Improved fiber washing in corn wet milling

Abstract

A method for increasing starch yield and / or gluten yield from corn kernels in a wet milling process is disclosed, the method comprising contacting a fiber-rich fraction of the milled kernels with an effective amount of SO2 and an effective amount of one or more hydrolytic enzymes during a fiber washing step, wherein at least one of the hydrolytic enzymes is selected from xylanases and / or cellulases.

Description

Technical Field

[0001] The present invention relates to a method for improving / enhancing starch and / or gluten yield from corn kernels by contacting the kernels with an enzyme composition comprising xylanases and / or cellulases during a wet milling process, preferably during fiber washing. Background Art

[0002] Conventional corn wet milling is a process designed for the recovery and purification of starch and several by-products, including germ, gluten (protein), and fiber. Fiber is the least valuable by-product, so the industry has invested a lot of effort to increase the yield of more valuable products, such as starch and gluten, while reducing the fiber fraction. High-quality starch is valuable because it can be used for a variety of commercial purposes after further processing into products such as dried starch, modified starch, dextrins, sweeteners, and alcohol. Gluten is commonly used in animal feed, such as corn gluten meal (about 60% protein) or corn gluten feed (about 20% protein).

[0003] The wet milling process can vary significantly depending on the specific milling equipment used, but the process generally includes: grain cleaning, steeping, milling, germ separation, secondary milling, fiber separation, gluten separation, and starch separation. After cleaning the corn kernels, they are typically softened by soaking in water or in a dilute SO2 solution under controlled time and temperature conditions. The kernels are then milled to break down the pericarp and separate the germ from the rest of the kernel. The remaining slurry, consisting primarily of fiber, starch, and gluten, is finely milled and screened in a fiber washing process to separate the fiber from the starch and gluten, and the gluten and starch are then separated, and the starch may be purified in a washing / filtration process.

[0004] It has been suggested to use enzymes in several steps of the wet milling process, such as using enzymes in the impregnation step of the wet milling process. It has been shown that commercial enzyme products (available from Novozymes A / S) is suitable for the first step of the wet milling process, a steeping step in which the corn kernels are soaked in water.

[0005] Recently, "enzymatic milling," a modified wet milling process that uses proteases to significantly reduce overall processing time during corn wet milling and eliminate the need for sulfur dioxide as a processing agent, has been developed. Johnston et al., Cereal Chem, 81, pp. 626-632 (2004).

[0006] US 6,566,125 discloses a method for obtaining starch from maize, the method involving soaking maize kernels in water to produce soaked maize kernels, milling the soaked maize kernels to produce a milled maize slurry, and incubating the milled maize slurry with an enzyme (eg, a protease).

[0007] US 5,066,218 discloses a method of grinding cereals, particularly corn, which comprises cleaning the cereals, soaking the cereals in water to soften them, and then grinding the cereals with cellulase.

[0008] WO 2002 / 000731 discloses a process for treating crop seeds, which comprises soaking the seeds in water for 1-12 hours, wet milling the soaked seeds, and treating the seeds with one or more enzymes, including an acid protease.

[0009] WO 2002 / 000911 discloses a process for starch gluten separation which comprises subjecting ground starch to an acid protease.

[0010] WO 2002 / 002644 discloses a process for washing a starch slurry obtained from the starch gluten separation step of a milling process, which process comprises washing the starch slurry with an aqueous solution comprising an effective amount of an acid protease.

[0011] WO 2014 / 082566 and WO 2014 / 082564 disclose cellulolytic compositions for use in wet milling.

[0012] WO 2016 / 095856 discloses compositions comprising xylanases and arabinofuranosidases and the use of these compositions in fiber washing in corn wet milling processes.

[0013] WO 2019 / 023222 discloses a wet milling process using GH5 xylanase and GH30 xylanase in combination with cellulase in the fiber washing step.

[0014] WO 2017 / 088820 discloses a process for improving starch release from fiber in corn wet milling by adding α-L-arabinofuranosidase (GH62) alone or in combination with xylanase (GH10) during the fiber washing step.

[0015] WO 2018 / 053220 discloses a fiber washing system as part of a wet milling process that is optimized to apply enzymes in the fiber washing step by using dedicated spaces / tanks for enzyme incubation.

[0016] Although the effects of using enzymes in corn wet milling have been studied in the art, there remains a need for improved techniques during steeping / soaking of the corn kernel, during milling of the corn kernel, and during starch gluten separation that can reduce the energy expenditure and costs associated with corn wet milling and provide increased starch and gluten yields. Summary of the Invention

[0017] In a first aspect, the present invention relates to a method for increasing starch yield and / or gluten yield from corn kernels in a wet milling process, the method comprising contacting a fiber-rich fraction of the milled kernels with an effective amount of SO2 and an effective amount of one or more hydrolytic enzymes during a fiber washing step, wherein at least one of the hydrolytic enzymes is selected from xylanases and / or cellulases.

[0018] definition

[0019] Definition of Enzyme:

[0020] Arabinofuranosidase / polypeptide with arabinofuranosidase activity: The term "arabinofuranosidase" refers to an α-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55), which catalyzes the hydrolysis of terminal non-reducing α-L-arabinofuranoside residues in α-L-arabinosides. The enzyme acts on α-L-arabinofuranosides, α-L-arabinans containing (1,3)- and / or (1,2)- and / or (1,5)-linkages, arabinoxylans, and arabinogalactans. α-L-arabinofuranosidase is also known as arabinosidase, α-arabinosidase, α-L-arabinosidase, α-arabinofuranosidase, polysaccharide α-L-arabinofuranosidase, α-L-arabinofuranoside hydrolase, L-arabinosidase, or α-L-arabinanase. Medium viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co., Wicklow, Ireland) can be used in a total volume of 200 μl at 40°C for 30 minutes, followed by Arabinofuranosidase activity is determined by arabinose analysis using HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Arabinofuranosidases can be found, for example, in the GH43, GH62, and GH51 families according to Henrissat, 1991, A classification of glycosyl hydrolases based on amino-acid sequences similarities, Biochem. J. 280:309-316, and Henrissat and Bairoch, 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

[0021] β-glucosidase / polypeptide with β-glucosidase activity: The term "β-glucosidase" means a β-D-glucoside glucohydrolase (EC 3.2.1.21) that catalyzes the hydrolysis of terminal non-reducing β-D-glucose residues and releases β-D-glucose. β-glucosidase activity can be determined according to the procedure of Venturi et al., 2002, J. Basic Microbiol. 42:55-66 using p-nitrophenyl-β-D-pyranoside as a substrate. One unit of β-glucosidase is defined as the activity of β-glucosidase at 25°C, pH 4.8 in a solution containing 0.01% 20% of 50 mM sodium citrate produces 1.0 micromole of p-nitrophenolate anion per minute from 1 mM p-nitrophenyl-β-D-pyranoglucopyranoside as a substrate.

[0022] β-Xylosidase / polypeptide with β-xylosidase activity: The term "β-xylosidase" means β-D-xyloside xylohydrolase (EC 3.2.1.37), which catalyzes the exohydrolysis of short β(1→4)-xylo-oligosaccharides to remove consecutive D-xylose residues from the non-reducing end. β-xylosidase activity was determined in 100 mM sodium citrate at pH 5 and 40°C using 1 mM p-nitrophenyl-β-D-xyloside as a substrate. One unit of β-xylosidase was defined as the activity of 1 mM p-nitrophenyl-β-D-xyloside at 40°C and pH 5 in a solution containing 0.01% 20% of 100 mM sodium citrate produces 1.0 micromole of p-nitrophenolate anion per minute from 1 mM p-nitrophenyl-β-D-xyloside.

[0023] Cellobiohydrolase / polypeptide with cellobiohydrolase activity: The term "cellobiohydrolase" means 1,4-β-D-glucan cellobiohydrolase (EC 3.2.1.91 and EC 3.2.1.176) which catalyzes the hydrolysis of 1,4-β-D-glycosidic linkages in cellulose, cellooligosaccharides, or any polymer containing β-1,4-linked glucose, releasing cellobiose from either the reducing (cellobiohydrolase I) or non-reducing (cellobiohydrolase II) ends of the chain (Teeri, 1997, Trends in Biotechnology 15:160-167; Teeri et al., 1998, Biochem. Soc. Trans. 26:173-178). Cellobiohydrolase activity can be determined according to the procedures described by Lever et al., 1972, Anal. Biochem. 47:273-279; van Tilbeurgh et al., 1982, FEBS Letters 149:152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters 187:283-288; and Tomme et al., 1988, Eur. J. Biochem. 170:575-581.

[0024] Cellulolytic enzymes or cellulase / polypeptides having cellulase activity or cellulolytic activity: The term "cellulolytic enzyme" or "cellulase" means one or more (e.g., several) enzymes that hydrolyze a cellulosic material, including any material comprising cellulose (e.g., fiber). Cellulolytic enzymes include one or more endoglucanases (EC 3.2.1.4), one or more cellobiohydrolases (EC 3.2.1.91 and EC 3.2.1.150), one or more β-glucosidases (EC 3.2.1.21), or a combination thereof. Two basic methods for measuring cellulolytic enzyme activity include: (1) measuring total cellulolytic enzyme activity, and (2) measuring individual cellulolytic enzyme activities (endoglucanase, cellobiohydrolase, and β-glucosidase), as described in Zhang et al., 2006, Biotechnology Advances 24:452-481. Total cellulolytic enzyme activity can be measured using insoluble substrates, including Whatman No. 1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, and the like. The most common assay for total cellulolytic activity is the filter paper assay using Whatman No. 1 filter paper as the substrate. This assay was developed by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Pure Appl. Chem. 59:257-68).

[0025] Cellulolytic enzyme activity can be determined by measuring the increase in sugar production / release during hydrolysis of a cellulosic material by one or more cellulolytic enzymes under the following conditions: 1-50 mg cellulolytic enzyme protein / g cellulose in pretreated corn stover (PCS) (or other pretreated cellulosic material) at a suitable temperature (such as 40°C-80°C, e.g., 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C) and at a suitable pH (such as 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0) for 3-7 days, compared to a control hydrolysis to which no cellulolytic enzyme protein is added. Typical conditions were: 1 ml reaction, washed or unwashed PCS, 5% insoluble solids (dry weight), 50 mM sodium acetate (pH 5), 1 mM MnSO4, 50°C, 55°C, or 60°C, 72 hours, by Sugar analysis was performed using HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

[0026] Endoglucanase: The term "endoglucanase" means endo-1,4-(1,3;1,4)-β-D-glucan 4-glucanohydrolase (EC 3.2.1.4), which catalyzes the endohydrolysis of 1,4-β-D-glucosidic bonds in cellulose, cellulose derivatives (such as carboxymethylcellulose and hydroxyethylcellulose), lichenin, mixed β-1,3 glucans such as cereal β-D-glucans or xyloglucans, and β-1,4 bonds in other plant materials containing cellulose components. Endoglucanase activity can be determined by measuring a decrease in substrate viscosity or an increase in reducing ends as determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24:452-481). For the purposes of the present invention, endoglucanase activity is determined according to the procedure of Ghose, 1987, Pure and Appl. Chem 59: 257-268, at pH 5, 40°C, using carboxymethylcellulose (CMC) as substrate.

[0027] Family 61 glycoside hydrolase: The term "Family 61 glycoside hydrolase" or "Family GH61" or "GH61" means a polypeptide belonging to glycoside hydrolase family 61 according to Henrissat, 1991, A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280:309-316, and Henrissat and Bairoch, 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696. The enzymes in this family were originally classified as a glycoside hydrolase family based on measurement of very weak endo-1,4-β-D-glucanase activity in one family member. The structure and mode of action of these enzymes are irregular, and they cannot be regarded as true glycosidases. However, based on their ability to enhance lignocellulose decomposition when used in combination with a mixture of cellulase or cellulase, they are retained in the CAZy classification. GH61 polypeptides have recently been classified as soluble polysaccharide monooxygenases (Quinlan et al., 2011, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 208: 15079-15084; Phillips et al., 2011, ACS Chem. Biol. [ACS Chemical Biology] 6: 1399-1406; Lin et al., 2012, Structure [Structure] 20: 1051-1061), and are referred to as " auxiliary activity 9 " or " AA9 " polypeptides.

[0028] Hydrolytic enzyme or hydrolase / polypeptide with hydrolase activity: "Hydrolase" refers to any catalytic protein that uses water to break down a substrate. Hydrolases include cellulases (EC 3.2.1.4), xylanases (EC 3.2.1.8), arabinofuranosidases (EC 3.2.1.55 (non-reducing end α-L-arabinofuranosidase); EC 3.2.1.185 (non-reducing end β-L-arabinofuranosidase), cellobiohydrolase I (EC 3.2.1.150), cellobiohydrolase II (EC 3.2.1.91), cellobiosidase (EC 3.2.1.176), β-glucosidase (EC 3.2.1.21), β-xylosidase (EC 3.2.1.37).

[0029] Xylanase / polypeptide with xylanase activity: The term "xylanase" means a 1,4-β-D-xylan-xylohydrolase (EC 3.2.1.8) that catalyzes the endohydrolysis of 1,4-β-D-xylosidic bonds in xylans. Xylanase activity can be detected at 0.01% at 37°C. Xylanase activity is determined in 200 mM sodium phosphate (pH 6) and 0.2% AZCL-arabinoxylan as a substrate. One unit of xylanase activity is defined as the production of 1.0 micromole of azurine per minute from 0.2% AZCL-arabinoxylan as a substrate in 200 mM sodium phosphate (pH 6) at 37°C. Xylanases can be found, for example, in the GH5, GH30, GH10, and GH11 families.

[0030] GH5 polypeptide: refers to a polypeptide having enzymatic activity, which is classified as a member of glycoside hydrolase family 5 in the Carbohydrate-Active Enzymes (CAZyme) database (http: / / www.cazy.org / ).

[0031] GH8 polypeptide: refers to a polypeptide having enzymatic activity, which is classified as a member of glycoside hydrolase family 5 in the Carbohydrate-Active Enzymes (CAZyme) database (http: / / www.cazy.org / ).

[0032] GH30 polypeptide: refers to a polypeptide having enzymatic activity, which is classified as a member of glycoside hydrolase family 30 in the Carbohydrate-Active Enzymes (CAZyme) database (http: / / www.cazy.org / ).

[0033] GH10 polypeptide: refers to a polypeptide having enzymatic activity that is classified as a member of glycoside hydrolase family 10 in the Carbohydrate-Active Enzymes (CAZyme) database available at http: / / www.cazy.org / . (Lombard, V.; Golaconda Ramulu, H.; Drula, E.; Coutinho, PM; Henrissat, B. (21 November 2013). “The carbohydrate-active enzymes database (CAZy) in 2013.” Nucleic Acids Research. 42(D1):D490-D495; Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (January 2009). “The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics.” Nucleic Acids Res. 37(Database Issue):D233-8).

[0034] The GH11 polypeptide refers to a polypeptide having enzymatic activity, which is classified as a member of glycoside hydrolase family 11 in the carbohydrate-active enzyme (CAZyme) database.

[0035] GH62 polypeptide: refers to a polypeptide having enzymatic activity, which is classified as a member of glycoside hydrolase family 62 in the carbohydrate-active enzyme (CAZyme) database.

[0036] GH43 polypeptide: refers to a polypeptide having enzymatic activity, which is classified as a member of glycoside hydrolase family 43 in the carbohydrate-active enzyme (CAZyme) database.

[0037] GH51 polypeptide: refers to a polypeptide having enzymatic activity, which is classified as a member of glycoside hydrolase family 51 in the carbohydrate-active enzyme (CAZyme) database.

[0038] Other definitions:

[0039] In this context, terms are used in a manner that is common to a person skilled in the art. Some of these terms are explained below:

[0040] Contact Time: For one or more enzymes to react with a substrate, the one or more enzymes must be in contact with the substrate. "Contact time" refers to the period of time during which an effective amount of one or more enzymes is in contact with at least a portion of the substrate material. During the contact time, the enzyme may not be in contact with all of the substrate material; however, mixing the one or more enzymes with the substrate material allows for the potential for enzyme-catalyzed hydrolysis of a portion of the substrate material during the contact time.

[0041] Corn kernels: Many varieties of corn kernels are known, including, for example, dent corn, flint corn, lemma corn, striped corn, sweet corn, waxy corn, and the like.

[0042] Some corn kernels have an outer covering called the pericarp that protects the germ inside the kernel. It repels water and water vapor and is undesirable to insects and microorganisms. The only area of ​​the kernel not covered by the pericarp is the tip cap, which is the point of attachment of the kernel to the cob.

[0043] Corn kernel or fraction of corn kernel: This term is used to describe corn kernels that have been subjected to the wet milling process. When the corn kernels are broken down and processed, all fractionated portions of the corn kernels are considered to be included when using this term. This term includes, for example, soaked kernels, ground kernels, corn kernel material, a first fraction, a second fraction, one or more fractions of corn kernel material, etc.

[0044] Corn kernel material: preferably used to refer to a material comprising fiber, gluten, and starch, preferably obtained by steaming and grinding the crop kernels and separating the material comprising fiber, gluten, and starch from the germ. As the corn kernel material moves through the fiber wash, it is separated into several fractions, including a first fraction (s) and a second fraction (f). Thus, "fractions of corn kernel material" and "one or more fractions of corn kernel material" refer specifically to these first fraction (s) and second fraction (f).

[0045] cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced ​​mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA, which is processed through a series of steps, including splicing, before appearing as mature, spliced ​​mRNA.

[0046] Coding sequence: The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon (e.g., ATG, GTG, or TTG) and ends with a stop codon (e.g., TAA, TAG, or TGA). The coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0047] Control sequences: The term "control sequences" means nucleic acid sequences necessary for the expression of a polynucleotide encoding a mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from a different gene) to the polynucleotide encoding the polypeptide, or native or exogenous to each other. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, these control sequences include a promoter, and transcription and translation termination signals. For the purpose of introducing specific restriction sites that facilitate connection of the control sequences to the coding region of the polynucleotide encoding the polypeptide, these control sequences may be provided with a plurality of linkers.

[0048] Expression: The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0049] Expression vector: The term "expression vector" means a linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.

[0050] Fragment: The term "fragment" means a polypeptide having one or more (eg, several) amino acids deleted from the amino and / or carboxyl terminus of a mature polypeptide, wherein the fragment has pectate lyase activity.

[0051] Germ: The "germ" is the only living part of the corn kernel. It contains the genetic information, enzymes, vitamins, and minerals necessary for the kernel to grow into a corn plant. In yellow dent corn, about 25% of the germ is corn oil. The endosperm, which is covered or surrounded by the germ, contains about 82% of the kernel's dry weight and is the source of energy (starch) and protein for seed germination. There are two types of endosperm, soft endosperm and hard endosperm. In hard endosperm, the starch is tightly packed together. In soft endosperm, the starch is loose.

[0052] Gluten: Gluten is a protein composed of two smaller proteins, glutenin and gliadin. "Gluten" herein refers to the majority of the protein found in corn kernels. The main products of gluten from corn wet milling are corn gluten meal (about 60% protein) and corn gluten feed (about 20% protein).

[0053] Grinding (grind or grinding): The term "grinding" refers to breaking down the corn kernels into smaller components.

[0054] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0055] Isolated: The term "isolated" means a substance that is in a form or setting not found in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance, including but not limited to any enzyme, variant, nucleic acid, protein, peptide, or cofactor, that is at least partially removed from one or more or all naturally occurring components associated with its properties; (3) any substance that has been modified by the hand of man relative to the substance found in nature; or (4) any substance that has been modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance).

[0056] Incubation time: The time that one or more fractions of corn kernel material are contacted with the hydrolytic enzyme during fiber washing without being screened.

[0057] In many preferred embodiments, the methods according to the present invention utilize a system comprising a space (V) or "incubator" within which the material is "left to be affected" by the enzyme, and in such cases, the incubation time can be determined by:

[0058]

[0059] Alternatively, if the flow into the incubator is expressed as volume per time unit:

[0060]

[0061] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form after translation and any post-translational modifications (such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.). It is known in the art that a host cell can produce a mixture of two of a plurality of different mature polypeptides (i.e., having different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus one host cell expressing a polynucleotide can produce different mature polypeptides (e.g., having different C-terminal and / or N-terminal amino acids) when compared to another host cell expressing the same polynucleotide.

[0062] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide.

[0063] Grinding Equipment: "Grinding Equipment" refers to all equipment used on a mill. The wet grinding process will vary depending on the available grinding equipment. Examples of grinding equipment include impregnation tanks, evaporators, screw presses, rotary dryers, dewatering screens, centrifuges, hydrocyclones, etc. The size and number of individual grinding equipment / grinding lines can vary on different mills, which will affect the grinding process. For example, the number of fiber washing screen units can vary, as can the size of the centrifuge.

[0064] Nucleic acid construct: The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified to contain a nucleic acid segment in a manner not originally found in nature, or is synthetic, and that contains one or more control sequences.

[0065] Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

[0066] Hold Time: The time allowed for one or more hydrolytic enzymes to react with corn kernels or fractions of corn kernels during a fiber washing process.

[0067] In some embodiments, the retention time is the period of time that the corn kernel material and one or more fractions thereof received in the first screen unit (S1) are contacted with an effective amount of one or more hydrolytic enzymes before leaving the fiber washing system again. During the retention time, the one or more fractions of the corn kernel material are incubated with the one or more hydrolytic enzymes in the space (V) and then leave the fiber washing system as part of the first fraction (s1) from the most upstream screen unit (S1) or as part of the second fraction (f4) from the most downstream screen unit (S4).

[0068] Retention time can preferably be estimated as the average duration that the solids spend in the fiber washing system as defined in the present invention. This can be estimated by the following relationship:

[0069]

[0070] Alternatively, if the inflow to the system is expressed in volume per time unit:

[0071]

[0072] The volume of the system is typically set equal to the sum of the volumes of all voids in the system; however, since the pipes in the system are typically made very small, it may be preferable to ignore the volume of the pipes.

[0073] Screened: The term "screened" or "screening" refers to the process of separating corn kernel material into a first fraction s and a second fraction f and moving these fractions from one screening unit to another. The non-screening period is a non-separation period provided for incubating the corn kernel material or its fractions with the enzyme.

[0074] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0075] For the purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as implemented in the Needleman program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably Version 3.0.0 or later). Version 6.1.0 is used.

[0076] The optional parameters used are gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle (obtained using the non-simplified option) labeled as "longest identity" is used as identity percentage and is calculated as follows: (identical residue x 100) / (total number of gaps in the alignment length-alignment).

[0077] Starch: The term "starch" refers to a complex polysaccharide made from plants

[0078] It is composed of glucose units that are widely present in plant tissues in the form of storage grains, composed of amylose and amylopectin and is represented by (C6H 10 O5)n (where n is any number) of any material.

[0079] Steeping or soaking: The term "steeping" means soaking the crop kernels with water and optionally SO2. DETAILED DESCRIPTION

[0080] It is an object of the present invention to provide a method for improving starch and gluten yields from a corn wet milling process.

[0081] In particular, the object of the present invention is to provide a method for improving the starch and / or gluten yield obtainable from corn kernels by treating the fiber fraction with a hydrolytic enzyme composition in a wet milling process, preferably during a fiber washing procedure. The inventors of the present invention have surprisingly found that enzymatic treatment of corn fiber in the presence of at least one xylanase and / or cellulase and an effective amount of SO2 increases the release of bound starch and gluten from the fiber and thereby improves the obtainable starch and / or gluten yield.

[0082] Wet grinding process:

[0083] Corn kernels are wet milled to open the kernels and separate them into their four main components: starch, germ, fiber, and gluten.

[0084] The wet milling process can vary significantly between mills, however conventional wet milling generally involves the following steps:

[0085] 1. Maceration

[0086] 2. Grinding

[0087] 3. Separate into streams containing:

[0088] i) germ; ii) fiber, iii) starch and gluten

[0089] 4. Wash the fibers, press and dry

[0090] 5. Starch / gluten separation, and

[0091] 6. Washing starch.

[0092] Maceration, grinding and germ separation

[0093] Corn kernels are softened by soaking them in water at a temperature of about 50°C, such as between about 45°C and 60°C, for a period of about 30 minutes to about 48 hours, preferably 30 minutes to about 15 hours, such as about 1 hour to about 6 hours. During the soaking period, the kernels absorb water, increasing their moisture content from 15% to 45%, and more than double in size. Optionally, 0.1% sulfur dioxide (SO2) and / or NaHSO3, for example, are added to the water to prevent excessive bacterial growth in the warm environment. As the corn swells and softens, the mild acidity of the steeping water begins to loosen the gluten bonds within the corn and release the starch. After the corn kernels are steeped, they are cracked open by grinding to release the germ. The germ contains corn oil. The germ is essentially separated from the denser mixture of starch, gluten, and fiber (the corn kernel material comprises fiber, starch, and gluten) by "floating" the germ segments, which are free of other substances, under strictly controlled conditions. This process serves to eliminate any adverse effects of trace amounts of corn oil in subsequent processing steps. The germ can then be dried and the oil extracted.

[0094] The corn kernel material comprising fiber, starch, and gluten is then separated into fiber, starch, and gluten fractions, such as in a fiber washing step.

[0095] Washing fibers, pressing and drying

[0096] In order to obtain maximum starch and gluten recovery while keeping any fiber in the final product to an absolute minimum, free starch and gluten must be washed from the fiber during processing. The free starch and gluten are separated from the fiber during screening (washing) and collected as ground starch. The remaining fiber is then pressed to reduce the water content.

[0097] Isolate starch gluten

[0098] The starch-gluten suspension, along with other starch gluten released from the fiber washing step (called mill starch), is separated into starch and gluten. Gluten has a lower density than starch. Gluten is easily spun out by passing mill starch through a centrifuge.

[0099] Detergent starch

[0100] The starch slurry from the starch separation step contains some insoluble protein and a lot of soluble matter. Before top quality starch (high purity starch) can be produced, it must be removed. In a hydrocyclone, the starch, which has only 1% or 2% protein left, is diluted, washed 8 to 14 times, rediluted and washed again to remove the last traces of protein and produce high quality starch, typically with a purity greater than 99.5%.

[0101] Products of wet grinding:Wet milling can be used to produce, but is not limited to, corn steep liquor, corn gluten feed, germ, corn oil, corn gluten meal, corn starch, modified corn starch, syrups (such as corn syrup), and corn ethanol.

[0102] One aspect of the present invention provides a method for increasing the total starch yield and / or gluten yield obtainable from corn kernels in a wet milling process, the method comprising: mixing corn kernels or fractions of corn kernels with an enzyme composition comprising an effective amount of one or more hydrolases, wherein at least one of the hydrolases is selected from the group consisting of: a xylanase polypeptide, and / or a cellulase polypeptide, or a combination thereof.

[0103] Some starch and / or gluten in the corn kernel or corn kernel fractions may be bound to the fiber fraction and never released during the wet milling process. However, the addition of a hydrolase (which may include any catalytic protein that can use water to break down substrates present in the corn kernel) may release some of the bound starch and / or gluten, thereby increasing the overall yield of starch and / or gluten in the wet milling process.

[0104] The present inventors have surprisingly found that the effect of adding hydrolytic enzymes such as cellulases and xylanases to the fiber-rich fraction of milled grain material, particularly during the fiber washing step, can be enhanced at elevated SO2 levels.

[0105] Thus, in a first aspect, the present invention relates to a method for increasing starch yield and / or gluten yield from corn kernels in a wet milling process, the method comprising contacting the milled corn kernels or fractions of the milled kernels, particularly fiber-rich fractions, with an effective amount of SO and an effective amount of one or more hydrolases, wherein at least one of the hydrolases is selected from xylanases and / or cellulases. Preferably, the contacting occurs during a fiber washing step.

[0106] In one embodiment, the method of the present invention increases the amount of starch and / or gluten released from the fiber during the wet milling process compared to a process in the absence / addition of SO2.

[0107] The specific procedures and equipment used in the wet milling process may vary, but the general principles of the process remain the same (see description of the wet milling process).

[0108] In a specific embodiment, the method of the present invention comprises the following steps:

[0109] a) soaking the corn kernels in water to produce soaked kernels;

[0110] b) milling the soaked kernels to produce milled kernels;

[0111] c) separating the germ from the milled kernels to produce a corn kernel material comprising fiber, starch, and gluten; and

[0112] d) subjecting the resulting fiber-containing corn kernel material to a fiber washing procedure to separate starch, gluten, and fiber;

[0113] wherein at least one xylanase and / or cellulase and an effective amount of SO2 are present / added before or during step d).

[0114] In order to obtain maximum starch and gluten recovery while keeping any fiber in the final product to an absolute minimum, free starch and gluten must be washed from the fiber fraction during processing. The fiber is collected, slurried, and sieved, typically after soaking, grinding, and separating the germ from the corn kernel, to recover any residual starch or gluten in the corn kernel material. This process is referred to herein as the fiber washing procedure.

[0115] In a preferred embodiment, said corn kernels or said fiber-rich fraction of corn kernels are mixed with said one or more hydrolyzing enzymes during the step of subjecting the corn kernel material to a fiber washing procedure.

[0116] According to the present invention, in order to maximize the effect of the hydrolytic enzymes during the fiber washing step, an enhanced effect is observed when an effective amount of SO2 is present during the fiber washing step. Typically, SO2 is added during the step of soaking the kernels, however, in downstream steps, such as the fiber washing step, the SO2 level will drop below the level required by the present method.

[0117] In one embodiment, SO2 is present / added during fiber washing in an amount of at least 400 ppm, at least 450 ppm, at least 500 ppm, at least 600 ppm, at least 700 ppm, at least 800 ppm.

[0118] In another embodiment, SO2 is present / added during fiber washing in an amount ranging from 400 ppm to 3000 ppm, 500 ppm to 2000 ppm, 600 ppm to 1500 ppm, such as 600 ppm to 1200 ppm.

[0119] In fiber washing program, the specific equipment used can change, but the main principle of the process remains unchanged.WO 2018 / 053220 describes a fiber washing system, which includes a dedicated enzyme incubation space / groove. Based on this disclosure and the general knowledge of those skilled in the art, it is possible to design a fiber washing system for providing enough incubation times for hydrolytic enzymes to play a role. In one embodiment, the fraction (for example, fiber-rich fraction) of the corn kernel or the corn kernel is allowed to react with the one or more hydrolytic enzymes for at least 15 minutes, such as at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes or at least 120 minutes.

[0120] In one embodiment, the fiber washing program comprises a fiber washing system optimized for the introduction of one or more hydrolases, wherein the fiber washing system comprises a space (V) configured to provide the following total reaction time (retention time) in the fiber washing system: at least 35 minutes (such as at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes or at least 120 minutes) and less than 48 hours (such as less than 40 hours, less than 36 hours, less than 30 hours, less than 24 hours, less than 20 hours, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours). In one embodiment, the total residence time in the fiber washing system is between 35 minutes and 48 hours, such as between 35 minutes and 24 hours, between 35 minutes and 12 hours, between 35 minutes and 6 hours, between 35 minutes and 5 hours, between 35 minutes and 4 hours, between 35 minutes and 3 hours, between 35 minutes and 2 hours, between 45 minutes and 48 hours, between 45 minutes and 24 hours, between 45 minutes and 1 hour, between 45 minutes and 6 hours, between 45 minutes and 5 hours, between 45 minutes and 4 hours, between 45 minutes and 3 hours, between 45 minutes and 2 hours, between 1-48 hours, between 1-24 hours, between 1-12 hours, between 1-6 hours, between 1-5 hours, between 1-4 hours, between 1-3 hours, and between 1-2 hours.

[0121] In one embodiment, the fiber washing system comprises:

[0122] - a plurality of screen units (S1 ... S4) fluidly connected in a countercurrent wash configuration; each screen unit being configured to separate the corn kernel material and the liquid stream into two fractions: a first fraction (s) and a second fraction (f), the second fraction (f) containing a higher amount of fiber measured in wt% compared to the first fraction (s);

[0123] - a space (V) arranged in the system and fluidically connected to receive the first fraction (s), the second fraction (f), or the mixed first and second fractions (s, f), preferably only the second fraction (f), and configured to provide an incubation time for one or both fractions received in the space; and to conduct the thus incubated one or both fractions to a downstream sieve unit (S4),

[0124] The system is configured to

[0125] -Introducing corn kernel material and liquid into the most upstream screen unit (S1)

[0126] - conducting the first fraction (s1) from the most upstream screen unit (S1) as a starch-containing product stream,

[0127] - introducing process water, preferably arranged to introduce process water into the most downstream screen unit (S4),

[0128] - The second fraction (f4) from the most downstream screen unit (S4) is conducted as washed corn kernel material, which contains lower amounts of starch and gluten than the initial corn kernel material.

[0129] -Introduction of hydrolytic enzymes into the system.

[0130] In one embodiment, the incubation time in the space (V) configured in the fiber washing system is at least 5 minutes (such as at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes or at least 120 minutes) and less than 48 hours (such as less than 40 hours, less than 36 hours, less than 30 hours, less than 24 hours, less than 20 hours, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours).

[0131] In one embodiment, the incubation time in the space (V) is between 35 minutes and 48 hours, such as between 35 minutes and 24 hours, between 35 minutes and hours, between 35 minutes and 6 hours, between 35 minutes and 5 hours, between 35 minutes and 4 hours, between 35 minutes and 3 hours, between 35 minutes and 2 hours, between 45 minutes and 48 hours, between 45 minutes and 24 hours, between 45 minutes and 12 hours, between 45 minutes and 6 hours, between 45 minutes and 5 hours, between 45 minutes and 4 hours, between 45 minutes and 3 hours, between 45 minutes and 2 hours, between 1-48 hours, between 1-24 hours, between 1-12 hours, between 1-6 hours, between 1-5 hours, between 1-4 hours, between 1-3 hours, and between 1-2 hours.

[0132] In one embodiment, the incubation temperature in the space (V) is between 25°C and 95°C, such as between 25°C and 90°C, between 25°C and 85°C, between 25°C and 80°C, between 25°C and 75°C, between 25°C and 70°C, between 25°C and 65°C, between 25°C and 60°C, between 25°C and 55°C, between 25°C and 53°C, between 25°C and 52°C, between 30°C and 90°C, between 30°C and 85°C, between 30°C and 80°C, between 30°C and 75°C, between 30°C and 70°C, between 30°C and 65°C, between 30°C and 60°C, between 30°C and 55°C, between 30°C and 53°C , between 30℃ and 52℃, between 35℃ and 90℃, between 35℃ and 85℃, between 35℃ and 80℃, between 35℃ and 75℃, between 35℃ and 70℃, between 35℃ and 65℃, between 35℃ and 60℃, between 35℃ and 55℃, between 35℃ and 53℃, between 35℃ and 52℃, between 39℃ and 90℃, between 39℃ and 85℃, between 39℃ and 80℃, between 39℃ and 75℃, between 39℃ and 70℃, between 39℃ and 65℃, between 39℃ and 60℃, between 39℃ and 55℃, between 39℃ and 53℃, between 39℃ and 52℃, such as between 46℃ and 52℃.

[0133] In addition, the size of the space (in m 3The incubation time (in units) is preferably configured to provide an incubation time of at least at least 5 minutes (e.g., at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least 120 minutes).

[0134] The space designated for incubation (V) preferably has a surface area of ​​at least 30 m 3 , at least 40m 3 , at least 50m 3 , at least 60m 3 , at least 70m 3 , at least 80m 3 , at least 90m 3 , at least 100m 3 , at least 110m 3 , at least 120m 3 , at least 130m 3 , at least 140m 3 , at least 150m 3 , at least 160m 3 , at least 170m 3 , at least 180m 3 , at least 190m 3 , at least 200m 3 , at least 210m 3 , at least 220m 3 , at least 230m 3 , at least 240m 3 , at least 250m 3 , at least 260m 3 , at least 270m 3 , at least 280m 3 , at least 290m 3 , at least 300m 3 , at least 400m 3 , or at least 500m 3 The incubation time can also be in more than one space V, with at least 100m 3 , at least 110m 3 , at least 120m 3 , at least 130m 3 , at least 140m 3 , at least 150m 3 , at least 160m 3 , at least 170m 3 , at least 180m 3 , at least 190m3 , at least 200m 3 , at least 210m 3 , at least 220m 3 , at least 230m 3 , at least 240m 3 , at least 250m 3 , at least 260m 3 , at least 270m 3 , at least 280m 3 , at least 290m 3 , at least 300m 3 , at least 400m 3 , at least 500m 3 The total volume or combined volume.

[0135] During the incubation time, the fluid received in the space V is preferably not filtered. Therefore, the fluid leaving the space V has the same composition, e.g. starch and fibers as the fluid received in the space V, although it preferably contains a higher proportion of starch that has been released from the fibers.

[0136] In order to ensure intimate contact between the enzyme and the fibers, the space V may preferably be configured for stirring the material contained in said space V, such as by comprising a rotor or an impeller.

[0137] The space V is preferably arranged downstream of the most upstream screen unit S1 and upstream of the most downstream screen unit S4; in particular, the space V is arranged to feed the fluid into the second most downstream screen unit S3.

[0138] Hydrolases suitable for use in the methods of the present invention

[0139] In one embodiment, the hydrolases suitable for use in the methods of the invention comprise one or more enzymes selected from the group consisting of cellulases (EC 3.2.1.4), xylanases (EC 3.2.1.8), arabinofuranosidases (EC 3.2.1.55 (non-reducing end α-L-arabinofuranosidase); EC 3.2.1.185 (non-reducing end β-L-arabinofuranosidase), cellobiohydrolase I (EC 3.2.1.150), cellobiohydrolase II (EC 3.2.1.91), cellobiosidase (EC 3.2.1.176), β-glucosidase (EC 3.2.1.21), β-xylosidase (EC 3.2.1.37), and protease (EC 3.4).

[0140] In one embodiment, the xylanase is selected from the group consisting of a GH5 polypeptide, a GH30 polypeptide, a GH10 polypeptide, a GH11 polypeptide, a GH8 polypeptide, or a combination thereof.

[0141] In another embodiment, these lytic enzymes comprise one or more cellulases.These cellulases can be selected from the group consisting of: endoglucanase (EG) and cellobiohydrolase (CBH) at least.More particularly, these one or more cellulases comprise one or more enzymes selected from the group consisting of: endoglucanase, cellobiohydrolase I, cellobiohydrolase II or its combination.

[0142] In one embodiment, the hydrolases further comprise an arabinofuranosidase. The arabinofuranosidase may be selected from the group consisting of a GH43 polypeptide, a GH62 polypeptide, a GH51 polypeptide, in particular a GH62 polypeptide.

[0143] In one embodiment, one or more hydrolases are expressed in an organism with a cellulase background, such as Trichoderma reesei. According to these embodiments, xylanase and or arabinofuranosidase polypeptides as defined herein are expressed together with endogenous cellulases from Trichoderma.

[0144] In one embodiment, an enzyme composition comprising one or more hydrolases may comprise cellulases expressed in Trichoderma reesei and other hydrolases added to the enzyme composition in purified or semi-purified form.

[0145] In one embodiment, one or more hydrolases are purified. These purified enzymes can be used in the enzyme compositions described in other embodiments of the present invention.

[0146] In one embodiment, the one or more hydrolases are in a liquid composition.The composition may be homogeneous or heterogeneous.

[0147] In one embodiment, the one or more hydrolases are in a solid composition.

[0148] In one embodiment, the effective amount of one or more hydrolases mixed with one or more fractions of corn kernel material is between 0.005-0.5 kg enzyme protein (EP) per metric ton (MT) of corn kernel entering the wet milling process, such as between 0.010-0.5 kg EP per MT of corn kernel, such as between 0.05-0.5 kg / MT of corn kernel, or between 0.075-0.5 kg / MT, or between 0.1-0.5 kg / MT of corn kernel, or between 0.005-0.4 kg / MT of corn kernel, or between 0.01-0.4 kg / MT of corn kernel, or between 0.05-0.4 kg / MT of corn kernel, or between 0.075-0.4 kg / MT of corn kernel, or between 0.1-0.4 kg / MT of corn kernel, or between 0.005-0.3 kg / MT of corn kernel, or between 0.01-0.3 kg / MT of corn kernel. MT corn kernels, or between 0.05-0.3 kg / MT corn kernels, or between 0.075-0.3 kg / MT or between 0.1-0.3 kg / MT corn kernels, or between 0.005-0.2 kg / MT corn kernels, or between 0.010-0.2 kg / MT corn kernels, or between 0.05-0.2 kg / MT corn kernels, or between 0.075-0.2 kg / MT, or between 0.1-0.2 kg / MT corn kernels, or such as between 0.075-0.10 kg / MT corn kernels, or between 0.075-0.11 kg / MT corn kernels.

[0149] In a preferred embodiment, the enzyme composition comprises cellulases obtained from a culture of Trichoderma reesei, such as a culture of Trichoderma reesei ATCC 26921. Suitable cellulases are available, for example, from Novozymes under the trade name

[0150] Polypeptides with xylanase activity

[0151] Xylanases are suitable for use in the methods according to the invention.The xylanase polypeptide may be selected from the families GH5, GH10, GH30, GH11 and GH8.

[0152] More specific embodiments relate to the method according to the invention, wherein the GH5 xylanase is selected from the group consisting of:

[0153] (a) a polypeptide having at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 1;

[0154] (b) a variant of the mature polypeptide of SEQ ID NO: 1, the variant comprising a substitution, deletion and / or insertion at one or more positions; and

[0155] (c) A fragment of the polypeptide of (a) or (b) having xylanase activity.

[0156] In one embodiment, the mature polypeptide is amino acids 25 to 551 of SEQ ID NO: 1.

[0157] Another particular embodiment relates to the method according to the invention, wherein the GH10 xylanase is chosen from the group consisting of:

[0158] (a) a polypeptide having at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 2;

[0159] (b) a variant of the mature polypeptide of SEQ ID NO: 2, the variant comprising a substitution, deletion and / or insertion at one or more positions; and

[0160] (c) A fragment of the polypeptide of (a) or (b) having xylanase activity.

[0161] In one embodiment, the mature polypeptide is amino acids 21 to 405 of SEQ ID NO:2.

[0162] Another particular embodiment relates to the method according to the invention, wherein the GH10 xylanase is chosen from the group consisting of:

[0163] (a) a polypeptide having at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 4;

[0164] (b) a variant of the mature polypeptide of SEQ ID NO: 4, the variant comprising a substitution, deletion and / or insertion at one or more positions; and

[0165] (c) A fragment of the polypeptide of (a) or (b) having xylanase activity.

[0166] In one embodiment, the mature polypeptide is amino acids 20 to 319 of SEQ ID NO:4.

[0167] Polypeptide having arabinofuranosidase activity

[0168] Another particular embodiment relates to the method according to the invention, wherein the GH62 arabinofuranosidase is chosen from the group consisting of:

[0169] (a) a polypeptide having at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 3;

[0170] (b) a variant of the mature polypeptide of SEQ ID NO: 3, the variant comprising a substitution, deletion and / or insertion at one or more positions; and

[0171] (c) A fragment of the polypeptide of (a) or (b) having xylanase activity.

[0172] In one embodiment, the mature polypeptide is amino acids 17 to 325 of SEQ ID NO:3.

[0173] Sources of polypeptides having xylanase activity

[0174] The polypeptide having xylanase activity can be obtained from microorganisms of any genus. For the purposes of the present invention, the term "obtained from" as used herein in conjunction with a given source shall mean that the polypeptide encoded by the polynucleotide is produced by the source or by a strain into which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0175] The polypeptide can be a bacterial polypeptide. For example, the polypeptide can be a Gram-positive bacterial polypeptide, such as a Bacillus, Chryseobacterium, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or a gram-positive bacterial polypeptide having pectate lyase activity. ccus), or Streptomyces polypeptides, or Gram-negative bacterial polypeptides, such as Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma polypeptides.

[0176] In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide.

[0177] In another aspect, the polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide.

[0178] In another aspect, the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide.

[0179] The polypeptide can be a fungal polypeptide. For example, the polypeptide can be a yeast polypeptide, such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; or a filamentous fungal polypeptide, such as an Acremonium, Agaricus, Alternaria, an Aspergillus, such as Aspergillus niger. niger), Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lenti nula), Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, such as Talaromycesleycettanus), Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria polypeptides.

[0180] Strains of these species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Culture Collection of Microorganisms (DSMZ), the Netherlands Center for the Study of Microorganisms (Centraalbureau Voor Schimmelcultures (CBS), and the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0181] The above-mentioned probe can be used from other sources, including from nature (for example, soil, compost, water etc.) separated microorganisms or directly from natural materials (for example, soil, compost, water etc.) DNA sample identification and obtain this polypeptide. The technology for directly isolating microorganisms and DNA from natural habitats is well known in the art. The polynucleotide encoding this polypeptide can then be obtained by similarly screening the genomic DNA or cDNA library of another microorganism or the DNA sample of mixing. Once the polynucleotide encoding polypeptide has been detected with one or more probes, then the polynucleotide can be separated or cloned by utilizing technology known to those of ordinary skill in the art (see for example, Sambrook et al., 1989, the same).

[0182] Enzyme composition

[0183] The enzyme compositions used in the methods according to the invention may comprise a xylanase polypeptide as the major enzymatic component, e.g., a monocomponent composition. Alternatively, these compositions may comprise multiple enzyme activities, such as one or more (e.g., several) enzymes selected from the group consisting of: cellobiohydrolases, cellulases, endoglucanases, and / or arabinofuranosidases.

[0184] The composition may be prepared according to methods known in the art and may be in the form of a liquid or dry composition.The composition may be stabilized according to methods known in the art.

[0185] The invention is further disclosed in the following numbered examples.

[0186] Example 1. A method for increasing starch yield and / or gluten yield from corn kernels in a wet milling process, the method comprising contacting a fiber-rich fraction of the milled kernels with an effective amount of SO2 and an effective amount of one or more hydrolytic enzymes during a fiber washing step, wherein at least one of the hydrolytic enzymes is selected from xylanases and / or cellulases.

[0187] Example 2. The method of Example 1, wherein the amount of starch and / or gluten released from the fiber during the wet milling process is increased compared to a process in which SO2 is not present / added.

[0188] Embodiment 3. The method according to any one of the preceding embodiments, comprising the following steps:

[0189] a) soaking the corn kernels in water to produce soaked kernels;

[0190] b) milling the soaked kernels to produce milled kernels;

[0191] c) separating the germ from the milled kernels to produce a corn kernel material comprising fiber, starch, and gluten;

[0192] d) subjecting the resulting fiber-containing corn kernel material to a fiber washing procedure to separate starch, gluten, and fiber;

[0193] wherein at least one xylanase and / or one or more cellulases and an effective amount of SO2 are present / added before or during step d).

[0194] Embodiment 4. The method of any one of embodiments 1 to 3, wherein SO2 is present / added during the fiber washing step (d) in an amount of at least 400 ppm, at least 450 ppm, at least 500 ppm, at least 600 ppm, at least 700 ppm, at least 800 ppm.

[0195] Embodiment 5. The method of any one of embodiments 1 to 4, wherein SO2 is present / added during fiber washing in an amount ranging from 400 ppm to 3000 ppm, 500 ppm to 2000 ppm, 600 ppm to 1500 ppm, such as 600 ppm to 1200 ppm.

[0196] Embodiment 6. The method of any one of the preceding embodiments, wherein the xylanase is selected from the group consisting of a GH5 polypeptide, a GH30 polypeptide, a GH10 polypeptide, a GH11 polypeptide, a GH8 polypeptide, or a combination thereof.

[0197] Embodiment 7. The method of any of the preceding embodiments, wherein the hydrolases comprise one or more cellulases, particularly cellulases obtained from Trichoderma, more particularly from Trichoderma reesei.

[0198] Embodiment 8. The method of embodiment 7, wherein the one or more cellulases comprise one or more enzymes selected from the group consisting of endoglucanase (EG) and cellobiohydrolase (CBH).

[0199] Embodiment 9. The method of embodiment 8, wherein the one or more cellulases comprise one or more enzymes selected from the group consisting of endoglucanases, cellobiohydrolases I, cellobiohydrolases II, or a combination thereof.

[0200] Embodiment 10. The method of any preceding embodiment, wherein the hydrolases further comprise an arabinofuranosidase.

[0201] Example 11 The method of Example 10, wherein the arabinofuranosidase is selected from the group consisting of a GH43 polypeptide, a GH62 polypeptide, and a GH51 polypeptide.

[0202] Embodiment 12. The method of any one of embodiments 3 to 9, wherein the fiber washing procedure comprises using a fiber washing system comprising a space (V) / tank configured to provide a total retention time in the fiber washing system of at least 35 minutes and less than 48 hours.

[0203] Embodiment 13. A method as described in any of the preceding embodiments, wherein the incubation time in the space (V) / tank configured in the fiber washing system is at least 5 minutes and less than 48 hours, such as between 35 minutes and 24 hours, between 35 minutes and hours, between 35 minutes and 6 hours, between 35 minutes and 5 hours, between 35 minutes and 4 hours, between 35 minutes and 3 hours, between 35 minutes and 2 hours, between 45 minutes and 48 hours, between 45 minutes and 24 hours, between 45 minutes and 12 hours, between 45 minutes and 6 hours, between 45 minutes and 5 hours, between 45 minutes and 4 hours, between 45 minutes and 3 hours, between 45 minutes and 2 hours.

[0204] Embodiment 14. The method of any of the preceding embodiments, wherein the incubation temperature is between 25°C and 95°C, such as between 25°C and 90°C, between 25°C and 85°C, between 25°C and 80°C, between 25°C and 75°C, between 25°C and 70°C, between 25°C and 65°C, between 25°C and 60°C, between 25°C and 55°C, between 25°C and 53°C, between 25°C and 52°C, between 30°C and 90°C, between 30°C and 85°C, between 30°C and 80°C, between 30°C and 75°C, between 30°C and 70°C, between 30°C and 65°C, between 30°C and 60°C, between 30°C and 55°C, between 30°C and 53°C ℃, between 30 ℃ and 52 ℃, between 35 ℃ and 90 ℃, between 35 ℃ and 85 ℃, between 35 ℃ and 80 ℃, between 35 ℃ and 75 ℃, between 35 ℃ and 70 ℃, between 35 ℃ and 65 ℃, between 35 ℃ and 60 ℃, between 35 ℃ and 55 ℃, between 35 ℃ and 53 ℃, between 35 ℃ and 52 ℃, between 39 ℃ and 90 ℃, between 39 ℃ and 85 ℃, between 39 ℃ and 80 ℃, between 39 ℃ and 75 ℃, between 39 ℃ and 70 ℃, between 39 ℃ and 65 ℃, between 39 ℃ and 60 ℃, between 39 ℃ and 55 ℃, between 39 ℃ and 53 ℃, between 39 ℃ and 52 ℃, preferably between 46 ℃ and 52 ℃.

[0205] Embodiment 15. The method of any of the preceding embodiments, wherein the level of SO2 present / added during fiber washing results in the same extraction yield of starch and gluten while reducing the contact time required between the hydrolytic enzyme and the milled corn kernel material compared to a process wherein the SO2 level is less than 400 ppm.

[0206] Embodiment 16. The method of any one of the preceding embodiments, wherein the one or more hydrolases are expressed in an organism with a cellulase background, such as Trichoderma reesei.

[0207] Embodiment 17. The method of any preceding embodiment, wherein the one or more hydrolases are purified.

[0208] Embodiment 18. The method of any one of the preceding embodiments, wherein the one or more hydrolases are in a liquid composition.

[0209] Embodiment 19. The method of any one of the preceding embodiments, wherein the one or more hydrolases are in a solid composition.

[0210] Embodiment 20. The method of any of the preceding embodiments, wherein the effective amount of one or more hydrolytic enzymes mixed / contacted with one or more fractions of the milled corn kernel material is between 0.005 kg and 0.5 kg enzyme protein per metric ton of corn kernels entering the wet milling process.

[0211] Embodiment 21. The method of any one of the preceding embodiments, wherein the source of SO2 is sodium metabisulfite (Na2S2O5) and / or added SO2 gas.

[0212] Embodiment 22. The method of any one of the preceding embodiments, wherein the xylanase is selected from the group consisting of:

[0213] (a) a polypeptide having at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 1;

[0214] (b) a variant of the mature polypeptide of SEQ ID NO: 1, the variant comprising a substitution, deletion and / or insertion at one or more positions; and

[0215] (c) A fragment of the polypeptide of (a) or (b) having xylanase activity.

[0216] Embodiment 23. The polypeptide of embodiment 22, wherein the mature polypeptide is amino acids 1 to 551 of SEQ ID NO: 1.

[0217] Embodiment 24. The method of any one of the preceding embodiments, wherein the xylanase is selected from the group consisting of:

[0218] (a) a polypeptide having at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 2;

[0219] (b) a variant of the mature polypeptide of SEQ ID NO: 2, the variant comprising a substitution, deletion and / or insertion at one or more positions; and

[0220] (c) A fragment of the polypeptide of (a) or (b) having xylanase activity.

[0221] Embodiment 25. The polypeptide of embodiment 24, wherein the mature polypeptide is amino acids 21 to 405 of SEQ ID NO: 2.

[0222] Embodiment 26. The method of any one of the preceding embodiments, wherein the arabinofuranosidase is selected from the group consisting of:

[0223] (a) a polypeptide having at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 3;

[0224] (b) a variant of the mature polypeptide of SEQ ID NO: 3, the variant comprising a substitution, deletion and / or insertion at one or more positions; and

[0225] (c) A fragment of the polypeptide of (a) or (b) having xylanase activity.

[0226] Embodiment 27. The polypeptide of embodiment 26, wherein the mature polypeptide is amino acids 17 to 325 of SEQ ID NO: 3.

[0227] Embodiment 28. The method of any one of the preceding embodiments, wherein the xylanase is selected from the group consisting of:

[0228] (a) a polypeptide having at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 4;

[0229] (b) a variant of the mature polypeptide of SEQ ID NO: 4, the variant comprising a substitution, deletion and / or insertion at one or more positions; and

[0230] (c) A fragment of the polypeptide of (a) or (b) having xylanase activity.

[0231] Embodiment 29. The polypeptide of embodiment 28, wherein the mature polypeptide is amino acids 20 to 319 of SEQ ID NO: 4.

[0232] Embodiment 30. The method of any preceding embodiment, wherein the cellulase is derived from Trichoderma reesei.

[0233] The present invention is further illustrated by the following examples.

[0234] Examples

[0235] Enzymes:

[0236] GH5 xylanase A : GH5 xylanase is derived from Chryseobacterium sp.-10696 and is disclosed as SEQ ID NO: 1

[0237] Cellulase A : A whole cellulase derived from Trichoderma reesei. The cellulase composition will contain all cellulase activities expressed in Trichoderma reesei (T. reesei); for example, endoglucanase and cellobiohydrolase.

[0238] Example 1:

[0239] The 10-g fiber assay was performed using a blend of Cellulase A and GH5 xylanase with either 400 ppm or 800 ppm bisulfite (HSO3 - ) and incubated at pH 4.0, 50°C for 150 minutes with 5% fiber dry matter at a dosage of 200 μg or 300 μg enzyme protein per gram fiber dry matter. The blend consisted of 8% GH5 xylanase A and 92% cellulase A based on enzyme protein. + +2HSO3 - After the reaction, sodium metabisulfite (Na2S2O5) was added to the buffer to generate bisulfite. As a comparison, a blend containing only 92% cellulase A and 8% GH5 xylanase A (without SO2) was included, including both a low dose (200 μg EP / g-ds fiber) and a high dose (300 μg EP / g-ds fiber). In the fiber assay, corn fiber with 17.77% residual starch and 9.88% residual protein was used as the substrate. The release of starch + gluten (dry matter) as well as individual proteins from corn fiber was measured under the following specified treatments.

[0240] Table 1. Starch and gluten yields with and without enzyme treatment

[0241]

[0242] Therefore, adding bisulfite to cellulase A+GH5 xylanase A can significantly improve the yield of starch+gluten and protein in corn wet milling process.

[0243] Example 2.

[0244] The 10-g fiber assay typically involves incubating a wet fiber sample obtained from a wet-milled plant in the presence of an enzyme under process-related conditions (pH 4, temperature of approximately 50°C) for a period of between 1 and 4 hours. After incubation, the fiber is transferred and pressed through a 75-micron sieve, and the filtrate, which is primarily composed of separated starch and gluten, is collected. Multiple washes are performed on the sieve, and the washes are collected along with the initial filtrate. The collected filtrate is allowed to stand overnight, allowing the insoluble matter to settle at the bottom of the flask. Most of the supernatant is removed by vacuum suction, and the remaining insoluble matter is then centrifuged in a 50 ml conical tube, after which the supernatant is decanted, leaving a wet insoluble precipitate. The wet insoluble precipitate is freeze-dried overnight to complete dryness. The insoluble dry matter is weighed to determine the percent yield of the insoluble matter, and the total nitrogen content (protein) is then analyzed by the Leco assay.

[0245] The 10-g fiber assay was performed using a blend comprising cellulase A and GH5 xylanase A, incubated at pH 4.0, 48°C for 240 minutes with 6.4% fiber dry matter at a dosage of 5000 μg enzyme protein per gram of fiber dry matter. The blend consisted of 10% GH5 xylanase A and 90% cellulase A based on enzyme protein. The blend was tested both on the substrate as is and on the substrate sheared (homogenized) for 3 minutes in a mixer. The homogenized substrate was treated with the enzyme blend with and without 1000 ppm SO2 (added from a 20-fold dilution of 1.48 g sodium metabisulfate dissolved in 50 ml of water), and without enzyme treatment. The substrate as is was treated with the enzyme blend under both conditions with and without 1000 ppm SO2, and twice without enzyme treatment, with and without 1000 ppm SO2. The weights of insoluble material (starch and gluten) and insoluble protein (gluten) released by the specific treatments are shown below.

[0246] Table 2. Results

[0247]

Claims

1. A method for increasing starch yield and / or gluten yield from corn kernels in a wet milling process, the method comprising the steps of: a) soaking the corn kernels in water to produce soaked kernels; b) milling the soaked kernels to produce milled kernels; c) separating the germ from the milled kernels to produce a corn kernel material comprising fiber, starch, and gluten; d) subjecting the resulting fiber-containing corn kernel material to a fiber washing procedure to separate starch, gluten, and fiber; wherein the fiber-rich fraction of the milled grain is contacted with an effective amount of SO2 and an effective amount of one or more hydrolytic enzymes during the fiber washing step, wherein at least one of the hydrolytic enzymes is selected from xylanases and / or cellulases, and wherein the SO2 is added in an amount of at least 400 ppm during the fiber washing step.

2. The method of claim 1, wherein SO2 is added during the fiber washing step in an amount of at least 450 ppm.

3. The method of claim 1, wherein SO2 is added in an amount of at least 500 ppm during the fiber washing step.

4. The method of claim 1, wherein SO2 is added in an amount of at least 600 ppm during the fiber washing step.

5. The method of claim 1, wherein SO2 is added in an amount of at least 700 ppm during the fiber washing step.

6. The method of claim 1, wherein SO2 is added in an amount of at least 800 ppm during the fiber washing step.

7. The method of claim 1 , wherein the amount of starch and / or gluten released from the fiber during the wet milling process is increased compared to a process without the addition of SO 2 .

8. The method of any one of claims 1 to 7, wherein SO2 is added during fiber washing in an amount ranging from 400 ppm to 3000 ppm.

9. The method of any one of claims 1 to 7, wherein SO2 is added during fiber washing in an amount ranging from 500 ppm to 2000 ppm.

10. The method of any one of claims 1 to 7, wherein SO2 is added during fiber washing in an amount ranging from 600 ppm to 1500 ppm.

11. The method of any one of claims 1 to 7, wherein SO2 is added during fiber washing in an amount ranging from 600 ppm to 1200 ppm.

12. The method of any one of claims 1 to 7, wherein the xylanase is selected from the group consisting of a GH5 polypeptide, a GH30 polypeptide, a GH10 polypeptide, a GH11 polypeptide, a GH8 polypeptide, or a combination thereof.

13. The method of any one of claims 1 to 7, wherein the hydrolase comprises one or more cellulases.

14. The method of claim 13, wherein the one or more cellulases comprise one or more enzymes selected from the group consisting of endoglucanase (EG) and cellobiohydrolase (CBH).

15. The method of claim 14, wherein the one or more cellulases comprise one or more enzymes selected from the group consisting of endoglucanase, cellobiohydrolase I, cellobiohydrolase II, or a combination thereof.

16. The method of any one of claims 1 to 7, wherein the hydrolase further comprises an arabinofuranosidase.

17. The method of claim 16, wherein the arabinofuranosidase is selected from the group consisting of a GH43 polypeptide, a GH62 polypeptide, and a GH51 polypeptide.

18. The method of any one of claims 1 to 7, wherein the fiber washing procedure comprises using a fiber washing system comprising a space V / tank configured to provide a total residence time in the fiber washing system of at least 35 minutes and less than 48 hours.

19. The method of claim 18, wherein the incubation time in the space V / tank configured in the fiber washing system is at least 5 minutes and less than 48 hours.

20. The method of claim 19, wherein the incubation time is between 35 minutes and 24 hours.

21. The method of claim 19, wherein the incubation time is between 35 minutes and 12 hours.

22. The method of claim 19, wherein the incubation time is between 35 minutes and 6 hours.

23. The method of claim 19, wherein the incubation time is between 35 minutes and 5 hours.

24. The method of claim 19, wherein the incubation time is between 35 minutes and 4 hours.

25. The method of claim 19, wherein the incubation time is between 35 minutes and 3 hours.

26. The method of claim 19, wherein the incubation time is between 35 minutes and 2 hours.

27. The method of claim 19, wherein the incubation time is between 45 minutes and 48 hours.

28. The method of claim 19, wherein the incubation time is between 45 minutes and 24 hours.

29. The method of claim 19, wherein the incubation time is between 45 minutes and 12 hours.

30. The method of claim 19, wherein the incubation time is between 45 minutes and 6 hours.

31. The method of claim 19, wherein the incubation time is between 45 minutes and 5 hours.

32. The method of claim 19, wherein the incubation time is between 45 minutes and 4 hours.

33. The method of claim 19, wherein the incubation time is between 45 minutes and 3 hours.

34. The method of claim 19, wherein the incubation time is between 45 minutes and 2 hours.

35. The method of claim 18, wherein the incubation temperature is between 25°C and 95°C.

36. The method of claim 35, wherein the incubation temperature is between 25°C and 90°C.

37. The method of claim 35, wherein the incubation temperature is between 25°C and 85°C.

38. The method of claim 35, wherein the incubation temperature is between 25°C and 80°C.

39. The method of claim 35, wherein the incubation temperature is between 25°C and 75°C.

40. The method of claim 35, wherein the incubation temperature is between 25°C and 70°C.

41. The method of claim 35, wherein the incubation temperature is between 25°C and 65°C.

42. The method of claim 35, wherein the incubation temperature is between 25°C and 60°C.

43. The method of claim 35, wherein the incubation temperature is between 25°C and 55°C.

44. The method of claim 35, wherein the incubation temperature is between 25°C and 53°C.

45. The method of claim 35, wherein the incubation temperature is between 25°C and 52°C.

46. ​​The method of claim 35, wherein the incubation temperature is between 30°C and 90°C.

47. The method of claim 35, wherein the incubation temperature is between 30°C and 85°C.

48. The method of claim 35, wherein the incubation temperature is between 30°C and 80°C.

49. The method of claim 35, wherein the incubation temperature is between 30°C and 75°C.

50. The method of claim 35, wherein the incubation temperature is between 30°C and 70°C.

51. The method of claim 35, wherein the incubation temperature is between 30°C and 65°C.

52. The method of claim 35, wherein the incubation temperature is between 30°C and 60°C.

53. The method of claim 35, wherein the incubation temperature is between 30°C and 55°C.

54. The method of claim 35, wherein the incubation temperature is between 30°C and 53°C.

55. The method of claim 35, wherein the incubation temperature is between 30°C and 52°C.

56. The method of claim 35, wherein the incubation temperature is between 35°C and 90°C.

57. The method of claim 35, wherein the incubation temperature is between 35°C and 85°C.

58. The method of claim 35, wherein the incubation temperature is between 35°C and 80°C.

59. The method of claim 35, wherein the incubation temperature is between 35°C and 75°C.

60. The method of claim 35, wherein the incubation temperature is between 35°C and 70°C.

61. The method of claim 35, wherein the incubation temperature is between 35°C and 65°C.

62. The method of claim 35, wherein the incubation temperature is between 35°C and 60°C.

63. The method of claim 35, wherein the incubation temperature is between 35°C and 55°C.

64. The method of claim 35, wherein the incubation temperature is between 35°C and 53°C.

65. The method of claim 35, wherein the incubation temperature is between 35°C and 52°C.

66. The method of claim 35, wherein the incubation temperature is between 39°C and 90°C.

67. The method of claim 35, wherein the incubation temperature is between 39°C and 85°C.

68. The method of claim 35, wherein the incubation temperature is between 39°C and 80°C.

69. The method of claim 35, wherein the incubation temperature is between 39°C and 75°C.

70. The method of claim 35, wherein the incubation temperature is between 39°C and 70°C.

71. The method of claim 35, wherein the incubation temperature is between 39°C and 65°C.

72. The method of claim 35, wherein the incubation temperature is between 39°C and 60°C.

73. The method of claim 35, wherein the incubation temperature is between 39°C and 55°C.

74. The method of claim 35, wherein the incubation temperature is between 39°C and 53°C.

75. The method of claim 35, wherein the incubation temperature is between 39°C and 52°C.

76. The method of claim 35, wherein the incubation temperature is between 46°C and 52°C.

77. The method of any one of claims 1 to 7, wherein the level of SO2 added during fiber washing results in the same extraction yield of starch and gluten while reducing the contact time required between the hydrolytic enzyme and the milled corn kernel material as compared to a process wherein the SO2 level is less than 400 ppm.

78. The method of any one of claims 1 to 7, wherein the one or more hydrolases are expressed in an organism with a cellulase background.

79. The method of claim 78, wherein the organism is Trichoderma reesei.

80. The method of any one of claims 1 to 7, wherein the one or more hydrolases are purified.

81. The method of any one of claims 1 to 7, wherein the one or more hydrolases are in a liquid composition.

82. The method of any one of claims 1 to 7, wherein the one or more hydrolases are in a solid composition.

83. The method of any one of claims 1 to 7, wherein an effective amount of one or more hydrolytic enzymes mixed / contacted with one or more fractions of said milled corn kernel material is between 0.005 kg and 0.5 kg of enzyme protein per metric ton of corn kernels entering the wet milling process.

84. The process of any one of claims 1 to 7, wherein the source of SO2 is sodium metabisulfite (Na2S2O5) and / or added SO2 gas.

85. The method of any one of claims 1 to 7, wherein the xylanase is selected from the group consisting of: (a) a polypeptide having at least 85% sequence identity to the mature polypeptide of SEQ ID NO: 1; (b) a variant of the mature polypeptide of SEQ ID NO: 1, the variant comprising a substitution, deletion and / or insertion at one or more positions; and (c) A fragment of the polypeptide of (a) or (b) having xylanase activity.

86. The method of claim 85, wherein the sequence identity is at least 86%.

87. The method of claim 85, wherein the sequence identity is at least 87%.

88. The method of claim 85, wherein the sequence identity is at least 88%.

89. The method of claim 85, wherein the sequence identity is at least 89%.

90. The method of claim 85, wherein the sequence identity is at least 90%.

91. The method of claim 85, wherein the sequence identity is at least 91%.

92. The method of claim 85, wherein the sequence identity is at least 92%.

93. The method of claim 85, wherein the sequence identity is at least 93%.

94. The method of claim 85, wherein the sequence identity is at least 94%.

95. The method of claim 85, wherein the sequence identity is at least 95%.

96. The method of claim 85, wherein the sequence identity is at least 96%.

97. The method of claim 85, wherein the sequence identity is at least 97%.

98. The method of claim 85, wherein the sequence identity is at least 98%.

99. The method of claim 85, wherein the sequence identity is at least 99%.

100. The method of claim 85, wherein the sequence identity is 100%.

101. The method of claim 85, wherein the mature polypeptide is amino acids 1 to 551 of SEQ ID NO:

1.

102. The method of any one of claims 1 to 7, wherein the xylanase is selected from the group consisting of: (a) a polypeptide having at least 85% sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) a variant of the mature polypeptide of SEQ ID NO: 2, the variant comprising a substitution, deletion and / or insertion at one or more positions; and (c) A fragment of the polypeptide of (a) or (b) having xylanase activity.

103. The method of claim 102, wherein the sequence identity is at least 86%.

104. The method of claim 102, wherein the sequence identity is at least 87%.

105. The method of claim 102, wherein the sequence identity is at least 88%.

106. The method of claim 102, wherein the sequence identity is at least 89%.

107. The method of claim 102, wherein the sequence identity is at least 90%.

108. The method of claim 102, wherein the sequence identity is at least 91%.

109. The method of claim 102, wherein the sequence identity is at least 92%.

110. The method of claim 102, wherein the sequence identity is at least 93%.

111. The method of claim 102, wherein the sequence identity is at least 94%.

112. The method of claim 102, wherein the sequence identity is at least 95%.

113. The method of claim 102, wherein the sequence identity is at least 96%.

114. The method of claim 102, wherein the sequence identity is at least 97%.

115. The method of claim 102, wherein the sequence identity is at least 98%.

116. The method of claim 102, wherein the sequence identity is at least 99%.

117. The method of claim 102, wherein the sequence identity is 100%.

118. The method of claim 102, wherein the mature polypeptide is amino acids 21 to 405 of SEQ ID NO:

2.

119. The method of claim 16, wherein the arabinofuranosidase is selected from the group consisting of: (a) a polypeptide having at least 85% sequence identity to the mature polypeptide of SEQ ID NO: 3; (b) a variant of the mature polypeptide of SEQ ID NO: 3, the variant comprising a substitution, deletion and / or insertion at one or more positions; and (c) A fragment of the polypeptide of (a) or (b) having arabinofuranosidase activity.

120. The method of claim 119, wherein the sequence identity is at least 86%.

121. The method of claim 119, wherein the sequence identity is at least 87%.

122. The method of claim 119, wherein the sequence identity is at least 88%.

123. The method of claim 119, wherein the sequence identity is at least 89%.

124. The method of claim 119, wherein the sequence identity is at least 90%.

125. The method of claim 119, wherein the sequence identity is at least 91%.

126. The method of claim 119, wherein the sequence identity is at least 92%.

127. The method of claim 119, wherein the sequence identity is at least 93%.

128. The method of claim 119, wherein the sequence identity is at least 94%.

129. The method of claim 119, wherein the sequence identity is at least 95%.

130. The method of claim 119, wherein the sequence identity is at least 96%.

131. The method of claim 119, wherein the sequence identity is at least 97%.

132. The method of claim 119, wherein the sequence identity is at least 98%.

133. The method of claim 119, wherein the sequence identity is at least 99%.

134. The method of claim 119, wherein the sequence identity is 100%.

135. The method of claim 119, wherein the mature polypeptide is amino acids 17 to 325 of SEQ ID NO:

3.

136. The method of any one of claims 1 to 7, wherein the xylanase is selected from the group consisting of: (a) a polypeptide having at least 85% sequence identity to the mature polypeptide of SEQ ID NO: 4; (b) a variant of the mature polypeptide of SEQ ID NO: 4, the variant comprising a substitution, deletion and / or insertion at one or more positions; and (c) A fragment of the polypeptide of (a) or (b) having xylanase activity.

137. The method of claim 136, wherein the sequence identity is at least 86%.

138. The method of claim 136, wherein the sequence identity is at least 87%.

139. The method of claim 136, wherein the sequence identity is at least 88%.

140. The method of claim 136, wherein the sequence identity is at least 89%.

141. The method of claim 136, wherein the sequence identity is at least 90%.

142. The method of claim 136, wherein the sequence identity is at least 91%.

143. The method of claim 136, wherein the sequence identity is at least 92%.

144. The method of claim 136, wherein the sequence identity is at least 93%.

145. The method of claim 136, wherein the sequence identity is at least 94%.

146. The method of claim 136, wherein the sequence identity is at least 95%.

147. The method of claim 136, wherein the sequence identity is at least 96%.

148. The method of claim 136, wherein the sequence identity is at least 97%.

149. The method of claim 136, wherein the sequence identity is at least 98%.

150. The method of claim 136, wherein the sequence identity is at least 99%.

151. The method of claim 136, wherein the sequence identity is 100%.

152. The method of claim 136, wherein the mature polypeptide is amino acids 20 to 319 of SEQ ID NO:

4.

153. The method of any one of claims 1 to 7, wherein the cellulase is derived from a Trichoderma strain.

154. The method of claim 153, wherein the Trichoderma strain is Trichoderma reesei.

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