AU6984400A - A method for the separation of flour - Google Patents

Description

WO 01/17363 PCT/DKOO/00494 1 A method for the separation of flour The present invention relates to the field of flour separa tion. The invention discloses a method for the separation of 5 flour, in particular wheat flour, compositions for the separa tion of flour and the use thereof. Further the invention re lates to a fraction consisting essentially of gluten, and a product comprising said fraction. 1o Background of the invention The industrial implications of flour and the products ob tained from its separation are receiving increasing attention. Flour may be separated into fractions of starch, gluten and fi bres. In the separation of flour the very nature of the flour 15 protein, i.e., gluten, as being water insoluble presents a challenge desirable to overcome. Gluten consists primarily of the proteins, glutenin and gliadin. Upon hydration and during processing gliadin and glu tenin interact to form a network. Proteins of wheat flour form 20 a network with disulphide bridges (S-S-bridges). The network is stronger, the more sulphur bridges are present. The network en traps carbon dioxide formed during fermentation creating the characteristic elasticity of the wheat flour dough. The elastic properties of gluten are due to the glutenin fraction and the 25 viscous properties come from the gliadin fraction. It is there fore a fact that the quality of the flour for the purpose of baking is highly dependent on the amount of gluten contained in the flour. Gluten may be added to flour of poor quality to im prove the baking properties of the flour. 30 Prior art attempts to separate wheat flour have been made. US 4,217,414, US 3,951,938 and UK 2 032 245 all describe exam- WO 01/17363 PCT/DKOO/00494 2 ples of methods for the separation of wheat flour and the re covery of wheat gluten. Here the separation of wheat flour into gluten and starch fractions are performed by the means of me chanical processing. The mechanical separation methods de s scribed in the references are centrifugation, screening, de canting or milling. The application of mechanical techniques of separation pro vided for fractions of gluten and starch. However, to obtain less contaminated fractions effort was directed toward improv io ing the purity of the fractions. The addition of enzymes to the flour or dough proved to be a successful way of achieving an improved separation. The added enzymes are capable of reacting with the flour and thereby im prove the separation quality of the gluten. is In the prior art numerous references disclose such use of gluten improving enzymes. Among those are Weegels et al. (Wee gels, P.L., Marseille, J.P., and Hamer, R.J., 1992, Starch/stArke 44, 2, pp. 44-48) who describe the use of enzymes as a processing aid in the separation of wheat flour into 20 starch and gluten. The enzymes mentioned are lipase, hemicellu lase and cellulase. Further, Christophersen et al. (Christophersen, C., Ander sen, E., Jakobsen, T. S., and Wagner, P., Starch/StArke, 1997, 49, pp. 5-12) describe the successful use of a xylanase to im 25 prove the yield of gluten and starch, without apparent negative effects on the gluten quality. Formerly it has not been possible to obtain gluten frac tions from prior art processes of flour separation having a high content of pure protein, such as gluten fractions consist- WO 01/17363 PCT/DKOO/00494 3 ing essentially of gluten, having a very low content of starch or fibres of the xylan or arabinoxylan type. Summary s It is an object of the present invention to provide for a method for the separation of flour, in particular wheat flour, into one gluten fraction and at least one other fraction, com prising the steps of: a) mixing the flour and a liquid to obtain a dough, io b) separating the dough into a fraction comprising gluten and at least one other fraction, c) recovering at least the gluten fraction, characterized in that an oxidoreductase is added at any of steps a) , b) or c). is In another aspect the invention relates to a method for the separation of flour into one gluten fraction and at least one other fraction, comprising the steps of: a) mixing the flour and a liquid and at least one oxidoreduc tase enzyme obtaining a dough, 20 b) separating the dough into a fraction comprising gluten and at least one other fraction, c) recovering at least the gluten fraction. It should be mentioned that the method can also be carried out as described above where the oxidoreductase(s) in question 25 is(are) added to the (dry) flour. If the oxidoreductase(s) in question has(have) been added to the flour the oxidoreduc tase(s) has(have) time to react with 02 (e.g., from the air or by addition of pure 02) to provide a flour composition with the desired gluten quality.

WO 01/17363 PCT/DKOO/00494 4 The present invention permits the separation of gluten from any quality of flour, in particular wheat flour, even from flour of poor quality, such as flour used for fodder. Thus advantages of the invention may include improved s yields, more pure gluten and/or higher quality of gluten. By the present method an improved separation of flour, in particular wheat flour, is obtained. The improvement in the separation of the flour increases the yield of gluten and starch fractions by providing a more efficient method of sepa 10 ration. Further according to the invention the separated gluten is of a higher quality, (i.e. less contaminated with other flour components, such as starch or fibres) than gluten frac tions obtained according to the prior art. Further the invention relates to a composition for the is separation of flour, in particular wheat flour, into one gluten fraction, and at least one other fraction, comprising at least one oxidoreductase enzyme. The compositions of the invention may be used for the sepa ration of flour, in particular wheat flour. 20 In a further aspect the invention relates to a flour compo sition comprising any of the oxidoreductases mentioned below. The gluten fraction obtained may be added to flour to in crease the gluten content, such as to enhance poor quality flour, and thereby improving the baking quality of the flour. 25 Drawings Fig. 1 shows an example of the steps of a process on wheat flour for preparation of a dough, starch extraction and the separation on sieves.

WO 01/17363 PCT/DKOO/00494 5 Fig. 2 shows an example of the steps of a process on wheat flour for preparation of a homogenized batter (thinned dough) and separation by the means of a decanter centrifuge. s Detailed description of the invention Due to the significance of the industrial applications of flour, in particular wheat flour, much attention is given to the development of separation methods for flour. The present invention reveals a method for the improved separation of io flour, in particular wheat flour, into starch and gluten by us ing oxidoreductase enzymes, whereby gluten of a high quality is obtained. The two main components of flour, such as wheat flour, are gluten and starch. After separation the vitality of gluten is 15 preserved due to controlled drying. When hydrated, the dried gluten must possess the same vitality as the fresh gluten. The vitality of the separated gluten may be determined by the means of gluten vitality tests, such as the farinograph test, or the SDS (sodium dodecyl sulphate) sedimentation test used to deter 20 mine the degree of denaturation of the gluten. The denaturation of the gluten may occur during the drying step, and great care must therefore be taken to ensure a gentle drying procedure. Yet, a further parameter correlating to the vitality of gluten is the protein dry matter ratio, describing the purity 25 of the gluten. The higher the amount of protein of dry matter, the better the quality of gluten. In the present context the term "protein" is meant to equal the term "gluten". The purity of the gluten may be determined by applying the Kjeldahl analy sis, and a dry matter determination analysis.

WO 01/17363 PCT/DKOO/00494 6 Gluten may be used in the food industry, such as in bakery products, pet foods, meat products and as mentioned above in flour fortification. In the case of the latter application it is important that the gluten creates a visco-elastical dough s ball, having good elasticity and extensibility properties, and that it is capable of cohering to a wide variety of products, thereby improving texture, strength and nutritional content of the food item in question. In the baking industry, a high vitality of gluten is corre 10 lated with a high baking quality, i.e., a high bread volume. The higher the denaturation degree, measured by the methods de scribed above, the lower the vitality of the gluten, and the lower the baking quality. Further, the higher the amount of protein of dry matter, measured as mentioned above, the better is the baking quality. By applying the method of the present invention it is pos sible to obtain a gluten fraction consisting essentially of gluten, i.e., higher purity of gluten fraction. In the present context the term "consists essentially of" is meant to define a 20 fraction wherein the content of gluten is at least 80 % protein of dry matter. In a preferred embodiment the gluten content is at least 90%, more preferred 95%, even more preferred 97% pro tein of dry matter, even more preferred 99 protein of dry mat ter, and in an even most preferred embodiment the gluten frac 25 tion is consisting of protein only (i.e., gluten only - 100%). The method of the invention additionally provides for an in creased yield of gluten from, e.g., wheat independent of the crop variety. The method may be applied to any kind of crop. However, espe 30 cially contemplated are wheat, but also crops such as corn, WO 01/17363 PCT/DKOO/00494 7 rice, sorghum bean, barley, rye, or fruit hulls are contem plated. Wheat s Modern wheat varieties are classified as winter wheat and spring wheat. Wheat varieties may be hard grained or soft grained. Hard grained varieties normally have a large content of gluten. The flour of the latter varieties is preferably used in the baking industry for making products, such as bread. In io case of the soft grained wheat varieties the flour thereof may be used for the production of cakes, biscuits, and fodder. Du rum wheat is hard grained and the flour is used for pasta prod ucts. Wheat in general may also be used for the manufacture of beer and whiskey. Independent of the wheat variety the present 15 method has improved the gluten yield obtainable from said soft varieties. Besides gluten another main wheat component is starch. Gluten Quality 20 Wheat starches are classified according to their degree of purity. The grades are denominated A and B, respectively. Grade-A starch has a particle size of 20-35 microns and grade-B starch has a particle size of 2-10 microns. Grade-A starch is a versatile product providing strength and a pleasing 25 texture to a variety of foods. Low levels of protein and fibre insure that grade-A starch gelatinise at low temperatures pro ducing a smooth paste. The paste may be applied to foods such as, baby food, soups, sauces, gravies, sour cream, and dips. Grade-B starch has a higher protein content and a lower den 30 sity.

WO 01/17363 PCT/DKOO/00494 8 Application of starches There are a vast number of areas in which starches may be applied, such as in glucose syrup production and in sweetening 5 production in general. According to this invention at least one other fraction is obtained, said fraction comprising starch and optionally other wheat constituents, such as fibres. Preferably, the at least one other fraction consists essentially of no gluten. 10 Accordingly, the other fraction(s) of the present invention consist of essentially no gluten. By the term "essentially" is meant that the gluten content in the other fractions are less than 20%, preferably less than 10%. In a more preferred embodi ment at least two fractions are obtained, one of said other is fractions being a starch fraction. The starch fraction is sub stantially free of gluten and also substantially free of wheat constituents, being an essentially pure starch fraction. It is an object of the invention to obtain a substantially pure starch fraction. 20 The method is conducted by mixing flour, in particular wheat flour, and a liquid, said liquid being acceptable in products intended for animal and human consumption, and an oxi doreductase enzyme. The mixing may be carried out using any suitable method 25 known in the art, which may be by the use of an electrically operated mixer. In a preferred embodiment of the invention wa ter is preferred as a liquid. The mixing may be conducted by mixing the flour and the en zyme in a first step, followed by the addition of the liquid.

WO 01/17363 PCT/DKOO/00494 9 Furthermore, the enzyme(s) of the invention may be in a dry form or in a liquid form, and may be applied to the flour as such, dependent on the requirement of the timing of the separa tion process as described below. s In one embodiment of the invention the flour is mixed with the enzyme(s) in a dry form. This method allows for the prepa ration of flour and enzyme(s) mixture that may not be further processed immediately after mixing, but may in fact be kept for use at a later point in time. As the process is desired to pro io ceed, in a second step the liquid may be added to the flour and enzyme(s) mixture. In another embodiment the flour may be mixed with the en zyme(s) in a liquid form. By doing so the enzyme reaction(s) may begin immediately after the mixing, and may thus be fully is complete by the time the flour separation process is meant to continue by, in a second step, adding the liquid. When adding the enzymes in a dry form the holding time for the flour and enzyme mixture may be up to 3 months, such as up to six months, or longer dependent on the type of enzymes applied and physical 20 parameters, such as storage conditions, humidity and tempera ture. By using this procedure the resting time may be mini mised, preferably eliminated once the actual separation process begins, due to the fact that the enzyme reaction by then may be partly or fully completed, again dependent on the parameters 2S mentioned above. In a further embodiment of the invention the flour and the liquid are mixed in a first step obtaining a slurry, and whereto in a second step the enzyme(s) are added. This provides for the initiation of the immediate action of the enzyme(s) ac 30 cording to the invention, in the wheat separation process.

WO 01/17363 PCT/DKOO/00494 10 Accordingly, the flour mixture (i.e., flour and enzymes) of the invention may be dry or it may be liquid. In case of the former it is advantageous that the enzyme preparation is a dry product, e.g., a non-dusting granulate, whereas in the latter 5 case the enzyme preparation may be in liquid form. For the enzyme reaction(s) of the invention to occur oxy gen must be present. The level of oxygen present must be suffi cient to ensure the reaction to take place, i.e., oxygen may not be the limiting factor of the chemical reaction. 10 According to the invention in one embodiment step a) may comprise mixing the flour and a liquid obtaining a dough, resting the dough, and adding at least one oxidoreductase en zyme to the dough. The time needed for resting the dough may be any suitable 15 time. The resting time may be dependent upon the method chosen to mechanically process the dough for the purpose of obtaining individual fractions of gluten and starch, or it may depend upon the crop (e.g., wheat) variety used. In yet another embodiment of the present invention step a) 20 may comprise mixing the flour and a liquid and the at least one oxidoreductase enzyme obtaining a dough, followed by resting the dough prior to the separating step b) of the invention. In a further embodiment of the invention the dough is di luted prior to the separating step b). The dilution of the 25 dough may be in the order of 1.5:1, preferably 2:1. The term "dough" in the present context is meant to be distinguished from the term "batter", the latter containing more liquid than dough, such as 3 to 4 times more liquid than dough. Accordingly, the diluted dough of the invention may be 30 referred to as batter.

WO 01/17363 PCT/DKOO/00494 11 In the present context, the term "oxidoreductase" in cludes enzymes capable of creating S-S (sulphur-sulphur) bridges in gluten, or making reduction and exchange between chains thereby creating a network. The oxidation reaction is as s follows: SH-0 2 -+ S-S and the reduction reaction is as follows: S-S -> SH. The enzymatic activity of the enzymes according to the invention may be determined by standard assays. According to the invention the enzymes may be selected from the group consisting of Peroxidase (EC 1.11.1.7), Tyrosinase io (EC 1.14.18.1), Catechol oxidase (1.10.3.1), Laccase (EC 1.10.3.2), Bilirubin oxidase (EC 1.3.3.5), (Glutathione oxidase = Sulfhydryl oxidase (EC 1.8.3.3), Glucose oxidase (EC 1.1.3.4), Pyranose oxidase (EC 1.1.3.10), Hexose oxidase (EC 1.1.3.5), L-amino acid oxidase (EC 1.4.3.2), Lysyl oxidase (EC is 1.4.3.13), xylitol oxidase, galactose oxidase (E.C. 1.1.3.9), alcohol oxidase (E.C. 1.1.3.13) alone or in combination. The oxidoreductase(s) in question should be added in an effec tive amount. Dependent on the flour and the purpose of the use of the 20 product, the enzymes may be applied alone or in combination. The enzymes of the invention may be applied to the flour mix ture alone or the enzymes may be applied in combination with another enzyme, such as an enzyme selected from the group con sisting of hemicellulase, cellulase, xylanase, proteases or de 2s hydrogenases. The pH value is preferably within a range suitable for the enzymatic activity. In one embodiment of the invention the dough has a pH value of between 4.5 and 8.0, preferably between 5.0 and 6.5. It is preferred that the pH is the non-regulated WO 01/17363 PCT/DKOO/00494 12 pH of the slurry and dough, and that no pH regulators are added. Further, according to the invention the temperature of the dough or slurry is preferably between 10-60 0 C, more pref s erably between 20-50 0 C, and most preferably between 35-45 0 C. When the mixing step is accomplished the dough obtained is subjected to a separating step. The separating step may be conducted by a variety of methods suitable for the separation of the dough of the inven 1o tion, i.e., the separating method may rely on differences in particle size between gluten and starch (fibres) and thus rely on particle weight. In one widely used embodiment of the invention the dough is separated by the means of centrifugation. According to this 15 method the dough is centrifuged thereby obtaining a heavy phase containing pure starch and a free flowing light phase contain ing the gluten. In another embodiment of the present invention the sepa rating process is performed by the means of screening. The 20 screening may be conducted by arranging at least a gluten screen, for obtaining the gluten fraction. The screen size may vary dependent on the nature of the material to be screened. For example the screen for gluten may have the size of 500 [, or 400 pi, or 200 pt, or 125 t .The screening method may 25 furthermore comprise two or more screens, the first for gluten, and the other(s) for one or more starch fractions and/or fibre fractions. It is of importance that the screens are adapted to the gluten particle size, which is regulated by the enzymatic 30 treatment during the mixing step.

WO 01/17363 PCT/DKOO/00494 13 The screening method is preferably conducted with diluted dough, whereby the starch and fibres are washed through the gluten particle network, leaving the gluten particles on the first screen. 5 In another embodiment of the present invention the sepa rating process is performed by the means of decanting. The de canting process may begin by homogenising the batter in a ho mogeniser. Here shear forces break up the matrix. After this the mixture is passed through a decanter centrifuge capable of 1o separating the dough into distinct phases, such as starch and gluten phases. The gluten phase may be further processed by ad ditional washing and centrifugation or screening. Yet another method of separating according to the inven tion may be air classification. In this method the wheat flour is is separated into fractions, i.e. starch and gluten, by passing the flour through a spiral air stream. The particles in the flour will separate according to size, resulting in starch and gluten fractions. This method may advantageously be applied to the separation of the stored flour and enzyme(s) mixture de 20 scribed earlier. In another aspect of the present invention separating process is by the means of a hydrocyclone. In the hydrocyclone apparatus the diluted mixture is applied to the top of a static cone shaped container. The mixture is rotating inside the con 25 tainer and the heavier particles will settle in the lower frac tion of the mixture, whereas the lighter particles will be pre sent in the top fraction of the mixture. The methods applied for the separating process may be one method, or it may be a combination of more processes.

WO 01/17363 PCT/DKOO/00494 14 The separating step is followed by a recovering step, wherein the gluten fraction is recovered. The gluten fraction thus obtained may be kept as a suspension of gluten in a liquid or it may subsequently be dried. The latter provides for the s option of processing and storing the gluten for later purposes. It is an object of the invention that the separated gluten maintains its characteristic properties, having properties identical to the original gluten. The drying step is especially crucial for the conservation of the gluten properties. Too io forceful a drying process may result in a considerable loss in gluten quality. According to the invention the gluten may be dried in a ring dryer, or it may be dried in a fluid bed dryer. In the ring dryer process the wet gluten is fed into a ring duct after is a size reduction in a disintegrator. Upon entering the ring duct the gluten is mixed with circulating gluten particles that are already partially dried. Dried gluten particles are removed from the ring by a manifold. The principle behind the fluid bed dryer is similar to the ring dryer, except the fluid bed dryer 20 is arranged horizontally and air is entering the bed from be low. In a preferred embodiment at least one starch fraction is obtained, which fraction(s) may be further processed as appli cable. 25 The present invention further relates to a composition for the separation of wheat flour into one gluten fraction and at least one other fraction, comprising at least one oxidore ductase enzyme. The composition is preferably suitable for mixing with 30 the flour as described above.

WO 01/17363 PCT/DKOO/00494 15 According to the invention the composition may comprise any of the oxidoreductase enzymes described above alone or in combination. Furthermore, the composition may comprise at least one other enzyme. Said other enzyme may be an enzyme for en s hancing the gluten separation, e.g., an enzyme with affinity to the non-starch carbohydrate fractions, fibres or soluble arabi noxylan fractions. The more specific combination of components for the com position according to the invention is dependent upon the type 1o of flour used for the separation, and upon the purpose of the application of gluten obtained by the invention. According to the invention the enzymes may be from fungal (including filamentous fungi and yeasts) or bacterial origin. The enzymes may be derived from the bacterial strain(s) is of strains of the order Actinomycetales, e.g., Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus (IFO 12382) or Streptoverticillum verticillium ssp. verticillium; strains of Bacillus sp., e.g., Bacillus pumilus (ATCC 12905), Bacillus stearothermophilus, Rhodobacter sphaeroides, Rhodomonas 20 palustri, Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958) or Pseudomonas fluorescens (NRRL B-11); or strains of Myxococcus sp., e.g., M. virescens. Further the enzymes may be derived from the fungi strains belonging to the subdivision: Deuteromycotina, class Hypho 25 mycetes, e.g., Fusarium, Humicola, Tricoderma, Myrothecium, Ver ticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia, Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM 2672), Humicola insolens, Trichoderma resii, Myrothecium verrucana (IFO 6113), Verticillum alboatrum, Verticillum dahlie, 30 Arthromyces ramosus (FERM P-7754), Caldariomyces fumago, Ulocla- WO 01/17363 PCT/DKOO/00494 16 dium chartarum, Embellisia allior Dreschlera halodes; strains belonging to the subdivision Basidiomycotina, class Basidiomy cetes, e.g., Coprinus, Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus f. microsporus (IFO 8371) , Coprinus s macrorhizus, Phanerochaete chrysosporium (e.g. NA-12) or Tram etes (previously called Polyporus), e.g., T. versicolor (e.g., PR4 28-A); or strains belonging to the subdivision Zygomycotina, class Mycoraceae, e.g., Rhizopus or Mucor, in particular Mucor hiemalis. 10 Laccases The laccase may be derived from a fungi such as Collybia, Fomes, Lentinus, Pleurotus, Aspergillus, Neurospora, Podospora, Phle bia, e.g., P. radiata (WO 92/01046), Coriolus sp., e.g. C. hir 15 situs (JP 2-238885) , or Botrytis. Specifically contemplated laccases are the laccases derived from a strain of Polyporus sp., in particular a strain of Poly porus pinsitus or Polyporus versicolor, or a strain of My celiophthora sp., e.g., M. thermophila or a strain of Rhizocto 20 nia sp., in particular a strain of Rhizoctonia praticola or Rhizoctonia solani, or a strain of a Rhus sp., in particular Rhus vernicifera. In a preferred embodiment of the invention the enzyme is a microbial laccase derived from a strain of genus Myceliophthora, 2S such as a strain of the species Myceliophthora thermophila, e.g., the purified laccase described in WO 95/33836 from Novo Nordisk, which is hereby incorporated by reference. In another preferred embodiment the enzyme is a laccase derived from a strain of the genus Polyporus, such as a strain WO 01/17363 PCT/DKOO/00494 17 of the species P. pinsitus laccase, especially the purified lac case described in WO 96/00290 from Novo Nordisk. Other laccases include a Scytalidium sp. laccase, such as the S. thermophilium laccase described in WO 95/33837 (from Novo s Nordisk Biotech inc.) or a Pyricularia sp. laccase, such as the Pyricularia oryzae laccase which can be purchased from SIGMA un der the trade name SIGMA no. L5510, or a Coprinus sp. laccase, such as a C. cinereus laccase, especially a C. cinereus IFO 30116 laccase, or a Rhizoctonia sp. laccase, such as a Rh. so 1o lani laccase, especially the neutral Rh. solani laccase de scribed WO 95/07988 (from Novo Nordisk A/S) having a pH optimum in the range from 6.0 to 8.5. Laccase may be added in an effective amount. In an embodiment (as shown in the examples) the laccase may be the above 15 mentioned Polyporus pinsitus laccase. A laccase may preferably be added in an amount of from 0.1 to 50 LACU/g DS flour, more preferably 0.2-10 LACU/g DS flour, even more preferably 0.5-5 LACU/g DS flour. 20 Bilirubin Oxidase Bilirubin oxidases may be derived from a strain of Myrothe cium sp., such as M. verrucaria. Bilirubin oxidase may be added in an effective amount. 2s L-amino acid oxidase L-amino acid oxidase may be derived from a starin of Tri choderma sp. such as Trichoderma harzianum, such as the L-amino acid oxidase described in WO 94/25574 (from Novo Nordisk A/S), or Trichoderma viride. 30 L-amino acid oxidase may be added in an effective amount.

WO 01/17363 PCT/DKOO/00494 18 Glucose Oxidase A suitable glucose oxidase may originate from Aspergillus sp., such as a strain of Aspergillus niger, or from a strain of 5 Cladosporium sp. in particular Cladosporium oxysporum, espe cially Cl. oxysporum CBS 163 described in WO 95/29996 (from Novo Nordisk A/S). Glucose oxidase may be added in an effective amount. As shown in the examples below the glucose oxidase may be derived io from Aspergillus niger. Glucose oxidase may preferably be added in amounts of 0.001-10,000 GODU/g DS flour, more preferably from 0.005-5,000 GODU/g DS flour, even more preferably from 0.01-2,000 GODU/g DS flour. 15 Hexose Oxidase A hexose oxidases may be derived from the red sea-weed Chondrus crispus (commonly known as Irish moss) (Sullivan and Ikawa, (1973), Biochim. Biophys. Acts, 309, p. 11-22; Ikawa, (1982), Meth. in Enzymol. 89, carbohydrate metabolism part D, 20 145-149) oxidises a broad spectrum of carbohydrates, such as D glucose, D-galactose, maltose, cellobiose, lactose, D-glucose 6 phasphate, D-mannose, 2-deoxy-D-glucole, 2-deoxy-D-galactose, D fucase, D-glucurnic acid, and D-xylose. Also the red sea-weed Iridophycus flaccidum produces easily extractable hexose oxi 25 dases, which oxidise several different mono- and disaccharides (Bean and Hassid, (1956), J. Biol. Chem, 218, p. 425; Rand et al. (1972, J. of Food Science 37, p. 698-710). Hexose oxidase may be added in an effective amount.

WO 01/17363 PCT/DKOO/00494 19 Xylitol Oxidase Another relevant oxidoreductase is xylitol oxidase (see, e.g., JP 80892242), which oxidises xylitol, D-sorbitol, D galactitol, D-mannitol and D-arabinitol in the presence of oxy 5 gen. A xylitol oxidase can be obtained from strains of Strepto myces sp. (e.g., Streptomyces IKD472, FERM P-14339). Xylitol Oxidase may be added in an effective amount. Sulfhydryl oxidase (SOX) 10 Glutathione oxidases or Sulfhydryl oxidases may be derived from Calodon and Cortinarius sp. (US patent no. 4,610,963); or a sulfhydryl oxidase from Aspergillus, in particular A. niger (US patent no. 5,529,926 and EP 321 811-Al), Aspergillus awamori or Aspergills sojae; or Penicillium, in particular Penicillium is ochrochloron. Sulfhydryl oxidase may be added in an effective amount. As shown in the examples below the sulfdryl oxidase may be derived from Penicillium ochrochloron. Sulfhydryl oxidase may prefera bly be added in amounts of 0.001-5 milli SOX/ g DS flour, more 20 preferably from 0.01-3 milli SOX/g DS flour, even more prefera bly from 0.1-2 milli SOX/g DS flour. Pyranose oxidase Examples of pyranose oxidases as described in JP 61177986 25 and include pyranose oxidases derived from strains of the genera Irpex, such as a strain from the species Irpex lacteus; Auricu lariea, such as a strain of the species Auricularia polytricha, in particular Auricularia polytricha (FERM-P 7119), Coprinus, such as a strain of the species Coprinus micaceus, in particular 30 Coprinus micaceus ATCC 20122; and Trametes, such as a strain of WO 01/17363 PCT/DKOO/00494 20 the species Trametes cinnabarinus, in particular Trametes cinna barinus IFO 6139. Pyronose oxidase may be added in an effective amount. 5 Peroxidase The peroxidase may be derived from plants (e.g., horseradish peroxidase) or microorganisms including fungi and bacteria such as a strain of Coprinus sp., such as Coprinus cinereus or Copri nus macrorhizus, or bacteria such as Bacillus, such as Bacillus 10 pumilus. Peroxidase may be added in an effective amount. The enzymes of the invention may be obtained from the mi croorganism in question by the use of any suitable technique. For instance, the enzyme preparation may be obtained by ferment ing a microorganism and subsequently isolating the enzyme con is taining preparation from the fermented broth or microorganism by methods known in the art. According to the invention a more pre ferred embodiment is the use of recombinant DNA techniques as known in the art. Such methods normally comprise the cultivation of a host cell transformed with a recombinant DNA vector capable 20 of expressing and carrying a DNA sequence encoding the enzyme in question. The host cell is grown in a culture medium under con ditions permitting the expression of the enzyme, and is followed by the recovery of the enzyme from the culture. Additionally the present invention relates to the use of 25 the composition as previously described. By the method of the invention at least two different fractions are obtained. There are various products that may comprise such fractions. In one embodiment the obtained gluten may be added to 30 wheat flour of poor quality, i.e., wheat flour having low glu- WO 01/17363 PCT/DKOO/00494 21 ten content. Accordingly, flour normally used for products, such as fodder may after fortification with gluten be used for the manufacture of products, such, as bread. Consequently, the present invention presents a broadening of the types of avail s able applications for flour having low gluten content. The gluten fraction obtained by the method of the inven tion is applicable in any of the applications mentioned above and in a further aspect the present invention relates to a glu ten fraction consisting essentially of gluten and to a product 1o comprising said fraction. Further, starches may be used in products of the adhe sive, gypsum, paper, corrugating, mining and food industries. Starch and starch products may also be used as adhesive com pounds, such as in the production of bags and adhesive tapes, 15 laminates and wound tubes, wallpaper and poster glues, abrasive paper. Other applications include components of concrete re tarders, sizing agents for synthetic, natural and mixed yarns in the textile industry, and thickeners for the printing of textiles. 20 In the pharmaceutical industry starch products may be used as disintegration agents in tablets and surgical glove powder. Within the ceramic industry field the addition of starch increases the strength of the ceramic products. Starches may also be applied to detergents for the purpose of being 25 dirt-deposit inhibitors. A completely different application is in the field of oil well drilling, wherein starch solutions may be used as agents to help seal drilling cores and to increase the viscosity of drilling mud and cooling water.

WO 01/17363 PCT/DKOO/00494 22 Yet another application of starch is for the use in water treatment plants serving the purpose of flocculating various aqueous suspensions. A further application is in the plastic industry, wherein s starches may be used to improve the biological degradation of plastic products. Currently one of the applications of starch is for paint stripping using wheat starch blasting. Wheat starch blasting is a user-friendly blasting process wherein wheat starch can be 1o used in systems designed for plastic media blasting (PMB), as well as systems specifically designed for wheat starch blast ing. The wheat starch abrasive media is a crystallised form of wheat starch that is non-toxic, biodegradable, and made from renewable resources. The media is similar in appearance to 15 plastic media, except that it is softer. Wheat starch is a plentiful natural resource that is biodegradable. Waste gener ated from this process may be treated in a bio-reactor using amylase enzymes. The wheat starch blasting process may be used for removing coatings from both metallic and composite materi 20 als. This process is easy to control. It may be used to selec tively remove from one and up to all coating layers. Wheat starch blasting does not cause fatigue to the substrate sur face, and it allows for moderate stripping rates, whilst main taining a gentle stripping action. 25 MATERIALS & MATHODS Enzymes: Polyporus pinsitus laccase: Disclosed in W096/00290 from Novo Nordisk (available on request from Novo Nordisk, Denmark) WO 01/17363 PCT/DKOO/00494 23 Aspergillus niger glucose oxidase (available on request from Novo Nordisk, Denmark). Penicillium ochrochloron sulfdryl oxidase (available from Novo Nordiks, Denmark) s GlutomaticO System (Perten) (for washing out gluten from flour). SOX units (Sulfhydryl Oxidase Unit): One sulfhydryl oxidase Unit is the amount of enzyme re 10 quired to deplete 1 micromole of 02 per minute from an assay mixture containing 30 mM L-cystein in 100 mM sodium phosphate at pH 6.0 and a temperature of 30 0 C. The oxygen was measured with an oxygen electrode conneted to an Oxi 3000 Oximeter (MTW). 15 Determination of Polyporus Laccase Activity (LACU) Laccase activity is determined from the oxidation of syrin galdazin under aerobic conditions. The violet colour produced is photometered at 530 nm. The analytical conditions are 19 micro M 20 syringaldazin, 23.2 mM acetate buffer, pH 5.5, 30 0 C, 1 minute reaction time. 1 laccase unit (LACU) is the amount of enzyme that catalyses the conversion of 1.0 micro mole syringaldazin per minute under these conditions. 25 Determination of glucose oxidase activity (GODU) 1 GODU is defined as the amount of enzyme which, under stan dard conditions, catalyses the formation of 1 micromole of H 2 0 2 per minute. The analytic method AF266 is available upon request from Novo Nordisk A/S). 30 WO 01/17363 PCT/DKOO/00494 24 Determination of peroxidase activity units (POXU) Peroxidase activity is measured in POXU/ml. (1 POXU (peroxi dase unit) is defined as the amount of enzyme that catalyses the conversion of 1 micro mole H 2 0 2 per minute in a system where 5 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonate] is oxidised in the presence of 1 mM H 2 0 2 , pH 7.0, at a temperature of 40 0 C.) A. The enzyme treatment a. 100 g of wheat flour (type Pelikkan 441/1 from Meneba Meel 10 BV, Holland) is mixed, using the Hobart mixer at speed III (high speed) with 70 mL of tap city water (ca. 20 'dH) in cluding added enzyme solution. The water temperature is 37 0 C + 2 0 C. The specific enzyme and dosage hereof is de scribed under each example. is b. The Hobart Mixer is applied on the pre-dough at speed III (high speed) for 2 minutes. c. The 170 g of dough rested for 8 to 40 minutes at 37 0 C in the mixing cup, which is placed in a water bath. Thereby the enzyme reaction time is the resting time plus the 20 mixing time (approximately 3 minutes) for all the tests. Some further enzyme reaction time is possible during the dough wash ing (see B. Preparation of gluten) below. However the dough is washed continuously with water at 37 0 C, so that the part of the enzyme that is not attached to the substrate is washed out 25 rather quickly. B. Preparation of gluten. The Glutomatic) System (Perten Instruments AB, Sweden) consists of Glutomatic 2200 mixing and gluten washing device 30 used for preparing a wet sample of gluten. This sample is at WO 01/17363 PCT/DKOO/00494 25 flourmills used for determining gluten quantity and quality quantified as the gluten index by sieve-centrifugation using a Glutomatic Centrifuge 2015. The index value characterizes the gluten as being weak, normal or strong. 5 The Glutomatic 2200 gluten washer consists of a washing chamber, and a powerful stirrer. The washing and stirring is performed by continuous addition of water. The wash water in cluding suspended starch particles leaves the washing chamber through an 88-micron filter at the same continuous flow rate at 1o which fresh water was added. 3. 20.0 g of dough prepared as described above is placed in the mixing cup and 4.2 mL water is added. 4. The stirring and continuous flow of water is performed for 30 minutes using a flow rate of 46.3 mL/minute. Water is heated to 37'C + 2*C was used. 5. The washed piece of gluten was weighed and evaluated visu ally for elasticity. 6. The piece of gluten was freeze-dried using conventional freeze-drying technique and weighed. The weight of dried 20 gluten in relation to the weight of the wet gluten sample is equivalent to the dry matter content. 7. The dried gluten was grinded to a fine powder using a mor tar for 15 minutes, and it was assayed by the micro baking test. 25 C. Assay for measuring gluten quality by micro baking tests. One to five days before the test begins portions of 12 g flour + gluten was equilibrated at 30 0 C. Solutions of salt and sugar were prepared. Also a solution of the yeast was prepared 30 (see the table below) and stored in refrigerator.

WO 01/17363 PCT/DKOO/00494 26 The mass and volume of ingredients for the single breads are the following: Ingredients Mass or volume Flour 12.0 g Water (from the city) 4.3 mL Yeast solution (40 g + 60 mL water) 1.2 mL Solution of 10.8 g sugar + 10.8 g 2.5 mL salt + 64.2 mL water Gluten (2 % w/w of the amount of 0.24 g flour) 5 2. To a thermo-equilibrated kettle (25-30 0 C) 12.24 g of flour including gluten, the yeast solution, the salt plus sugar solution and water is added (in this order) . The kettle is the mixer device for the Micro Mixer type NSI-33R that is used. 3. The kettle was mounted on the micro-mixer and kneading is 10 performed for 3.0 minutes. 4. 18.0 g of the dough is weighed and kneaded by hand. The temperature in the dough must be 27-29*C. The dough is flatten out and shaped using a long rolling pin and it rested for 15 minutes at 28-29?C. is 5. It is again rolled and shaped using the special long roll ing pin and it rested for 10 minutes at 280C. 6. Finally it is rolled out again and shaped to bread using the special long rolling pin. The dough is then placed in a 37 mL baking tin and placed in a conditioning cabinet at 20 32.00C, and 86 % relative humidity (RH) for 45 minutes. 7. It was then baked in the oven at 2300C for 13 minutes.

WO 01/17363 PCT/DKOO/00494 27 8. After cooling the volume of the bread was weighed and meas ured using a "mini-PUP size" volume-measuring device. The baking result was presented as the specific bread volume (mL/g). The higher the specific bread volume the better is s the baking effect of the gluten. In the following examples the enzyme treatment, the prepa ration of gluten and the assay for measuring gluten quality by micro baking tests were used. The enzymes used and the results 1o of the tests are mentioned under each example. Experimentals The following are examples of methods of separation of wheat flour using enzymes. Fig. 1 is an illustration of the i5 steps in one of the possible separation processes and Fig. 2 is an example of a decanter process for the separation of wheat flour. Example 1 20 5 kg of wheat flour is mixed with 3.5 L of water having at temperature of 25"C. The enzyme of Polyphenol oxidase - Lac case is added. The dough is resting for approximately 8 minutes followed by the addition of 5 L of water. The dough suspension is mixed 25 for approximately 18 minutes. After mixing the dough suspension is diluted with 4 L of water and is circulated for 20 minutes. The diluted dough suspension is then separated on screens by addition of water. The fractions obtained are gluten, hemicellulose and 30 starch fractions.

WO 01/17363 PCT/DKOO/00494 28 Example 2 200 kg of wheat flour is mixed with 400 L of water. The enzyme of Polyphenol oxidase-Laccase is added. 5 The dough is continuously pumped into a homogeniser at 30-50 bar and is homogenised at a temperature of approximately 35 0 C. After homogenising, the mixture is passed through a de canter centrifuge separating the mixture into a supernatant fraction and a pellet fraction. The supernatant comprises the 1o gluten and the pellet comprises the starch. The supernatant is kept in a tank at a pH of approximately 7.0, and is then passed onto a 150 p1 sieve. The gluten of the supernatant is thereby separated from the effluent. The wet gluten is freeze dried and milled. 15 The A-starch (sediment) is liquefied and subsequently saccharified and made into syrup. Example 3 Test of laccase 20 Enzyme treatment, preparation of gluten and baking test are shown in table 1. The washing of the gluten was performed at approximately 220C. Enzyme Type Dosage Resting Dry mat- Baking of time, ter of test enzyme minutes wet glu- Specific activ- ten, % volume ity. w/w of bread, WO 01/17363 PCT/DKOO/00494 29 LACU/g mL/g Dry mat ter of flour Laccase Poly- 1.1 40 33.3 3.29 porus pinsitus Laccase Poly- 3.0 40 32.2 3.33 porus pinsitus No 0 40 33.9 3.26 enzyme (average of 3 tests) Table 1. Effect of laccase treatment of the flour. A clear effect of the laccase treatment was seen, as il lustrated by the dosage response trial. 5 Example 4 Test of sulfhydryl oxidase: The specific activity of the Penicillium ochrochloron sulfhydryl oxidase was 5.12 SOX/A-280. A preparation having an activity of 6.45 SOX/mL was used. 1o Enzyme treatment, preparation of gluten and baking test are shown in table 2. The washing of the gluten was performed at approximately 37.7-38.1 0 C Enzyme Type Dosage Resting Dry mat- Baking of en- time, ter of test zyme ac- minutes wet glu- Specific tivity. ten, % volume WO 01/17363 PCT/DKOO/00494 30 w/w of Milli bread, SOX/g mL/g Dry mat ter of flour Sulfhy- Penicil- 1.0 10 34.0 3.36 dryl lium oxidase ochro chloron Sulfhy- Penicil- 1.0 45 33.9 3.38 dryl lium oxidase ochro chl oron No 0 40 33.9 3.26 enzyme (average of 3 tests) Table 2. Effect of sulfhydryl oxidase The wet gluten produced by use of sulfhydryl oxidase was found extraordinary elastic when evaluated by the visual test 5 Example 5 Test of glucose oxidase: Enzyme treatment, preparation of gluten and baking test io are shown in table 3. The washing of the gluten was performed at 36.1-37.8*C WO 01/17363 PCT/DKOO/00494 31 The enzyme preparation Novozymm 771 (batch no. OGN 00002) (As pergillus niger GOX) was analyzed to 1639 GODU/g according to the internal analyses procedure EAL-SM-0244 (available on re quest from Novo Nordisk). 5 Enzyme Type Dosage Resting Dry Baking of time, matter test enzyme minutes of Specific activ- wet volume ity. gluten, of % w/w bread, GODU/g mL/g Dry mat ter of flour Glucose Novozym 0.010 10 33.4 3.35 oxidase 771 from (average Asper- of 2 gillus tests) niger Glucose Novozym 0.010 45 34.4 3.35 oxidase 771 from (average Asper- of 2 gillus tests) niger Glucose Novozym 0.100 10 34.1 3.28 oxidase 771 from (average Asper- of 2 gillus tests) niger WO 01/17363 PCT/DKOO/00494 32 Glucose Novozym 0.100 45 33.9 3.28 oxidase 771 from (average Asper- of 2 gillus tests) niger No 0 40 33.9 3.26 enzyme (average of 3 tests) Table 3. Effect of glucose oxidase An increasing effect of the specific volume of the bread is clearly seen as a result of the treatment with the glucose oxidase. 5

Claims (32)

1. A method for the separation of flour into one gluten frac tion and at least one other fraction, comprising the steps of: s a) mixing the flour and a liquid to obtain a dough, b) separating the dough into a fraction comprising gluten and at least one other fraction, c) recovering at least the gluten fraction, characterized in that an oxidoreductase is added at any of steps a), b) or 10 c).

2. A method of claim 1, comprising the steps of: a) mixing the flour and a liquid and at least one oxidoreduc tase enzyme obtaining a dough, is b) separating the dough into a fraction comprising gluten and at least one other fraction, c) recovering at least the gluten fraction. 20

3. The method according to claims 1 or 2, wherein the step a) comprises al) mixing the flour and the at least one oxidoreductase en zyme obtaining a mixture, and a2) adding the liquid to the mixture. 25

4. The method according to claims 1-3, wherein the step a) comprises a3) mixing the flour and the liquid, followed by a4) adding the at least one enzyme. 30 WO 01/17363 PCT/DKOO/00494 34

5. The method of claims 1-4, wherein the flour is wheat flour.

6. The method according to claims 1-5, wherein the gluten s fraction consist essentially of gluten.

7. The method according to claim 1-5, wherein the other fractions consist of essentially no gluten. 10

8. The method according to any of the preceding claims, wherein one of the other fractions comprises starch.

9. The method according to any of the preceding claims, wherein the dough is resting prior to separation. 15

10. The method according to any of the preceding claims, wherein the dough is diluted prior to separation.

11. The method according to any of the preceding claims, 20 wherein the dough is homogenised prior to separation.

12. The method according to any of the preceding claims, wherein the dough is separated by the means of sieving. 25

13. The method according to any of the preceding claims, wherein the dough is separated by the means of a decanter centrifuge. WO 01/17363 PCT/DKOO/00494 35

14. The method according to any of the preceding claims, wherein the dough is separated by the means of a hydrocyc lone. s

15. The method according to any of the preceding claims, wherein the recovered gluten fraction is subsequently dried.

16. The method according to any of the preceding claims, io wherein the enzymes are selected from the group consisting of peroxidase, laccase, glutathione oxidase, glucose oxi dase, pyranose oxidase, hexose oxidase, L-amino acid oxi dase and lysyl oxidase alone or in combination. is

17. The method according to claim 15, wherein the enzymes are selected in combination with an enzyme selected from the group consisting of hemicellulase, cellulase, xy lanase, proteases and dehydrogenases. 20

18. The method according to any of the preceding claims, wherein the enzymes are from fungal or bacterial origin.

19. The method according to any of the preceding claims, wherein the step a) comprises mixing the flour and a liq 25 uid obtaining a dough, resting the dough, and adding at least one oxidoreductase enzyme to the dough.

20. The method according to claim 1-5, wherein the dough has a pH value of between 4.5 and 8.0. 30 WO 01/17363 PCT/DKOO/00494 36

21. The method according to claim 1-5, wherein the dough has a temperature of between 10 and 60 0 C.

22. A composition for the separation of flour into one glu s ten fraction and at least one other fraction, comprising at least one oxidoreductase enzyme.

23. The composition according to claim 22, wherein the oxi doreductase enzyme is selected from the group consisting 10 of peroxidase (EC 1.11.1.7), tyrosinase (EC 1.14.18.1), catechol oxidase (1.10.3.1), laccase (EC 1.10.3.2), bilirubin oxidase (EC 1.3.3.5), (glutathione oxidase = sulfhydryl oxidase (EC 1.8.3.3), glucose oxidase (EC 1.1.3.4), pyranose oxidase (EC 1.1.3.10), hexose oxidase 15 (EC 1.1.3.5), L-amino acid oxidase (EC 1.4.3.2), Lysyl oxidase (EC 1.4.3.13), xylitol oxidase, galactose oxidase (E.C. 1.1.3.9), alcohol oxidase (E.C. 1.1.3.13) alone or in combination. 20

24. The composition according to claim 22, comprising at least two oxidoreductase enzymes.

25. The composition according to claim 22, wherein the at least one enzyme is an enzyme other than an oxidoreductase 25 enzyme.

26. The composition according to claim 25, wherein the at least one enzyme is selected from the group consisting of hemicellulase, cellulase, xylanase, proteases and dehydro 30 genases. WO 01/17363 PCT/DKOO/00494 37

27. The use of a composition as defined in any of the pre ceding claims for the separation of wheat flour into one gluten fraction and at least one other fraction. 5

28. The use of the composition according to claim 27, in which the enzymes are derived from bacterial strains.

29. The use of the compositions according to claim 28, in 10 which the enzymes are derived from fungi strains.

30. A gluten fraction consisting essentially of gluten.

31. A product comprising a fraction as defined in claim 30. 15

32. A flour composition comprising an oxidoreductase for the use for the method of any of claims 1-21.