049. A still further aspect of the invention encompasses SOUCCS. an insoluble fibre fraction, wherein the Said fraction consists 0061. A still further aspect of the invention relates to a primarily of cell wall components with a relative lower use of a protein fraction, as described, in food application as hemicellulose content compared to the original cleaned cereal bran, Substantially free of gluten and Starch (<1% on a texturizer, emulsifier, fat binder and fat replacer. dry matter basis) and with a high water holding capacity (>6 0062) A further aspect of the invention relates to a use of g watering dry product). a protein fraction, as described, as a raw material for the extraction of Soluble high-molecular weight non-Starch 0050. A further aspect of the invention relates to a soluble polysaccharides. hemicellulose fraction, wherein the Said fraction consists primarily of medium molecular weight hemicellulose pref 0063 A still further aspect of the invention relates to a erably above 20 kDa (>40%) of the groups arabinoxylans use of a protein fraction, as described, in food applications from wheat, rye, rice and triticale, and beta-glucans from oat as a foam Stabilising agent, whipping agent, water binder, and barley, which also contains proteins (<10%) and gelling agent, and as a dietary Supplement rich in Soluble monosaccharides (<10%), and is Substantially free of gluten dietary fibre (beta-glucans and arabinoxylans) with associ ated health benefits Such as cholesterol-reducing effects of and starch (<1% on dry matter basis). the beta-glucans. 0051. A still further aspect of the invention relates to a Soluble oligosaccharide fraction, wherein the Said fraction 0064. A further aspect of the invention relates to a use of consists primarily of low molecular weight hemicellulose a protein fraction, as described, as an additive or ingredient sub-units of below about 20 kDa (>40%) of the groups in foods Such as baked products, processed meats, dairy arabinoxylans from wheat, rye, rice and triticale, and beta products, Soups and Sauces, high protein drinks and health glucans from oat and barley, which also contains proteins drinkS. (<10%), monosaccharides (<20%), lignans and related phe 0065. A still further aspect of the invention relates to a nolics (<5%), and is Substantially free of gluten and starch use of a fibre fraction, as described, in feed and food (<1% on dry matter basis). applications to replace other insoluble fibrous products as a texturizing and water binding additive in processed foods 0.052 A further aspect of the invention relates to a protein particularly meat products, and as a Source of dietary fibre fraction, wherein the oil can be optionally removed by in breakfast , baked products and health products, or conventional organic Solvent extraction or preferably by as a raw material for further processing to extract remaining Supercritical carbon dioxide extraction to yield an oil frac cellulose, hemicellulose, lignin and lignans. tion and a defatted protein fraction. 0066. A further aspect of the invention relates to a use of 0.053 A still further aspect of the invention relates to a a Soluble hemicellulose, as described, in feed and food protein fraction, wherein the oil can be optionally removed applications as a gellant, thickener, foam Stabilizer, emulsi by conventional organic Solvent extraction or preferably by fier, water binder, and as a dietary Supplement rich in Soluble Supercritical carbon dioxide extraction to yield an oil frac dietary fibre, and in chemical applications, or as a raw tion and a defatted protein fraction. material for further processing to obtain other functional hemicelluloses. 0054) A further aspect of the invention relates to an insoluble dietary fibre used for recovery of cellulose, hemi 0067. A still further aspect of the invention relates to a cellulose, lignin and lignans. use of a Soluble hemicellulose, as described, as an additive or ingredient in foods Such as baked products, processed 0.055 A still further aspect of the invention relates to a meats, dairy products, Soups and Sauces, high protein drinkS germ containing Sterols known to reduce the uptake of and health drinks. cholesterol in humans and intact E complex, Sterols, lecithins, phospholipids and glycolipids. 0068 A further aspect of the invention relates to a use of a Soluble oligosaccharide, as described, in feed and food 0056. A further aspect of the invention relates to a applications as a functional Soluble dietary fibre or low defatted germ rich protein produced in accordance with the calorie Sweetener, or as a raw material for further processing invention. to extract lignans and associated phenolics Such as ferulic acid, or as a feedstock for industrial fermentation. 0057. A still further aspect of the invention relates to an aleurone-rich oil produced in accordance with the invention. 0069. A still further aspect of the invention relates to a use of a Soluble oligosaccharide, as described, in confec 0.058 A further aspect of the invention relates to a tionery formulations in combination with glucose or other defatted aleurone-rich protein produced. Sugar SyrupS and further concentrated to produce moisture 0059 A still further aspect of the invention relates to a Stable products. protein fraction, wherein proteases are incorporated in to the 0070 A further aspect of the invention encompasses use Said fraction in Wet State and at controlled temperature and of a Soluble oligosaccharide, as described in claim 19, in US 2005/0O896O2A1 Apr. 28, 2005

food and biomedical applications as a combined Source of important production constraints and created interesting lignans and fermentable oligosaccharides for the conversion opportunities to extract and Separate new components from of lignans into active cancer-reducing agents Such as entero cereal branS. Furthermore, the inventors have developed a lactones. simple method in which wet milling is combined with 0071. A still further aspect of the invention relates to a enzymatic treatment using food grade commercial Xylanases use of a Sugar fraction, as described, in feed, food and and/or beta-glucanases, and cheap industrial Separation pro industrial fermentation applications as an energy Source, CCSSCS. flavouring agent and binding agent. 0081) Essentially, this invention allows one to economi 0.072 A further aspect of the invention relates to a set up cally produce fractions derived Substantially from the germ, for carrying out the process, wherein it comprises a hydroly endosperm and aleurone cells, hemicellulose, oligosaccha sis vessel, a wet mill, a heat eXchange for enzymatic ride and insoluble dietary fibre. inactivation, decanters, a holding tank, an ultra-filter, and EXAMPLE 1. optionally at least an evaporator, and dryers. 0082) Wheat bran produced from short milling (SMB) 0073. A still further aspect of the invention relates to a set and conventional milling (CMB) processes were used in this up for carrying out the process, wherein it comprises a trial. Bran Sample of 25 kg was transferred to a mixing tank hydrolysis vessels, a wet mill, a heat eXchange for enzymatic and Sequentially hydrolysed at temperatures varying from inactivation, decanters, a holding tank, an ultra-filter, and 70° C. at the first stage with O-amylase to 60° C. in the optionally evaporators, and dryers. Second Stage with amyloglucosidase for a total hydrolysis time of 3 h. During this period the reaction mixture was 0.074. In a further embodiment of the process of the intermittently wet milled to increase in Surface area and invention, the enzymatic treatment is carried out at a pH of dispersion of soluble components. The pH of the reaction 4 to 7.5 and at a temperature of from 50 to 90° C., at an mixture was Set at neutral initially and then decreased down enzymatic activity of 200 to 1500 IU/g of Substrate. to 4.5 with acetic acid in the Second Stage. In addition of 0075. In a further embodiment of the process the enzy maximising the enzymatic activity the acidic pH allowed matic treatment is carried out at a pH of 4 to 7, preferably partial Solubilization of the phytates present in the bran. 4.5-5.5, and at a temperature of from 35 to 80 C., at an 0083. At the end of the enzymatic hydrolysis and wet enzymatic activity of at least 1 IU/g of Substrate, preferably milling Step the enzymes contained in the reaction mixture 200 to 1500 IU/g of substrate. were inactivated by wet heating through a heat exchange and quickly cooled down to room temperature. DESCRIPTION OF THE PREFERRED 0084. The hydrolysed bran solution was then put through EMBODIMENTS a two-phase decanter to separate the insoluble (fibre and 0.076 It is widely known and accepted that when cereals aleurone fractions) from the soluble fraction. are milled with the purpose of producing flour the most 0085. The soluble fraction was fed to a separator so that nutritious part of the grain is diverted into the by-product, the heavy phase containing mostly the germ components i.e. cereal bran. Despite the fact that cereal branS are rich in could be separated from the light phase containing mainly proteins, oils, and minerals its use in the food components from the remaining endosperm found in the industry and high value feed industry is rather limited. bran. The light fraction, which was contained high levels of 0077. There is now developed an industrial process, Sugar, was processed in an ultrafilter having a 50 kDa which makes possible the separation of fractions of different membrane in order to Separate low molecular weight Sugars nature from various cereal bran, produce high value protein, and a protein fraction. Soluble non-Starch carbohydrates, and optionally oil frac 0086 All soluble protein fractions, i.e. heavy and light tions, and extract virtually all insoluble fibre as a Separate phases, were blended together and finally processed through fraction. The resulting low-fibre protein and Sugar fractions Spray drying. The Sugar fraction was concentrated by as well as the insoluble fibre fraction have much broader vacuum evaporation at mild temperature (40 to 60° C) until market applications and greater value than the original bran. a 75% sugar concentration was achieved. The fibre fraction 0078 Various methods of extraction and fractionation of was dried in a conventional laboratory oven, but in an hemicelluloses from cereal brans have been developed in the industrial process this can be carried out by a number of past. Equally, various methods for the extraction of valuable different dryers, i.e. tumble drier, ring drier, fine grinder, etc. proteins and insoluble dietary fibre from cereal brans have 0087 Average chemical composition of the brans and been disclosed. their respective fractions are shown below in Table 1. 0079 The problem is that when one combines the use of commercial cereal brans, which contain large quantities of TABLE 1. Soluble components Such as Starch, Soluble proteins, pen Dry tosans, oils, etc., and a simple extraction proceSS Such Sample matter Protein Oil Fibre Ash NNE* * * Solubles eventually become contaminants of the main prod uct Streams, and therefore have to be removed. This is a CMB* 90.8 15.7 4.1 45.4 5.5 29.3 costly procedure and in many cases jeopardizes the market CMB fractions of the process value of the non-hemicellulose components. Protein phase 92.9 31.8 7.7 1.1 7.9 515 Fibre 92.8 13.6 3.0 76.9 4.1 2.4 0080. By utilising previously cleaned bran as the pre SMB** 89.1 14.3 2.3 23.7 3.2 56.5 ferred raw material, the inventors have overcome many US 2005/0O896O2A1 Apr. 28, 2005

fibre fractions, the latter Still containing Substantially high TABLE 1-continued amounts of aleurone proteins. It could be of interest for Same applications to Separate, at least partly, the aleurone proteins Dry from the bran pericarp (fibre) and recover Such proteins in Sample matter Protein Oil Fibre Ash NNE * * * the same fraction as the endosperm-rich fraction for SMB fractions of the process instance. Protein phase 93.9 27.8 1.5 O.9 3.4 66.4 0096. A trial was set up in the same way as described in Fibre 94.3 22.5 4.1 64.8 1.6 7.0 EXAMPLE 2, except that a cocktail of polysaccharidases *Conventional milling bran containing both high cellulase and Xylanase activities was **Short milling bran added together with the amyloglucosidases, and let to work ***Non-nitrogen extracts for 3 h. Temperature and pH conditions were kept unchanged. The resulting reaction mixture was further EXAMPLE 2 treated exactly as described in EXAMPLE 2. 0097. The inclusion of polysaccharidase during the 0088. Wheat bran produced from conventional milling hydrolysis Step had a positive effect with regards to aleurone was Subjected to enzymatic treatment and wet milling as protein extraction and protein recovery as measured by the described in Example 1. The hydrolysed bran was fraction mass balance and protein content. The protein content in the ated using a two-phase decanter into an insoluble (combined endosperm-rich fraction increased from 28.7% (without fibre and aleurone) and a Soluble fraction. polysaccharidases) to 34.7% (with polysaccharidases) and 0089. The soluble fraction was fed into a separator for the overall protein recovery was increased by 35% when fractionation using centrifugal forces thus producing two polysaccharidase was added. phases. The germ-rich phase was washed with water and fed again to the Separator to remove the exceSS Solubles. The EXAMPLE 4 resulting protein fraction was kept as Such or mixed with evaporated liquid whey on a 1:1 ratio (dry matter basis). 0098. The colour of protein ingredients can be of impor tance particularly in Some food and feed applications. Milk 0090 The endosperm-rich wheat fraction, which con products Such as caseinates, whey powder and whey protein tained high levels of Sugar, was processed in to an ultrafilter concentrate has a light colour and Soy protein concentrate in order to Separate low molecular weight SugarS and a have a light brown colour. These products are the main protein fraction. ingredients in high value feeds Such as calf milk replacer. 0.091 All soluble protein fractions, i.e. germ and But, in Same food applications Such as Sausage and ham endosperm-rich phases and the mixtures with whey, were burger despite the fact the inclusion level is much lower, Spray dried Separately. The Sugar fraction was concentrated colour can Still play an important role in the product accept by vacuum evaporation at mild temperature (t=60° C) until ability. 75 Brix was achieved. The fibre fraction was oven dried. 0099. The technical feasibility of bleaching the germ-rich 0092. The additional washing carried out on both germ fraction was assessed by two means. 1. Solely alkali and rich protein and fibre fraction was very effective to decrease hydrogen peroxide bleaching, and 2. Alkali-free peroxidase the amount of light Soluble contaminants from each fraction, and hydrogen peroxide bleaching. and therefore increase the relative content of valuable com ponents. 0100) 1. 10 (ten) g samples of germ-rich fraction were incubated in 1 L beakers containing 100 ml water. Samples 0.093 Compositional data indicates that germ and were dispersed with stirring and ca. 0.25 ml NaOH added endosperm-rich protein fractions have a different relative until pH 12 was reached. Solutions were warmed at 50 C. content of protein and oil. Protein and oil content from the and 3.5, 5 and 10 ml of 30% HO were added to different former were 48.6 and 18.6%, respectively and those from flasks to provide uptake levels 10, 15 and 30% HO on the latter were 28.7% and 1.5%, respectively. The insoluble weight basis of germ-rich fraction. Mixtures were stirred for phase containing primarily the bran pericarp (fibre) and the 1 h and neutralise with acetic acid. aleurone proteins had 86.4% fibre and 12.6% protein. The chemical composition of the germ-rich phase-whey mix 0101 Full bleaching was achieved with 15 and 30% was 31.5% protein, 9.8% oil and 37% lactose. HO. Sample treated with 10% HO was only partly bleached. All alkali bleached samples became darker with 0094. A further important observation was that the spray drying. dried germ-rich fraction containing 18.6% oil was Substan tially more resistant to oxidation () compared 0102 2. 10 (ten) g sample of germ-rich fraction was to the original wheat bran. The original wheat bran Started incubated in 1 L beaker containing 100 ml water. Samples getting rancid after 3 weeks of Storage. Despite the fact that were dispersed with stirring and ca. 0.25 ml NS 51004 no exogenous anti-oxidants were added to the germ-rich Novozymes per-oxidase was added. Solution was warmed at fraction it only started going off after 12 weeks of Storage. 50° C. and 3.5 of 30% HO was added to the flask, i.e. 10% HO on weight basis of germ-rich fraction, and the mixture EXAMPLE 3 stirred for 2 hrs. 0.095 Previous examples illustrate the use of starch 0103) The peroxidase-hydrogen peroxide bleaching hydrolysing enzymes and wet milling followed by various was effective, consumed less chemicals and no darkening of Separation Steps in order to yield both protein, Sugar and the Sample was observed after drying. US 2005/0O896O2A1 Apr. 28, 2005

EXAMPLE 5 despite the amount of enzyme used. Extraction rates of 3.1, 32.0, 32.8, 33.1, 33.8 and 34.2%, respectively, were 0104. Amongst the various end-uses of the germ-rich obtained from control, 0.1, 0.25, 0.5, 1 and 2% treatments, fraction of EXAMPLES 14 one could describe meat prod respectively. ucts Such as hamburgers, Sausages and meatballs. In Such end-uses germ-rich fraction could replace meat, Soy protein EXAMPLE 7 concentrate and isolate, but also milk casein and caseinates, to mention just a few. It is therefore important to test the 0115) A similar trial to that described above was carried overall performance of the germ-rich fraction with regards out with a purified endo 1,4-beta Xylanase (pentosanase) at to emulsifying and binding capacity, taste, etc. two levels of inclusion: 0.25 and 0.5% (w/w basis). 0105. A trial set up to test the feasibility of incorporating 0116 Extraction rates were also high, and increased from various germ-rich fractions extracted from wheat bran into 3.1 (control treatment-no enzyme) to 28.6 and 26.1%, a traditional meat ball recipe consisted of meat, garlic, when 0.25 and 0.5% pentosanases were added, respectively. premix and water. EXAMPLE 8 0106 The following spray dried fractions were tested: 0117 Cleaned bran with the same specification as described in Examples 6 and 7 was used in a large-scale trial. 0107 Germ-rich fraction extracted from short mill The objective was to validate a process using Standard ing wheat bran-(I) industrial equipments, quantify process parameters, deter 0.108 Germ-rich fraction extracted from conven mine extraction rates of the various fractions, and ultimately tional milling wheat bran-(II) characterize the end products. 0109) 1:1 mix of whey and II, on dry matter basis 0118 Cleaned bran (80 kg) was incubated in hydrolysis (III). tanks containing 500 l water. The pH was adjusted to 5.5 and a purified endo 1,4-beta Xylanase (pentosanase) was added 0110 Meatball recipes were tested without germ-rich at 0.5% (w/w basis). The reaction mixture was continuously fraction (control recipe) or with 2.5% inclusion of samples stirred and intermittently wet milled while kept at 40 C. for I, II or III. Meatballs were analysed for weight loss, taste, 90 min. The hydrolysis/wet milling treatment was termi texture and colour after frying. The results are described in nated by heating up the reaction mixture to 90° C. for 2 min the table 2 below. in a heat exchange device. TABLE 2 0119) The inactivated hydrolysate was pumped through a commercial two-phase decanter where the insoluble phase Weight loss (insoluble dietary fibre) was separated from the solubles. after frying The insoluble phase was dried and further milled in a Recipe tested (%) Colour Texture commercial fine grinder using indirect heat. Control (meat, garlic, 23.4 Reference Reference premix and water) 0120) The solubles were then pumped through another Control + 2.5% of I 21.3 Slightly Slightly tougher two-phase decanter where a heavy phase (aleurone protein darker Control + 2.5% of II 20.8 Similar Similar rich fraction) was separated from a light phase containing Control + 2.5% of III 18.0 Similar Slightly more the extracted hemicellulose fraction in the form of both tender Soluble hemicelluloses and oligosaccharides. The protein rich phase was spray dried. 0121 The hemicellulose fraction was further separated 0111. The overall conclusion was that the samples per by size exclusion using an ultrafiltration unit whereby the formed well as additives in a meat ball recipe, and were large molecular size fraction (Soluble hemicellulose) was particularly interesting as they all decreased the weight loSS Separated from the Small molecular size fraction (oligosac after frying. charides and Sugars). The resulting fractions were further processed by Spray drying into a fine powder or alternatively EXAMPLE 6 evaporating the exceSS Water until 25% water content was 0112 Laboratory scale trials were carried out on cleaned achieved. wheat bran to test extraction rates using Xylanase treatment. The cleaned wheat bran used as a raw material contained 0.122 The following yields of insoluble dietary fibre, less than 1% starch and at least 50% and 70% of the protein hemicellulose, oligosaccharides and protein-rich fractions and oil, respectively, originally found in the Starting material were obtained from cleaned wheat bran: 51.0, 26.1, 17.3 and had been removed. 7.7%, respectively. 0113 Ten (10) g of cleaned bran were incubated in 150 EXAMPLE 9 ml water, the pH adjusted to 5.5 with acetic acid and an 0123. An insoluble fibre fraction extracted according to enzyme cocktail containing pentosanase and hemicellulase the procedure described in Example 8 was characterized activities was added at the following concentrations: 0 with focus on its potential use as a Source of dietary fibre and (control), 0.1, 0.25, 0.5, 1 and 2% (w/w basis). Reaction texturizer in food applications. mixtures were kept at 40 C. for 120 min. The treatment was 0.124 Typical composition was as following: dry matter terminated by inactivating the enzymes at 80 C. for 30 min. 95%, cell wall components 75%, protein 11%, soluble 0114) Results indicated relatively high extraction rates Sugars 3% (of which at least 75% is glucose), fat 4% and compared to the control treatment (no enzyme added) minerals 1.5%. US 2005/0O896O2A1 Apr. 28, 2005

0125 The water holding capacity (WHC), of primary can be used as a casein replacer in the production of Sausage, importance when assessing the usefulness of insoluble Spreads, dressings, etc., as well. dietary fibres, of the above-described product was 8.6 g of water/g Sample on dry basis. For comparison purposes the 0.136. In the feed industry, the germ-rich fraction is an WHC of wheat bran in the range of 3.5 g/g and that of ideal ingredient for high value feeds Such as calf milk cleaned wheat bran is 7.5 g/g. This indicates the improved replacer, Starter feeds for calves, piglets and chicks, fish water absorption of the fibre after cell wall components have feeds and pet food. In food applications it can Substantially been partly removed. Other commercial dietary fibres replace the use of Soy proteins (texturized Soy, concentrate extracted from wheat and Sugar beet have WHC of 6.3 and isolate), potato protein, hydrolysed gluten, high quality and 7.9 g/g, respectively. fishmeal, plasma protein, and dry milk products Such whey protein concentrate, whey and skimmed milk. EXAMPLE 10 0.137 The germ rich protein is of great interest as a 0.126 The protein fraction, which contains substantial functional food ingredient, especially in the case of rye, amounts of aleurone proteins, produced as described in primarily because it contains rye germ oil. Example 8, have a very interesting chemical composition, functionality and is an Ideal raw material for further pro 0138 Endosperm-Rich Fraction cessing. 0.139. This protein fraction is mainly derived from the 0127. A typical composition of the protein fraction is: dry residual endosperm proteins in cereal bran. It contains matter 98%, protein 40%, sugar 3%, fat 18%, non-Sugar 25-40% protein, much of which is highly soluble. It is also carbohydrates 32% and minerals 5%. particularly rich in soluble dietary fibre pentosans (>35%), 0128. In order to determine the effect of protease treat has a high water holding capacity and has a light colour. ment on the functionalities of the protein fraction, a protein 0140. The endosperm-rich fraction can be used in the Sample was Subjected to a mild protease treatment and the food industry in baked products, processed meats, dairy Samples analysed for dry matter and protein Solubility, products, Soups and Sauces, high protein drinks and health emulsifying capacity and emulsifying Stability. drinkS. It is a valuable Source of non-Starch polysaccharides, 0129. The results are shown in Table 3, and clearly which are excellent soluble dietary fibre and water-binding indicate the possibilities to further improve Some important materials. In feed applications, it can partly replace gluten, functionalities of the protein fraction. Soy and milk proteins as an ingredient to calf milk replacer, piglet Started feed and fish feed. TABLE 3 0.141. The emulsifying, water binding and foam stabilis Protein fraction treated ing properties are equivalent or better than those of other Parameters analysed Protein fraction with protease commercial proteins like caseinates, Soy protein concentrate Dry matter solubility (%) 19.7 38.1 and modified . The endosperm-rich protein is Protein solubility (%) 18.4 55.5 very Suitable to be used as an ingredient in milk replacer Emulsifying capacity (%) 52.5 90.6 formulae (both for humans and calves), Sauces, mayonnaise, Emulsifying stability (%) 47.5 86.0 dressings etc. 0.142 Because it contains high amounts of pentosans and 0130 Characterization & End-Uses asSociated ferulic acid there are extra health and functional benefits. In the cosmetic industry the Stabilising, emulsify 0131 Germ-Rich Fraction ing and water holding properties are ideal. 0132) The high protein content of the germ-rich fractions 0.143 A combination of the endosperm rich protein and makes it an ideal Substitute for existing expensive proteins Soluble hemicellulose is interesting in a number of food and from animal and vegetable origin. Additionally, the germ biomedical applications, because of the emulsifying effect of rich fraction because of the nature of its protein, the presence the endosperm rich protein and the Soluble pentosans and the of high quality oil, phospholipids and Sterols also exhibit thickening effects of the soluble hemicellulose. interesting functionalities Such as emulsification, texture and binding, and health benefits associated with cholesterol 0144. The endosperm-rich fraction is high in pentosan control in humans. hemicelluloses, mainly arabinoxylans in rye and wheat, or beta-glucans in the case of oat and barley. The claimed 0133) One can list, as examples, the following existing health benefits are therefore as described for arabinoxylans products, which can be replaced by the germ-rich fraction in and beta-glucans (see below). the food industry: 0145 Aleurone-Rich Fraction 0134) Animal protein: casein and caseinates, plasma protein and egg white Vegetable proteins: Soy protein 0146 This is the protein that is derived primarily from concentrates and isolates, texturized Soy, hydrolysed the aleurone cell layer and is both a functional and nutri gluten and potato protein. tionally valuable material, rich in essential amino acids. 0135 Generally, the above products can be used as meat 0147 In the food business it would be used as an emul extenders and texturizer ingredients in hamburger, Sausage, sifier, foam Stabilizer and texturiser. In addition there is high and meat balls production to mention a few. The products potential as a protein Supplement. US 2005/0O896O2A1 Apr. 28, 2005

0148. Insoluble Fibre Fraction industrial fermentation Since it produces fewer waste prod ucts. Ethanol and citric acid industries are therefore ideal 014.9 The insoluble fibre fraction can be used as an interesting Source of fibre in food applications. The main consumers of very large quantities of Such a product. The end-uses as food would be as a texturizing and water binding production of Single-cell protein for the feed and food additive in processed foods particularly meat products, and markets can also be considered. as a Source of dietary fibre in breakfast cereals, baked 0157 Soluble Hemicellulose products and health products. Specifically the high water 0158. This is the major cell wall non-cellulose polysac binding capacity and beneficial effect on bowel function charide in cereal bran. It can be produced with a medium to makes it an interesting product for the biomedical market. high molecular weight and high Solubility (the combination 0150. This is the remaining fibre after the soluble com of these two properties is the powerful aspect). Because the ponents (first step process) and a proportion of hemicellu product is a pentosan (arabino-Xylan) it is low calorie and lose (Second step process) has been removed from cereal beneficial for gut health. The product can be produced with bran. The insoluble fibre fraction is a cleaned cereal fibre or without ferulate side chains or free ferulic acid and other containing low levels of . Because the fibre has antioxidants and is a free flowing cream powder. already been partially “digested’ enzymatically, many ben 0159. Due to its composition and high water binding eficial compounds derived from the cell wall are available to capacity it is ideal to use as a thickener, gellant, Stabilizer, the gut for absorption. Soluble dietary fibre and fat replacer. A non-gellant form of 0151. The insoluble fibre has high water binding capac Soluble hemicellulose can also be produced. As a thickener ity, i.e. typically 100% higher than that of wheat bran. This and gellant it is interesting in the food industry as an additive provides increased gut transit (digesta flow). The remaining in Soups, margarine, deserts, pates, Sauces etc. AS a Stabilizer pentosans are more accessible to the gut wall (cholesterol it is a cheaper alternative or modified Starch (made from reducing) due to the fractionation process. wheat, maize etc), modified cellulose, gums (guar gum and carrageenan gum), alginates (seaweed), gelatin (cheap but 0152 Because of the increased availability of lignin type problem with BSE) and pectin (fruit peel & Sugar beet). materials and other antioxidants within the fibre, various Finally, it has a good potential in drinkS because it is an health benefits can be claimed. Specifically the lignans and excellent Source of Soluble dietary fibre alongside its Stabi polyphenolics from rye are known to mimic estrogens lizing properties. (female hormones), and more recently have been found to help preventing various types of cancers. This has been 0160 It is possible to supply the pentosan with ferulate verified for rye products. Side chains, and in this form the Substance will gel in combination with oxygen and enzyme. AS Such it is an 0153. Additionally the insoluble fibre is also a good raw interesting material, for example, for wound dressings as it material for the further extraction (enzymatically) of lignins, will keep the skin in a hydrated State and therapeutic agents ferulic acids, lignans etc., which are natural antioxidants and potential anticancer agents. These can be used in many can be added. biomedical and "cosmetic pharmaceutical” applications 0.161 From rye and wheat, this is almost exclusively Such as lotions, creams and moisturizers. The ferulic acid is arabino-Xylan (pentosan) hemicellulose. This is readily fer an effective UV absorber and as Such can be used in a mented in the colon, is low calorie to the human and is SUSCCC. reported to generate butyrate as a short chain fatty acid (SCFA) end product after fermentation. This is the most 0154 Insoluble dietary fibres are rich in accessible lign “healthy” SCFA according to recent studies as it is a anS and residual pentosans/hemicelluloses. Bacteria present preferred Source of energy to epithelial cells lining the colon. in the colon convert plant lignans to mammalian lignan, enterolactone, using hemicelluloses as a fermentation The health benefits of adding an enriched, available Source medium. These compounds mimic estrogens and appear to of arabinoxylan to the diet may therefore be far-reaching. have a tangible, demonstrable effect on the Suppression of 0162 This fraction is a perfect soluble dietary fibre, with hormone related cancers, e.g., breast, Ovarian and proStrate all of the concomitant health benefits. Arabinoxylans are cancers. Rye insoluble dietary fibre Specifically contains the also thought to be excellent binding Sites for Secondary bile lignans Secoisolariciresinol (SECO) and matairesinol acids, as a consequence of the rigidity of parts of the (MAT), which are known precursors of enterolactone. The molecular chain and the occurrence of relatively hydropho insoluble dietary fibre from wheat also contains these lign bic domains on the polymer. This is thought to reduce any ans, but the effect is not demonstrated in wheat. It is potential carcinogenic effects. In addition, this fraction con important to State that in this fraction the lignans are tains ferulic ester Side chains to a proportion of the polymer, Supplied in an accessible form, as the cell wall is already with concomitant free radical Stabilising and anti-oxidant partially enzymatically digested, along with their natural properties. Synergistic partners, the arabinoxylan hemicelluloses remaining on the fibre. 0163. It is important to emphasise that the arabinoxylan concentrated in this fraction is not normally available to the 0155 Sugar Fraction gut and colon if presented as a part of a normal diet or even 0156 This is the glucose produced from enzymatic deg from conventional bran. radation of residual Starch of the bran and is a more pure 0164. In oat and barley, this fraction is rich in beta-glucan product compared to molasses. It can be used in feed and with all of the documented beneficial effects of this polysac food applications as an energy Source, flavouring agent and charide. There is a tangible benefit in Supplying a concen binding agent. Alternatively, it is ideal as a feedstock for trated beta-glucan of this nature as the normal ingestion of US 2005/0O896O2A1 Apr. 28, 2005

oats does not supply sufficient material for the full effects to extracted without using any Solvent, and it contains no be realised. Purified beta-glucan can be purchased but is preservatives or additives. It is a good Source of poly and very expensive because of the extensive purification regime. mono unsaturated fat, has a good flavour, is rich in Vitamin It is important to realise that this high purification is required E and can be Suspended easily. As a flavouring component to remove the chemicals utilised in the extraction process. It it is good in wheat or rye based products (cereals, baking is Suspected that natural Synergistic partner compounds are goods, biscuits etc), deserts, ice creams etc. It can also be removed in Such a process, whilst these materials should still useful as an ingredient in fat and oil formulations, etc. be present in the present process fraction. with natural . 0.165 Oligosaccharide Syrup 0.175 Rye germ oil is particularly rich in naturally occur ring B-Sitosterol, a cholesterol lowering compound and 0166 This is derived from the hemicellulose fraction and , a cholesterol “burner'. These materials can be is a 100% soluble dietary fibre of low molecular weight and classified as “natural Synergistic partners', an important low Viscosity. The oligosaccharide Syrup can be produced factor in the functional food area. This massively increases with lignans, ferulic acid and other antioxidants and is the potential of the oil as a value-added neutraceutical extremely Soluble and hygroscopic. ingredient in foods Such as margarines and Spreads. 0167. It has a high potential in the drinks industry as it 0176 Germ oil is also a good UV blocker and therefore has low Viscosity, is a good Source of dietary fibre and gives together with ferulic acid it can be ideal as a component of good mouth feel and texture. a Sun tan lotion. Its emulsifying properties make it very 0.168. In combination with glucose syrup it could be used Suitable as an emulsion Stabilizer and as an emollient as a Sweetener and energy Source for drinks, cereal bars etc. ingredient for skin creams. AS it is rich in ferulic acid, pentosans and Solubilised lignans, one can also claim the related health benefits. It is 0177) Defatted Protein Fractions very important to Supply lignans in the presence of pentosan 0.178 This is the protein that remains after the oil has oligomers if the full cancer prevention effect is to be been extracted from full fat germ- and aleurone-rich frac expressed and realised: precisely the Situation in this frac tions and has at least 60% protein content. It is also a good tion. functional protein, has an extremely high fat binding capac 0169. The combination of glucose syrup and oligosac ity and can be easily upgraded enzymatically to increase charide Syrup is also ideal in applications where one requires Solubility, emulsion and foam stabilisation properties. increased dietary fibre content and increased water binding 0179 The product is an excellent stabiliser for water in capacity without thickening. Soluble oligosaccharide can oil emulsions and is interesting as a meat texturiser or also be used in confectionery formulations in combination extender in Sausages, burgers, patees etc. The defatted with glucose or other Sugar SyrupS and further concentrated protein fraction is a functional protein that can easily replace to produce moisture Stable products. Soya proteins and contains phospholipids, natural lecithins 0170 The oligosaccharide syrup is the low molecular and glycolipids. weight fraction of the Solubilised arabinoxylans along with 0180. It has a high potential in cosmetic formulation as an other low molecular weight components Solubilised from the emulsion Stabiliser because it contains natural lecithins. cell wail. This includes dissolved lignin fragments, phenolic compounds Such as ferulic acid and lignans. AS with the 0181. One preferred embodiment of a plant for carrying insoluble dietary fibre, the presence of lignans with arabi out the invention on cereal bran is shown in the attached noxylan gives rise to claims for cancer preventative roles for drawing, wherein rye and wheat derived fractions. In this case, the arabinoxy 0182 FIG. 1 shows a set-up for carrying out a preferred lans are present as oligomers and the lignans are very embodiment of the invention; and available in the Syrup with a potential high accessibility for the gut. This should increase the rate of conversion of the 0183 FIG.2 shows the fractionation of the cereal bran in plant lignans to enterolactone with a potentially larger an over-view. impact on cancer prevention. 0.184 One preferred embodiment of a plant for carrying 0171 Furthermore, the presence of high concentrations out the invention related to Separation of cereal bran or of oligomeric arabinoxylan provides a ready fermentation cleaned cereal bran is shown in the attached drawing, FIG. substrate for the production of beneficial SCFAS such as 1 wherein 1 denotes a Suspension and hydrolysis vessel to butyrate, with benefits as described for the hemicellulose which a wet mill 2 is connected. The reaction mixture is fraction. intermittently pumped through the wet mill 2 (from 1 to 3 times). The hydrolyzate is then optionally inactivated in a 0172 This fraction, especially in the case of rye, is heat eXchange 3 and transferred to a two-phase decanter 4, probably the most interesting in the present context being an which decanter 4 separates the insoluble (insoluble fibre) excellent Source of arabinoxylans, lignans and phenolic from the soluble phase. The insoluble phase having a dry antioxidants in Very accessible forms along with relevant matter content of about 35% is dried to approximately 95% Synergistic partner compounds. In oat and barley, it is a good dry matter in a ring drier 5. The Soluble phase, having a dry Source of low molecular weight beta-glucan fragments. matter content of approximately 3%, is pumped through another two-phase decanter 7, or optionally a Separator, via 0173 Germ Oil a holding tank 6, in which two-phase decanter 7 protein-rich 0.174. The germ oil is derived from the germ rich protein fraction is separated off. The protein-rich fraction is option and is a high quality food grade oil and ingredient. It can be ally enzymatically treated for improved functionality in US 2005/0O896O2A1 Apr. 28, 2005

hydrolysis vessel 8, and then dried to about 95% dry matter 7. A process according to claim 5, wherein cereal bran in spray drier 9. The soluble (liquid) phase from the two substantially free of both in water or less polar solvents phase decanter 7, having a dry matter content of approxi Soluble compounds are derived from wheat, rice, barley, oat, mately 3%, is allowed to pass an ultra filter 10 having a rye or triticale. molecular cut between 20 and 100 kD, preferably between 8. A process according to claim 1, wherein the combina 20 and 50 kD, which will depend on different product tion of intermittent wet milling with enzymatic treatment is requirements. The retentate (fraction retained in the ultra arranged to increase the rate of enzymatic hydrolysis of the filter) from ultrafilter 10 is optionally enzymatically treated substrate thereby improving the overall hydrolysis perfor for improved functionality in hydrolysis vessel 11, and then mance and the Subsequent Separation of the various fractions dried to about 95% dry matter in spray drier 12 or optionally by density/Solubility and molecular size. evaporated to a Syrup concentration of at least 75% Solids in 9. A proceSS according to claim 5, wherein the enzymatic an evaporator. The permeate fraction (not retained in the treatment is carried out using at least one non-Starch degrad ultrafilter) from ultrafilter 10 is preferably evaporated to a able polysaccharidase in the form of cellulases, hemicellu Syrup concentration of at least 75% Solids in an evaporator lases mainly Xylanases, beta-glucanases, and pectinases, and 13. optionally phytases. 10. A process according to claim 9, wherein the enzymatic 1. A process for the wet fractionation of cereal bran treatment is accomplished by using Xylanases with high beta components, wherein bran is first Subjected to a combination 1-4-Xylanase (pentosanase) and/or beta-glucanase activity. of enzymatic treatment with enzymes of the group Starch 11. A protein fraction derived substantially from the germ and phytate-hydrolysing enzymes, and aqueous wet milling, and produced according to claim 1, wherein the Said fraction followed by an optional Step of enzyme inactivation by wet contains at least 35% protein and 10% oil on dry matter basis heat treatment, and a Subsequent Step whereby the insoluble and exhibits a high emulsifying capacity and an increased phase containing a cleaned bran consisting of both pericarp Shelf life with regards to resistance to oxidation compared to and aleurone fractions are separated by centrifugal forces the original bran, and that the Said fraction contains less than into an aqueous phase containing a germ-rich fraction and a 5% fibre. further aqueous phase containing residual endosperm com 12. A protein fraction derived substantially from the ponents, and that the proteins contained in the endosperm residual endosperm and produced according to claim 1, rich fraction are concentrated. wherein the Said fraction contains at least 25% protein and 2. A process according to claim 1, wherein cereal brans 10%. Sugar and less than 3% oil and 3% fibre, and at least are the fibrous-residue resulting from a primary grain mill 25% Soluble high-molecular weight non-Starch polysaccha ing, i.e. after the Separation of the endosperm fraction, of rides of the groups beta-glucans for barley and oat and wheat, rice, barley, oat, rye and triticale, and having variable arabinoxylans for wheat, rice, rye and triticale. chemical compositions, presence of anti-nutritive factors, 13. A protein fraction according to claim 12, wherein and presence of various anatomical fractions, i.e. pericarp, liquid whey is incorporated in to the Said fraction at levels germ, and residual endosperm. varying from 20 to 80% by weight on dry matter basis, and 3. A proceSS according to claim 1, wherein the enzymatic that the final mixture is dried. treatment is accomplished using a Starch degrading enzyme 14. An insoluble fibre fraction produced according to of the group of amylases and amyloglucosidases. claim 1, wherein the Said fraction consists of cell wall 4. A process according to claim 1, wherein a further components of bran in an amount of at least 85% and enzymatic treatment is carried out using at least one non aleurone proteins in an amount of at least 10%, and Sub Starch degrading polysaccharidase in the form of cellulases, Stantially free of gluten and Starch, and with a high water hemicellulases mainly Xylanases, beta-glucanases, and pec holding capacity of at least 6 g water/g dry product. tinases, and/or phytases. 15. A Sugar fraction produced according to claim 1, 5. A process for the wet fractionation of cereal bran wherein the Said fraction is originated primarily from the Substantially free of Soluble compounds produced according residual endosperm and it contains more than 65%. Sugars, to claim 1, wherein Such cleaned bran is Subjected to a Such as glucose, maltose and malto-triose on dry matter combination of enzymatic treatment with Specific enzymes basis. of the group Xylanase and/or beta-glucanase under Strictly 16. A protein fraction derived substantially from the controlled hydrolysis conditions, and intermittent wet mill aleurone cells and produced according to claim 5, wherein ing, followed by an optional Step of enzyme inactivation by the said fraction contains at least 35% protein and 10% oil, wet heat treatment. less than 5% insoluble fibre on dry matter basis, substan 6. A process according to claim 5, wherein the inactivated tially free of gluten and Starch and with a high emulsifying hydrolysate is then fractionated by centrifugal forces into an capacity. insoluble phase containing primarily cellulose, lignin, leSS 17. An insoluble fibre fraction produced according to accessible hemicellulose, residual aleurone cells and cell claim 5, wherein the Said fraction consists primarily of cell wall bound proteins, and an aqueous phase containing wall components with a relative lower hemicellulose content Soluble hemicellulose, oligosaccharides, Sugars and pro compared to the original cleaned cereal bran, Substantially teins, and that the aqueous phase is further separated by free of gluten and starch (<1% on dry matter basis) and with centrifugal force into protein-rich fraction and a carbohy a high water holding capacity (>6 g water/g dry product). drate-rich fraction, and that the carbohydrate-rich fraction is 18. A Soluble hemicellulose fraction produced according further Separated by Size exclusion technique into a hemi to claim 5, wherein the Said fraction consists primarily of cellulose-rich fraction (medium molecular size fraction) and medium molecular weight hemicellulose preferably above an oligosaccharide-rich fraction (Small molecular size frac 20 kDa in an amount of at least 40% of the groups arabi tion). noxylans from wheat, rye, rice and triticale, and beta US 2005/0O896O2A1 Apr. 28, 2005

glucans from oat and barley, which also contains proteins in 33. The use of a fibre fraction, as described in claim 14, an amount of less than 10% and monosaccharides in an in feed and food applications to replace other insoluble amount of less than 10%, and is substantially free of gluten fibrous products as a texturizing and water binding additive and Starch in an amount of less than 1% on dry matter basis. in processed foods particularly meat products, and as a 19. A Soluble oligosaccharide fraction produced according Source of dietary fibre in breakfast cereals, baked products to claim 5, wherein the Said fraction consists primarily of and health products, or as a raw material for further pro low molecular weight hemicellulose sub-units of below cessing to extract remaining cellulose, hemicellulose, lignin about 20 kDa in an amount of at least 40% of the groups and lignans. arabinoxylans from wheat, rye, rice and triticale, and beta 34. The use of a soluble hemicellulose, as described in glucans from oat and barley, which also contains proteins in claim 18, in feed and food applications as a gellant, thick an amount of less than 10%, monosaccharides in an amount ener, foam Stabilizer, emulsifier, water binder, and as a of less than 20%, lignans and related phenolics in an amount dietary Supplement rich in Soluble dietary fibre, and in of less than 5%, and is substantially free of gluten and starch chemical applications, or as a raw material for further in an amount of less than 1% on dry matter basis. processing to obtain other functional hemicelluloses. 20. A protein fraction according to claim 11, wherein the 35. The use of a soluble hemicellulose, as described in oil can be optionally removed by conventional organic claim 18, as an additive or ingredient in foods Such as baked Solvent extraction or preferably by Supercritical carbon products, processed meats, dairy products, Soups and Sauces, dioxide extraction to yield an oil fraction and a defatted high protein drinks and health drinkS. protein fraction. 21. A protein fraction according to claim 16, wherein the 36. The use of a soluble oligosaccharide, as described in oil can be optionally removed by conventional organic claim 19, in feed and food applications as a functional Solvent extraction or preferably by Supercritical carbon Soluble dietary fibre or low calorie Sweetener, or as a raw dioxide extraction to yield an oil fraction and a defatted material for further processing to extract lignans and asso protein fraction. ciated phenolicS Such as ferulic acid, or as a feedstock for 22. An insoluble dietary fibre according to claim 14, used industrial fermentation. for recovery of cellulose, hemicellulose, lignin and lignans. 37. The use of a soluble oligosaccharide, as described in 23. A germ oil produced in accordance with claim 1 claim 19, in confectionery formulations in combination with containing Sterols known to reduce the uptake of cholesterol glucose or other Sugar Syrups and further concentrated to in humans and intact Vitamin E complex, Sterols, lecithins, produce moisture Stable products. phospholipids and glycolipids. 38. The use of a soluble oligosaccharide, as described in 24. A defatted germ rich protein produced in accordance claim 19, in food and biomedical applications as a combined with claim 1. Source of lignans and fermentable oligosaccharides for the 25. An aleurone-rich oil produced in accordance with conversion of lignans into active cancer-reducing agents claim 1. Such as enterolactones 26. A defatted aleurone-rich protein produced in accor 39. The use of a sugar fraction, as described in claim 15, dance with claim 1. in feed, food and industrial fermentation applications as an 27. A protein fraction according to claim 11, wherein energy Source, flavouring agent and binding agent. proteases are incorporated in to the Said fraction in Wet State 40. A Set up for carrying out the process according to and at controlled temperature and pH conditions, and the claim 1, wherein it comprises a hydrolysis vessel, a wet mill, resulting protein hydrolysate has enhanced functionalities a heat eXchange for enzymatic inactivation, decanters, a Such as Solubility, emulsifying and foaming capacities. holding tank, an ultra-filter, and optionally at least an 28. The use of a protein fraction, as described in claim 11, evaporator, and dryers. in feed and food applications to replace other protein prod 41. A Set up for carrying out the process according to ucts from vegetable and animal Sources. claim 5, wherein it comprises a hydrolysis vessels, a wet 29. The use of a protein fraction, as described in claim 11, mill, a heat eXchange for enzymatic inactivation, decanters, in food application as a texturizer, emulsifier, fat binder and a holding tank, an ultra-filter, and optionally evaporators, fat replacer. and dryers. 30. The use of a protein fraction, as described in claim 12, as a raw material for the extraction of Soluble high-molecu 42. A proceSS according to claim 1, wherein the enzymatic lar weight non-Starch polysaccharides. treatment is carried out for less than 3 hours at a pH of 4 to 31. The use of a protein fraction, as described in claim 12, 7.5 and at a temperature of from 50 to 90° C., at an in food applications as a foam Stabilising agent, whipping enzymatic activity of at least 1 IU/g of Substrate, preferably agent, Water binder, gelling agent, and as a dietary Supple 200 to 1500 IU/g of substrate. ment rich in Soluble dietary fibre (beta-glucans and arabi 43. A proceSS according to claim 5, wherein the enzymatic noxylans) with associated health benefits Such as choles treatment is carried out for less than 3 hours at a pH of 4 to terol-reducing effects of the beta-glucans. 7, preferably 4.5-5.5, and at a temperature of from 35 to 80 32. The use of a protein fraction, as described in claim 12, C., at an enzymatic activity of at least 1 IU/g of Substrate, as an additive or ingredient in foods Such as baked products, preferably 200 to 1500 IU/g of substrate. processed meats, dairy products, Soups and Sauces, high protein drinks and health drinkS.