Method for improving oil yield from germ in wet milling process

Abstract

The invention provides a method for improving oil yield from germ in a wet milling process, the method comprising mixing a process stream comprising corn germ with an enzyme composition comprising an effective amount of one or more hydrolytic enzymes, wherein at least one of the hydrolytic enzymes is a xylanase polypeptide selected from the group consisting of: a GH5, a GH10, a GH30, a GH11 polypeptide.

Description

Technical Field

[0001] This invention relates to a method for improving the total oil yield from germ in a wet milling process. Background Technology

[0002] Conventional corn wet milling is a process designed for the recovery and purification of starch and several byproducts, including germ, gluten, starch, fiber, and oil.

[0003] Fiber is the lowest-value byproduct, so industry has devoted considerable effort to increasing 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 further processed into products such as dried starch, modified starch, dextrin, sweeteners, and alcohols for various commercial purposes. Gluten is commonly used in animal feed, such as corn gluten meal (approximately 60% protein) or corn gluten feed (approximately 20% protein).

[0004] The wet milling process can vary significantly depending on the specific milling equipment used, but it typically includes: grain cleaning, soaking, milling, germ separation, secondary milling, fiber separation, gluten separation, and starch separation. After cleaning the corn kernels, they are typically softened by soaking them in water or a diluted 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, undergoes fine milling and screening during fiber washing to separate the fiber from the starch and gluten, followed by gluten and starch separation, and starch purification can be achieved during washing / filtration.

[0005] The use of enzymes in several steps of the wet milling process has been recommended, such as the impregnation step. Commercial enzyme products have been shown. (Available from Novozymes A / S) The first step suitable for wet milling is the soaking step of immersing corn kernels in water.

[0006] Recently, "enzymatic milling" has been developed, a modified wet milling process that uses proteases to significantly reduce the total processing time during corn wet milling and eliminate the need for sulfur dioxide as a processing agent. Johnston et al., Cereal Chem, 81, pp. 626-632 (2004).

[0007] US 6,566,125 discloses a method for obtaining starch from corn, which involves soaking corn kernels in water to produce soaked corn kernels, milling the soaked corn kernels to produce milled corn slurry, and incubating the milled corn slurry with an enzyme (e.g., protease).

[0008] US 5,066,218 discloses a method for grinding grains (especially corn) that involves cleaning the grains, soaking them in water to soften them, and then grinding them with a cellulase.

[0009] WO 2002 / 000731 discloses a process for processing crop grains, which includes soaking the grains in water for 1-12 hours, wet milling the soaked grains, and treating the grains with one or more enzymes (including acidic proteases).

[0010] WO 2002 / 000911 discloses a process for separating starch gluten, which includes subjecting milled starch to acidic proteases.

[0011] WO 2002 / 002644 discloses a process for washing starch slurry to obtain starch gluten separation step of a self-grinding process, the process comprising washing the starch slurry with an aqueous solution containing an effective amount of acidic protease.

[0012] WO 2014 / 082566 and WO 2014 / 082564 disclose cellulose decomposition compositions for use in wet milling.

[0013] Methods for increasing oil extraction from starch-containing materials have been previously disclosed, for example, in WO 92 / 20777, which discloses the addition of acid-stabilized fungal proteases during the process of starch fermentation into ethanol.

[0014] Although the effects of using enzymes in wet milling of corn have been studied in the art, there is still a need for improved enzyme technologies that can reduce energy expenditure and costs associated with wet milling of corn and provide increased oil yield, during the soaking / soaking of corn kernels, during the milling of corn kernels, and in the separation of starch and glutenin. Attached Figure Description

[0015] Figure 1 Schematic diagram of enzyme treatment, component analysis, and component quality determination of germ. Summary of the Invention

[0016] This invention relates to a method for improving the oil yield from germ in a wet milling process, the method comprising mixing a process stream containing corn germ with an enzyme composition containing an effective amount of one or more hydrolytic enzymes, wherein at least one of the hydrolytic enzymes is a xylanase polypeptide selected from the group consisting of GH5, GH10, GH30, and GH11 polypeptides.

[0017] Definition of enzyme:

[0018] Arabinofuranosylase / peptide with arabinofuranosylase activity: The term "arabinofuranosylase" refers to α-L-arabinofuranoside arabinofuranohydrolase (EC3.2.1.55), which catalyzes the hydrolysis of terminal non-reducing α-L-arabinofuranoside residues in α-L-arabinoside. This enzyme acts on α-L-arabinofuranoside, α-L-arabinanan containing (1,3)- and / or (1,2)- and / or (1,5)- bonds, arabinosylxylan, and arabinogalactan. α-L-arabinofuranosylase is also known as arabinosylase, α-arabinosylase, α-L-arabinosylase, α-arabinofuranosylase, polysaccharide α-L-arabinofuranosylase, α-L-arabinofuranoside hydrolase, L-arabinosylase, or α-L-arabinananase. Medium-viscosity wheat arabinosyl xylan (Megazyme International Ireland, Ltd., Bray, Co., Wicklow, Ireland) can be prepared at a total volume of 200 μl per ml of 100 mM sodium acetate (pH 5) and incubated at 40°C for 30 minutes, followed by... The activity of arabinofuranase was determined by HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA) through analysis of arabinose. Arabinofuranases can be found in families such as GH43, GH62, and GH51.

[0019] β-glucanase / peptide with β-glucanase activity: The term “β-glucanase” covers peptides having β-1,6-glucanase activity and / or exo- and / or endo-β-1,3-glucanase activity. As used herein, “peptide having β-1,6-glucanase activity and / or exo- and / or endo-β-1,3-glucanase activity” means that the peptide exhibits at least one of these activities, but may also have any combination of these activities, including all of them. The term “exo- and / or endo-β-1,3-glucanase” covers peptides having both exo- and / or endo-β-1,3-glucanase activity, exo- and / or endo-β-1,3-glucanase activity, and peptides having a mixture of β-1,3(4) and / or β-1,4(3)-glucanase activity. Preferably, the polypeptide having β-1,6-glucanase activity and / or exonuclease and / or endo-β-1,3-glucanase activity is selected from members of the following glycoside hydrolase families: GH30 (e.g., GH30_3), GH5 (e.g., GH5_15), GH16, GH55 (e.g., GH55_3), GH64, and GH131 (e.g., GH131A and GH131B).

[0020] On one hand, "β-glucanase" refers to a polypeptide possessing β-1,6-glucanase activity, specifically a 6-β-D-glucanase (EC 3.2.1.75) that catalyzes the random hydrolysis of the (1→6)-bond in (1→6)-β-D-glucan. Besides acting on 1,6-oligo-β-D-glucosides, members of this enzyme family also act on lutein and lithochrin. These β-glucanases include members of the GH30_3, GH5_15, and GH131A and GH131B families. For the purposes of this invention, β-1,6-glucanase activity was determined according to the procedure described in the examples. On one hand, the β-glucanase polypeptide possesses at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the β-1,6-glucanase activity of the mature polypeptide of SEQ ID NO:7. β-glucosidase / polypeptide with β-glucosidase activity: The term "β-glucosidase" refers to beta-D-glucoside glucohydrolase (EC3.2.1.21), which catalyzes the hydrolysis of terminal non-reducing β-D-glucose residues, releasing β-D-glucose. β-glucosidase activity can be determined using p-nitrophenyl-β-D-glucopyranoside as a substrate according to the procedure described by Venturi et al., 2002, J. Basic Microbiol. [Journal of Basic Microbiology] 42:55-66. One unit of β-glucosidase is defined as the concentration of 0.01% glucosidase at 25°C and pH 4.8. 1.0 μmol of p-nitrophenol anion was generated per minute from 1 mM p-nitrophenyl-β-D-glucopyranoside as a substrate in 50 mM sodium citrate.

[0021] β-xylosidases / peptides with β-xylosidase activity: The term "β-xylosidase" refers to β-D-xyloside xylohydrolase (EC 3.2.1.37), which catalyzes the exolytic hydrolysis of short β(1→4)-xylooligosaccharides to remove consecutive D-xylose residues from the non-reducing end. It can be used in formulations containing 0.01%... β-xylosidase activity was determined using 1 mM p-nitrophenyl-β-D-xyloside as a substrate in 100 mM sodium citrate at pH 5 and 40 °C. One unit of β-xylosidase was defined as the activity of β-xylosidase at 40 °C and pH 5 in a solution containing 0.01% sodium citrate. 1.0 μmol of p-15-nitrophenol anion is generated per minute from 1 mM p-nitrophenyl-β-D-xyloside in 20 mM sodium citrate.

[0022] Cellobiose hydrolases / peptides with cellobiose hydrolytic activity: The term "cellobiose hydrolase" refers to 1,4-β-D-glucan-cellobiose hydrolases (EC3.2.1.91 and EC3.2.1.176) that catalyze the hydrolysis of 1,4-β-D15-glycosidic bonds in cellulose, cellooligosaccharides, or any polymer containing β-1,4-linked glucose, releasing cellobiose from the reducing end (cellobiose hydrolase I) or non-reducing end (cellobiose hydrolase II) of the chain (Teeri, 1997, Trends in Biotechnology 15:160-167; Teeri et al., 1998, Biochem. Soc. Trans. 26:173-178). Cellobiose hydrolase activity can be determined according to the procedures described below: Lever et al., 1972, Anal. Biochem. [Analytical Biochemistry] 47:273-279; van Tilbeurgh et al., 1982, FEBS Letters [European Federation of Biochemical Societies Letters] 149:152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters [European Federation of Biochemical Societies Letters] 187:283-288; and Tomme et al., 1988, Eur. J. Biochem. [European Journal of Biochemistry] 170:575-581.

[0023] Cellulases or cellulase / peptides with cellulase activity or cellulolytic activity: The term “cellulase” or “cellulase” means one or more (e.g., several) enzymes that hydrolyze cellulose materials, including any material containing cellulose (such as fiber). Cellulases include one or more endoglucanases (EC 3.2.1.4), one or more cellobiases (EC 3.2.1.91 and EC 3.2.1.150), one or more β-glucosidases (EC 3.2.1.21), or combinations thereof. Two basic methods for measuring cellulase activity include: (1) measuring total cellulase activity, and (2) measuring individual cellulase activity (endoglucanase, cellobiase, and β-glucosidase), as described in Zhang et al., 2006, Biotechnology Advances, 24:452-481. Insoluble substrates, including Whatman No. 1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, and pretreated lignocellulose, can be used to measure total cellulase activity. The most common assay for total cellulase activity is the filter paper assay using Whatman No. 1 filter paper as the substrate. This assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Pure Appl. Chem. 59:257-68).

[0024] Cellulase activity can be determined by measuring the increase in sugar production / release during the hydrolysis of cellulose material by one or more cellulases under the following conditions: 1-50 mg cellulase protein / g of cellulose in pretreated corn stalks (PCS) (or other pretreated cellulose material), at a suitable temperature (e.g., 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 (e.g., 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 with control hydrolysis without added cellulase protein. Typical conditions are: 1 ml reaction, washed or unwashed PCS, 5% insoluble solids (dry weight), 50 mM sodium acetate 5 (pH 5), 1 mM MnSO4, 50°C, 55°C, or 60°C, 72 hours, via Sugar analysis was performed using HPX-87H column chromatography (Broker Laboratories, Hercules, California, USA).

[0025] Endoglucanase: The term "endoglucanase" refers to endo-1,4-(1,3;1,4)-β-D-glucan-4-glucan hydrolase (EC 3.2.1.4), which catalyzes the endo-hydrolysis of β-1,4-β-D-glycosidic bonds in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichen polysaccharides, mixed β-1,3-glucans such as cereal β-D-glucan or xyloglucan, and other plant materials containing cellulose components. Endoglucanase activity can be determined by measuring the decrease in substrate viscosity or the increase in reducing ends as determined by reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24:452-481). For the purposes of this invention, endoglucanase activity was determined using carboxymethyl cellulose (CMC) as a substrate at pH 5 and 40°C, following the procedure described in Ghose, 1987, Pure and Appl. Chem. 59:257-268.

[0026] Family 61 Glycoside Hydrolases: The terms "family 61 glycoside hydrolases," "family GH61," or "GH61" refer to polypeptides belonging to the glycoside hydrolases 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. Enzymes in this family were initially classified as glycoside hydrolases based on measurements of very weak endo-1,4-β-D-glucanase activity in one family member. These enzymes have irregular structures and modes of action, and they cannot be considered true glycosidases. However, they are retained in the CAZy classification based on their ability to enhance the breakdown of lignocellulose when used in combination with cellulases or mixtures of cellulases. The GH61 polypeptide was recently classified as a soluble polysaccharide monooxygenase (Quinlan et al., 2011, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 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 referred to as "auxiliary activity 9" or "AA9" polypeptide.

[0027] Hydrolytic enzymes or hydrolases with hydrolytic activity: "Hydrolytic enzyme" refers to any catalytic protein that uses water to break down substrates. Hydrolytic enzymes include cellulase (EC 3.2.1.4), xylanase (EC 3.2.1.8), arabinofuranylase (EC 3.2.1.55 (non-reducing terminal α-L-arabinofuranylase); EC 3.2.1.185 (non-reducing terminal β-L-arabinofuranylase), cellobiase I (EC 3.2.1.150), cellobiase II (EC 3.2.1.91), cellobiase (EC 3.2.1.176), β-glucosidase (EC 3.2.1.21), and β-xylosidase (EC 3.2.1.37).

[0028] Xylanases / peptides with xylanase activity: The term "xylanase" refers to 1,4-β-D-xylan-xylohydrolase (EC 3.2.1.8), which catalyzes the endo-hydrolysis of the 1,4-β-D-xylosidic bonds in xylan. Xylanase activity can be maintained at 0.01% at 37°C. X-100 and 200 mM sodium phosphate (pH 6) were determined using 0.2% AZCL-arabinosylxylan as a substrate. One unit of xylanase activity was defined as the production of 1.0 μmol azurine per minute from 0.2% AZCL-arabinosylxylan as a substrate at 37 °C and pH 6 in 200 mM sodium phosphate (pH 6). Xylanases can be found in, for example, the GH5, GH30, GH10, and GH11 families.

[0029] GH5 polypeptide: refers to a polypeptide with enzymatic activity, which is classified as a member of the glycoside hydrolase family 5 in the carbohydrate active enzyme (CAZyme) database. http: / / www.cazy.org / ).

[0030] GH30 polypeptide: refers to a polypeptide with enzymatic activity, which is classified as a member of the glycoside hydrolase family 30 in the carbohydrate active enzyme (CAZyme) database (http: / / www.cazy.org / ).

[0031] GH10 polypeptide: refers to a polypeptide with enzymatic activity. http: / / www.cazy.org / It is classified as a member of the glycoside hydrolase family 10 in the database of available carbohydrate-active enzymes (CAZyme). (Lombard, V.; Golaconda Ramulu, H.; Drula, E.; Coutinho, PM; Henrissat, B. (November 21, 2013). “The carbohydrate-active enzymes database (CAZy) in 2013”. Nucleic Acids Research. 42(D1):D490-D495; Cantalel 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 AcidsRes. 37(database issue):D233-8.)

[0032] GH11 polypeptide refers to a polypeptide with enzymatic activity, which is classified as a member of the glycoside hydrolase family 11 in the carbohydrate active enzyme (CAZyme) database.

[0033] GH62 polypeptide: refers to a polypeptide with enzymatic activity, which is classified as a member of the glycoside hydrolase family 62 in the carbohydrate active enzyme (CAZyme) database.

[0034] GH43 polypeptide: refers to a polypeptide with enzymatic activity, which is classified as a member of the glycoside hydrolase family 62 in the carbohydrate active enzyme (CAZyme) database.

[0035] Other definitions

[0036] Corn kernels: Various types of corn kernels are known, including, for example, dent corn, flint corn, hulled corn, striped corn, sweet corn, waxy corn, etc.

[0037] Some corn kernels have an outer covering called the pericarp, which protects the germ inside the kernel. It is waterproof and resistant to 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 attachment point between the kernel and the cob.

[0038] Corn kernel material: preferably a substance comprising fiber, gluten, and starch, preferably achieved by steaming and milling the crop kernels and separating the substance comprising fiber, gluten, and starch from the germ. As the corn kernel material moves through a fiber wash, it is separated into several fractions, including a first fraction (s) and a second fraction (f). Therefore, "fractions of corn kernel material" and "one or more fractions of corn kernel material" refer to these first fractions (s) and second fractions (f).

[0039] Dehydration: Dehydration refers to any process that removes excess water from corn fibers.

[0040] The germ: The germ is the only surviving part of a 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, approximately 25% of the germ is corn oil. The endosperm, covered or surrounded by the germ, contains approximately 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 loosely packed.

[0041] Gluten: Gluten is a protein composed of two smaller proteins—glutenin and gliadin. In this article, "gluten" refers to the majority of proteins found in corn kernels. The main products of wet-milled gluten from corn are corn gluten meal (approximately 60% protein) and corn gluten feed (approximately 20% protein).

[0042] Grinding: The term "grinding" refers to breaking down corn kernels into smaller components.

[0043] Insoluble matter: In this context, “insoluble matter” and “insoluble solids” are used interchangeably; it is defined as material that can pass through a 75 μm sieve and is insoluble in water.

[0044] Grinding equipment: "Grinding equipment" refers to all equipment used on a pulverizer. The wet grinding process will vary depending on the available grinding equipment. Examples of grinding equipment can be impregnation tanks, evaporators, screw presses, rotary dryers, dewatering screens, centrifuges, hydrocyclones, etc. The size and number of each grinding unit / grinding line can vary on different pulverizers, which will affect the grinding process. For example, the number of fiber washing screen units can vary, and the size of the centrifuge can also vary.

[0045] Screening: The term "screening" refers to the process of separating corn kernel material into primary fractions (s) and secondary fractions (f), and moving these fractions from one screen unit to another. A screen unit can be, for example, a pressure-feed screen / feed pressure screen, where material is fed through a nozzle or a rotating screen, where the material is forced through the screen by gravity. Examples of such screens include DSM screens and ICM screens.

[0046] The non-selection period is a non-separation period provided for incubating maize kernel material or its fractions with enzymes.

[0047] Sequence identity: The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0048] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48:443-453) is used to determine the degree of sequence identity between two amino acid sequences. This algorithm is implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Trends in Genetics] 16:276-277) (preferably version 3.0.0 or later). Version 6.1.0 is used.

[0049] The optional parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix. Nieder's output (obtained using the non-simplification option) marked "longest identity" is used as the identity percentage and calculated as follows: (identical residues x 100) / (alignment length - total number of vacancy in alignment).

[0050] Starch: The term "starch" refers to a complex polysaccharide produced by plants.

[0051] Composed of glucose units in the form of storage granules, widely found in plant tissues, and composed of amylose and amylopectin, and represented as (C6H). 10 O5)n (where n is any number) any material.

[0052] Impregnation or soaking: The term "impregnation" refers to soaking crop grains in water and optionally SO2. Detailed Implementation

[0053] One object of the present invention is to provide a method for improving the oil yield from corn germ in a wet milling process.

[0054] Wet milling process:

[0055] The corn kernels are wet-milled to open them up and separate them into their four main components: starch, germ, fiber, and gluten.

[0056] The wet milling process can vary significantly between milling operations; however, conventional wet milling typically includes the following steps:

[0057] 1. Impregnation

[0058] 2. Grinding

[0059] 3. Separate into streams containing the following:

[0060] i) germ; ii) fiber; iii) starch and gluten

[0061] 4. Wash the fibers, press and dry.

[0062] 5. Starch / gluten separation, and

[0063] 6. Wash the starch.

[0064] Soaking, grinding and separating the germ

[0065] Corn kernels are softened by soaking them in water at a temperature of about 50°C, for example, between about 45°C and 60°C, for about 30 minutes to about 48 hours, preferably 30 minutes to about 15 hours, for example, about 1 hour to about 6 hours. During soaking, the kernels absorb water, increasing their moisture content from 15% to 45%, and more than doubling in size. Optionally, 0.1% sulfur dioxide (SO2) and / or NaHSO3 are added to the water to prevent bacterial overgrowth in the warm environment. As the corn swells and softens, the mild acidity of the soaking water begins to loosen the gluten bonds within the corn and release starch. After soaking, the corn kernels crack open to release the germ. The germ contains corn oil. The germ is essentially separated from the heavier-density mixture of starch, gluten, and fiber by “floating” the germ segments, which contain no other substances, under closely controlled conditions. This method is used to eliminate any adverse effects of trace amounts of corn oil in subsequent processing steps.

[0066] Wash fibers, press and dry

[0067] To achieve maximum starch and gluten recovery while keeping any fiber in the final product to an absolute minimum, free starch and gluten must be washed out of the fiber during processing. Free starch and gluten are separated from the fiber during screening (washing) and collected as milled starch. The remaining fiber is then pressurized to reduce its viscosity.

[0068] Separate starch glutenin

[0069] The starch-gluten suspension from the fiber washing step (called milled starch) is separated into starch and gluten. Gluten has a lower density than starch. Gluten is easily separated by passing the milled starch through a centrifuge.

[0070] Washing starch

[0071] The starch slurry from the starch separation step contains some insoluble protein and a lot of soluble matter. This must be removed before top-quality starch (high-purity starch) can be produced. In a hydrocyclone, the starch, with only 1% or 2% protein remaining, is diluted, washed 8 to 14 times, re-diluted, and washed again to remove the last traces of protein and produce high-quality starch, typically with a purity greater than 99.5%.

[0072] Products of wet milling: Wet milling can be used to produce (but is not limited to) corn extract, corn gluten feed, germ, corn oil, corn gluten meal, corn starch, modified corn starch, syrups (such as corn syrup), and corn ethanol.

[0073] In a conventional wet milling process for corn, germ separation (separation of germ from starch / gluten and fiber) and germ drying are performed after milling. Oil can then be extracted from the dried germ.

[0074] The inventors have observed that contacting / incubating germ with one or more xylanases increases the yield of oil extracted from germ.

[0075] Therefore, in a first aspect, the present invention relates to a method for improving the oil yield from germ in a wet milling process, the method comprising mixing a process stream containing germ with an enzyme composition containing an effective amount of one or more hydrolytic enzymes, wherein at least one of the hydrolytic enzymes is a xylanase polypeptide selected from the group consisting of: GH5, GH10, GH30, GH11 polypeptides.

[0076] In one embodiment, the method according to the invention releases more oil from the germ compared to a process without enzyme treatment.

[0077] More specifically, the present invention relates to a wet milling method comprising the following steps:

[0078] a) Soak corn kernels in water to produce soaked kernels;

[0079] b) Grind these soaked seeds to release the germ from the seeds;

[0080] c) Separating the germ from these soaked and milled grains; and

[0081] d) subject the germ to enzyme treatment with at least one xylanase polypeptide selected from the group consisting of: GH5, GH10, GH30, GH11 xylanase.

[0082] In one embodiment, the embryo is

[0083] During or after step b) according to the invention, preferably during step c) or after these germs have been separated, they are mixed with one or more xylanases.

[0084] In one particular embodiment, the germ is mixed with one or more xylanases after step c).

[0085] Sufficient time should be allowed for enzyme treatment and an effective amount should be used. A technician will be able to determine this based on the wet milling conditions and the specific enzyme being applied. In one embodiment, the germ is allowed to react with the one or more xylanases for at least 45 minutes, for example, at least 1 hour, such as at least 2 hours, such as at least 2.5 hours, such as at least 3 hours. Preferably, the xylanase polypeptide is present in an amount preferably 0.0005 to 1.5 mg enzyme protein / g DS seed, preferably 0.001 to 1 mg enzyme protein / g DS seed, preferably 0.002 to 0.5 mg enzyme protein / g DS seed, preferably 0.003 to 0.4 mg enzyme protein / g DS seed.

[0086] In one embodiment, these hydrolases comprise at least one xylanase and at least one arabinofuranase. The arabinofuranase is GH62 or GH43 arabinofuranase, preferably GH62 arabinofuranase.

[0087] In one embodiment, these hydrolases comprise at least one β-glucanase. In one embodiment, the β-glucanase is GH5 β-glucanase, particularly GH5_15 β-glucanase.

[0088] In another embodiment, the xylanase is at least one GH5 xylanase, particularly GH5_21 xylanase.

[0089] In another embodiment, the xylanase is GH10 xylanase.

[0090] In another embodiment, the xylanase is GH11 xylanase.

[0091] In another embodiment, the xylanase is GH30 xylanase, particularly GH30_8 xylanase.

[0092] These hydrolases may further include cellulases. For example, xylanases and / or arabinofuranosaccharides may be expressed in Trichoderma host organisms, particularly Trichoderma reesei host organisms, and host-produced cellulases may be included in the enzyme composition.

[0093] Regardless of the type of filamentous fungal organism that can be used as a source of cellulase, preferably, the cellulase is selected from at least one or more endoglucanases and one or more cellobiases. In particular, these cellulases may be selected from endoglucanase (EG), cellobiase I (CBH I), cellobiase II (CBH II), GH61, β-glucosidase, or combinations thereof.

[0094] In one embodiment, the cellulase may contain at least CBH I, CBH II, and EG I.

[0095] In one specific embodiment, the xylanase is selected from the group consisting of:

[0096] (a) A polypeptide having at least 75%, at least 80%, 85%, for example 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 with the mature polypeptide of SEQ ID NO:1;

[0097] (b) A variant of the mature polypeptide of SEQ ID NO:1, comprising substitution, deletion, and / or insertion at one or more sites; and

[0098] (c)(a) or (b) fragments of the polypeptide with xylanase activity;

[0099] Preferably, the xylanases of a), b) and c) have at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% xylanase activity of the mature polypeptide of SEQ ID NO:1.

[0100] In one embodiment, the mature polypeptide is amino acids 21 to 405 of SEQ ID NO:1.

[0101] In another specific embodiment, the arabinofuranase is selected from the group consisting of:

[0102] (a) A polypeptide having at least 75%, at least 80%, 85%, for example 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 with the mature polypeptide of SEQ ID NO:2;

[0103] (b) A variant of the mature polypeptide of SEQ ID NO:2, comprising substitution, deletion, and / or insertion at one or more sites; and

[0104] (c)(a) or (b) fragments of the polypeptide having arabinofuranyl glycosidase activity;

[0105] Preferably, the arabinofuranases of a), b) and c) have at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the mature polypeptide of SEQ ID NO:2.

[0106] In one embodiment, the mature polypeptide is amino acids 17 to 325 of SEQ ID NO:2.

[0107] In another specific embodiment, the xylanase is selected from the group consisting of:

[0108] (a) A polypeptide having at least 75%, at least 80%, 85%, for example 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 with the mature polypeptide of SEQ ID NO:4;

[0109] (b) A variant of the mature polypeptide of SEQ ID NO:4, comprising substitution, deletion, and / or insertion at one or more sites; and

[0110] (c)(a) or (b) fragments of the polypeptide with xylanase activity;

[0111] Preferably, the xylanases of a), b) and c) have at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% xylanase activity of the mature polypeptide of SEQ ID NO:4.

[0112] In one embodiment, the mature polypeptide is amino acids 1 to 551 of SEQ ID NO:4.

[0113] In another specific embodiment, the xylanase is selected from the group consisting of:

[0114] (a) A polypeptide having at least 75%, at least 80%, 85%, for example 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 with the mature polypeptide of SEQ ID NO:5;

[0115] (b) A variant of the mature polypeptide of SEQ ID NO:5, comprising substitution, deletion, and / or insertion at one or more sites; and

[0116] (c)(a) or (b) fragments of the polypeptide with xylanase activity;

[0117] Preferably, the xylanases of a), b) and c) have at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% xylanase activity of the mature polypeptide of SEQ ID NO:5.

[0118] In one embodiment, the mature polypeptide is amino acids 28 to 417 of SEQ ID NO:5.

[0119] In another specific embodiment, the xylanase is selected from the group consisting of:

[0120] (a) A polypeptide having at least 75%, at least 80%, 85%, for example 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 with the mature polypeptide of SEQ ID NO:6;

[0121] (b) A variant of the mature polypeptide of SEQ ID NO:6, comprising substitution, deletion, and / or insertion at one or more sites; and

[0122] (c)(a) or (b) fragments of the polypeptide with xylanase activity;

[0123] Preferably, the xylanases of a), b) and c) have at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% xylanase activity of the mature polypeptide of SEQ ID NO:6.

[0124] In one embodiment, the mature polypeptide is amino acids 30 to 212 of SEQ ID NO:6.

[0125] In another specific embodiment, the β-glucanase is selected from the group consisting of:

[0126] (a) A polypeptide having at least 75%, at least 80%, 85%, for example 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 with the mature polypeptide of SEQ ID NO:7;

[0127] (b) A variant of the mature polypeptide of SEQ ID NO:7, comprising substitution, deletion, and / or insertion at one or more sites; and

[0128] (c)(a) or (b) fragments of the polypeptide with β-glucanase activity;

[0129] Preferably, the β-glucanases of a), b) and c) have at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% β-glucanase activity of the mature polypeptide of SEQ ID NO:7.

[0130] In one embodiment, the mature polypeptide is amino acids 17 to 408 of SEQ ID NO:7.

[0131] In one particular embodiment of the invention, the germ is dehydrated / dried before being contacted with the enzyme.

[0132] Another embodiment of the claimed method includes the step of extracting oil from the germ.

[0133] The invention is further disclosed in the following numbered paragraphs.

[0134] Paragraph 1. A method for improving the oil yield from germ in a wet milling process, the method comprising mixing a process stream containing corn germ with an enzyme composition containing an effective amount of one or more hydrolytic enzymes, wherein at least one of the hydrolytic enzymes is at least one xylanase polypeptide selected from the group consisting of: GH5, GH10, GH30, GH11 polypeptides.

[0135] Paragraph 2. The method as described in paragraph 1, wherein the amount of oil released from the germ is increased compared to the method without the addition of xylanase.

[0136] Paragraph 3. The method as described in any of the preceding paragraphs, comprising the following steps:

[0137] a) Soak corn kernels in water to produce soaked kernels;

[0138] b) Grind these soaked seeds to release the germ from the seeds;

[0139] c) Separating the germ from these soaked and milled grains; and

[0140] The germ is subjected to enzyme treatment with at least one xylanase polypeptide selected from the group consisting of: GH5, GH10, GH30, GH11 xylanase.

[0141] Paragraph 4. The method as described in any of the preceding paragraphs, wherein the germ is mixed with the one or more xylanases during or after step b) as described in paragraph 3, preferably during step c) or after the germ has been separated.

[0142] Paragraph 5. The method as described in any of the preceding paragraphs, wherein the germ is mixed with the one or more xylanases after step c) as described in paragraph 3.

[0143] Paragraph 6. The method as described in any of the preceding paragraphs, wherein the germ is allowed to react with the one or more xylanases for at least 45 minutes, at least 1 hour, such as at least 2 hours, such as at least 2.5 hours, such as at least 3 hours.

[0144] Paragraph 7. The method as described in any of the preceding paragraphs, wherein the hydrolytic enzyme is present in an amount preferably 0.0005 to 1.5 mg enzyme protein / g DS grain, preferably 0.001 to 1 mg enzyme protein / g DS grain, preferably 0.002 to 0.5 mg enzyme protein / g DS grain, preferably 0.003 to 0.4 mg enzyme protein / g DS grain.

[0145] Paragraph 8. The method as described in any of the preceding paragraphs, wherein the hydrolytic enzymes comprise at least one xylanase and at least one arabinofuranase.

[0146] Paragraph 9. The method as described in any of the preceding paragraphs, wherein the hydrolytic enzymes comprise at least one β-glucanase.

[0147] Paragraph 10. The method as described in any of the preceding paragraphs, wherein these hydrolytic enzymes further comprise cellulase.

[0148] Paragraph 11. The method as described in paragraph 9, wherein the cellulases are selected from endoglucanase, cellobiase I, cellobiase II, GH61, β-glucosidase, or combinations thereof.

[0149] Paragraph 12. The method as described in paragraph 11, wherein these cellulases comprise at least an endoglucanase and a cellobiase.

[0150] Paragraph 13. The method as described in paragraph 12, wherein these cellulases comprise at least cellobiase I, cellobiase II, and endoglucanase I.

[0151] Paragraph 14. As described in paragraphs 9 to 13, wherein these cellulases are derived from the Trichoderma genus, particularly Trichoderma reesei.

[0152] Paragraph 15. The method as described in any of the preceding paragraphs, wherein the xylanase is selected from the glycosyl hydrolase family GH5, GH30, GH10, GH11.

[0153] Paragraph 16. The method described in paragraph 15, wherein the xylanase is GH10 xylanase.

[0154] Paragraph 17. The method as described in paragraph 15, wherein the xylanase is GH5 xylanase, particularly GH5_21 xylanase.

[0155] Paragraph 18. The method described in paragraph 15, wherein the xylanase is GH11 xylanase.

[0156] Paragraph 19. The method as described in paragraph 15, wherein the xylanase is GH30 xylanase, particularly GH30_8 xylanase.

[0157] Paragraph 20. The method as described in paragraphs 8 through 19, wherein the arabinofuranase is GH62 or GH43 arabinofuranase.

[0158] Paragraph 21. The method as described in any one of paragraphs 9 to 20, wherein the β-glucanase is GH5β-glucanase, particularly GH5_15β-glucanase.

[0159] Paragraph 22. The method as described in any of the preceding paragraphs, wherein the xylanase is selected from the group consisting of:

[0160] (a) A polypeptide having at least 75%, 80%, 85%, for example 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 with the mature polypeptide of SEQ ID NO:1;

[0161] (b) A variant of the mature polypeptide of SEQ ID NO:1, comprising substitution, deletion, and / or insertion at one or more sites; and

[0162] (c)(a) or (b) fragments of the polypeptide with xylanase activity;

[0163] Preferably, the xylanases of a), b) and c) have at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% xylanase activity of the mature polypeptide of SEQ ID NO:1.

[0164] Paragraph 23. The method as described in paragraph 22, wherein the mature polypeptide is amino acids 21 to 405 of SEQ ID NO:1.

[0165] Paragraph 24. The method as described in any one of paragraphs 8 to 23, wherein the arabinofuranase is selected from the group consisting of:

[0166] (a) A polypeptide having at least 75%, at least 80%, 85%, for example 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 with the mature polypeptide of SEQ ID NO:2;

[0167] (b) A variant of the mature polypeptide of SEQ ID NO:2, comprising substitution, deletion, and / or insertion at one or more sites; and

[0168] (c)(a) or (b) fragments of the polypeptide having arabinofuranyl glycosidase activity;

[0169] Preferably, the arabinofuranases of a), b) and c) have at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the mature polypeptide of SEQ ID NO:2.

[0170] Paragraph 25. The method as described in paragraph 24, wherein the mature polypeptide is amino acids 17 to 325 of SEQ ID NO:2.

[0171] Paragraph 26. The method as described in any of the preceding paragraphs, wherein the xylanase is selected from the group consisting of:

[0172] (a) A polypeptide having at least 75%, at least 80%, 85%, for example 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 with the mature polypeptide of SEQ ID NO:4;

[0173] (b) A variant of the mature polypeptide of SEQ ID NO:4, comprising substitution, deletion, and / or insertion at one or more sites; and

[0174] (c)(a) or (b) fragments of the polypeptide with xylanase activity;

[0175] Preferably, the xylanases of a), b) and c) have at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% xylanase activity of the mature polypeptide of SEQ ID NO:4.

[0176] Paragraph 27. The method as described in paragraph 26, wherein the mature polypeptide is amino acid 1 to 551 of SEQ ID NO:4.

[0177] Paragraph 28. The method as described in any of the preceding paragraphs, wherein the xylanase is selected from the group consisting of:

[0178] (a) A polypeptide having at least 75%, at least 80%, 85%, for example 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 with the mature polypeptide of SEQ ID NO:5;

[0179] (b) A variant of the mature polypeptide of SEQ ID NO:5, comprising substitution, deletion, and / or insertion at one or more sites; and

[0180] (c)(a) or (b) fragments of the polypeptide with xylanase activity;

[0181] Preferably, the xylanases of a), b) and c) have at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% xylanase activity of the mature polypeptide of SEQ ID NO:5.

[0182] Paragraph 29. The method as described in paragraph 28, wherein the mature polypeptide is amino acids 28 to 417 of SEQ ID NO:5.

[0183] Paragraph 30. The method as described in any of the preceding paragraphs, wherein the xylanase is selected from the group consisting of:

[0184] (a) A polypeptide having at least 75%, at least 80%, 85%, for example 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 with the mature polypeptide of SEQ ID NO:6;

[0185] (b) A variant of the mature polypeptide of SEQ ID NO:6, comprising substitution, deletion, and / or insertion at one or more sites; and

[0186] (c)(a) or (b) fragments of the polypeptide with xylanase activity;

[0187] Preferably, the xylanases of a), b) and c) have at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% xylanase activity of the mature polypeptide of SEQ ID NO:6.

[0188] Paragraph 31. The method as described in paragraph 30, wherein the mature polypeptide is amino acids 30 to 212 of SEQ ID NO:6.

[0189] Paragraph 32. The method as described in any one of paragraphs 9 to 31, wherein the β-glucanase is selected from the group consisting of:

[0190] (a) A polypeptide having at least 75%, at least 80%, 85%, for example 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 with the mature polypeptide of SEQ ID NO:7;

[0191] (b) A variant of the mature polypeptide of SEQ ID NO:7, comprising substitution, deletion, and / or insertion at one or more sites; and

[0192] (c)(a) or (b) fragments of the polypeptide with β-glucanase activity;

[0193] Preferably, the β-glucanases of a), b) and c) have at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% β-glucanase activity of the mature polypeptide of SEQ ID NO:7.

[0194] Paragraph 33. The method as described in paragraph 32, wherein the mature polypeptide is amino acids 17 to 408 of SEQ ID NO:7.

[0195] Paragraph 34. The method as described in any of the preceding paragraphs, wherein the germ is dehydrated / dried.

[0196] Paragraph 35. The method as described in any of the preceding paragraphs, further comprising the step of extracting oil from the germ.

[0197] The invention is further illustrated in the following examples.

[0198] Example

[0199] The enzymes used in the example

[0200] GH10 xylanase from *Talaromyces leycettanus*. Disclosed herein as SEQ ID NO:1 (mature polypeptide amino acids 21 to 405).

[0201] GH62 arabinofuranylase from *Talaromyces phinophilus*. Disclosed herein as SEQ ID NO:2 (mature polypeptide amino acids 17 to 325).

[0202] A metalloproteinase from *Thermoascus aurantiacus*. Disclosed herein as SEQ ID NO:3 (mature polypeptide amino acids 1 to 177).

[0203] GH5_21 xylanase from a species of the genus *Chryseobacterium* sp. is disclosed herein as SEQ ID NO:4 (mature polypeptide amino acids 1 to 551).

[0204] GH30_8 xylanase from Bacillus sp.-18423 is disclosed herein as SEQ ID NO:5 (mature polypeptide amino acids 28 to 417).

[0205] GH11 xylanase from *Geobacillus stearothermophilus*. Disclosed herein as SEQ ID NO:6 (mature polypeptide amino acids 30 to 212).

[0206] GH5_15 endoglucanase from *Trichoderma astroviride*. Disclosed herein as SEQ ID NO:7 (mature polypeptide amino acids 17 to 408).

[0207] β-glucanase activity assay

[0208] β-glucanase activity was determined by measuring the concentration of reducing sugars (RS) released by β-glucanase after hydrolysis of a suitable β-glucan substrate. The activities of GH16β-1,3(4)-glucanase and GH64β-1,3-glucanase were determined using CM-Lycopodium (β-1,3-glucan, P-CMPAC, Megazyme). The activities of GH5_15β-1,6-glucanase and GH30_3β-1,6-glucanase were determined using lecithin (β-1,6-glucan, YP15423, carbon synthase). RS concentrations were measured using a p-hydroxybenzoic acid hydrazide (PHBAH) assay suitable for 96-well microplates. In this assay, the reaction between the reducing ends of the C6 and C5 sugars and PHBAH resulted in the formation of hydrazones, which are intensely yellow and detectable by absorbance measurement at 410 nm.

[0209] Enzymatic hydrolysis of β-glucan substrates

[0210] Enzymatic hydrolysis was initiated in a hard-shell 96-well PCR plate (HSP-9631, Bio-Rad) by combining 80 μL of 2.5 g / L β-glucan substrate, 10 μL of appropriately diluted enzyme sample, and 10 μL of 50 mM glucono delta-lactone (GDL). GDL was added to inhibit β-glucosidase activity in the expressed host background. Each incubation mixture (total volume 100 μL) contained 2 g / L substrate enzyme and 5 mM GDL (pH 5.0) in 50 mM sodium acetate buffer. The plate was sealed with aluminum sealing tape (Costar #6570, Corning) and incubated for 10 min in a 50°C thermal cycler (Mastercycler pro S, Eppendorf) before cooling to 10°C.

[0211] To generate protein dose profiles, each enzyme sample was serially diluted eight times with a 2-fold buffer at pH 5.0 (50 mM sodium acetate buffer), and each enzyme dose was typically measured in triplicate. Each plate included two sets of glucose standards, 0.0625–1 mM and 0.3125–5 mM. Glucose standards were prepared by diluting a 10 mM stock glucose solution in 50 mM sodium acetate buffer at pH 5.0. The processing of each glucose standard (100 μL) was similar to that of the samples.

[0212] Example 1: Using GH10 xylanase and GH62 arabinofuranase on corn germ after the first milling Oil extraction yield of buds.

[0213] Substrate and Experimental Preparation: Industrially produced dehydrated, wet-milled germ from maize kernels was used as the substrate. The xylanase used was GH10 xylanase, disclosed herein as SEQ ID NO:1, and GH62 arabinofuranase, disclosed herein as SEQ ID NO:2. The xylanase and arabinofuranase were expressed in *Trichoderma reesei* through two separate fermentations, followed by mixing. Therefore, the mixture contained all the cellulases typically expressed in *Trichoderma reesei*.

[0214] A dry solids (DFS) measurement of 51.79% was observed on the substrate. This DFS measurement was derived from moisture balance. Approximately 200 g of germ was weighed, yielding 0.1 g DFS / flask. An enzyme concentration of 10 g enzyme product / kg DS was used as the dosage. The use of xylanase and arabinofuranosaccharidase was compared with known processes that add protease to increase oil yield. The protease used was a wild-type protease derived from *Thermophilus orangeus*, disclosed in WO 2003 / 048353 and disclosed herein as SEQ ID NO:3. The flask containing the germ and enzyme was adjusted to a final DFS of 2.6% with tap water.

[0215] Table 1. Sample Setup

[0216]

[0217]

[0218] The flask was incubated at 48°C for 24 hours with continuous mixing. After 24 hours of incubation, the germ slurry was poured onto a filtrate funnel, allowing the liquid portion to flow through and leaving only the germ solids. After dehydration, the germ was placed in a 50°C oven to promote drying.

[0219] Data Measurement: The generated data conforms to the official analysis method ( http: / / www.eoma.aoac.org / ).

[0220] • Crude protein AOAC 990.03

[0221] • Fat (acid hydrolyzed) AOAC 954.02

[0222] • Fiber (coarse) AOCS Ba 6a-05

[0223] The analysis followed MWL FD 039 based on AOCS Ba 6a-05. A small amount of sample was weighed and placed in a membrane bag and sealed. The bag and sample were placed in a container that was treated with various chemicals to dissolve the material leaching from the bag. After repeated washing and rinsing, the bag was dried and weighed again. The material remaining in the bag was reported as coarse fibers.

[0224] Table 2. Analysis Data

[0225]

[0226]

[0227] Oil extraction:

[0228] The dried corn germ is placed in the hopper of a pressing device. This device has a screw, which acts as a press and squeezes the oil out of the germ. The oil flows through a trough below the screw and is collected in a beaker or suitable container.

[0229] The recovered oil was weighed. Oil recovery was determined based on this weight measurement. The amount of germ entering the hopper was recorded, and enzyme effectiveness was assessed based on the difference between oil and germ weight. The results of oil recovery are shown in Table 3 below.

[0230] Table 3. Oil Recovery

[0231]

[0232] Example 2. A comparative test of xylanase diversity according to the method of the present invention: washing germ with xylanase.

[0233] Five enzymes belonging to the GH enzyme family—GH5_15, GH5_21, GH10, GH11, and GH30_8—were tested for use in germ treatment. Of these, GH5_15 is a β-glucanase, while the other four are xylanases. Background cellulases were also present in each enzyme treatment. Background cellulases were obtained as all cellulases produced by *Trichoderma reesei* strain Tv30. Figure 1 As shown, the enzyme treatment consisted of incubating 1 L of corn germ slurry (136 g corn germ sample, moisture content: 46.4%) with 760 μg total enzyme protein / g dry solids at 48°C and pH 4 for 4 hours. A control was also included as a comparison. After incubation, the germ was washed with 2 L of water through a 75 μm sieve to separate the coarse germ from the fine solids. The germ samples were oven-dried and analyzed for crude fiber, protein, and fat content using standardized AOCS (American Oil Chemists' Society) methods: Ba 6a-05, 990.03, and 2003.05. Starch analysis was performed by dilute acid hydrolysis. The baseline (enzyme-free) treatment of the germ samples showed the following content on a dry weight basis: 16.6% crude fiber, 8.4% starch, 10.9% crude protein, and 44.1% crude fat.

[0234] The mass of each analyte in the corresponding washed germ sample is calculated using the following formula:

[0235] Mass of analyte = Mass of dried germ × % of dry weight of analyte

[0236] For example,

[0237] Protein quality, M 蛋白质 =M 胚芽 ×% protein dry weight

[0238] For enzyme treatment, the reduction in the amount of analyte is calculated using the following formula:

[0239] % reduction in analyte mass = [analyte mass (enzyme treatment - control) / analyte mass of control]

[0240] ×100%

[0241] For example, the percentage reduction in protein mass of enzyme-treated germ is the relative difference in protein mass between enzyme-treated and control germs relative to the control germ. For instance,

[0242] % reduction in germ protein quality = (enzyme treatment - control) / M 蛋白质 / M of the control embryo 蛋白质 ×100%

[0243] Enzyme treatment of germ releases fiber, starch, and protein, which are then separated into a fine solids slurry fraction. Table 4 shows the percentage reduction in germ, fiber, starch, protein, and fat mass of residual coarse germ by various enzyme treatments, relative to a no-enzyme control. Enzyme treatment releases significant amounts of fiber, starch, and protein during incubation, which are then separated into a fine solids slurry fraction. Recovering the released starch and protein from the fine solids slurry in downstream corn wet milling processes will improve the yield of these byproducts.

[0244] When enzyme treatment was used, the change in crude fat mass was minimal. Removal of fiber, starch, and protein yielded a fine solids slurry fraction, which increased the proportion of crude fat in the enzyme-treated germ samples, as shown in Table 5. Therefore, the oil yield of the enzyme-treated germ samples will also be improved.

[0245] Table 4. Percentage reduction in dry weight, crude fiber, starch, protein, and fat in residual crude germ after enzyme treatment

[0246]

[0247] Table 5. Percentage of crude fat in residual crude germ after enzyme treatment

[0248]

[0249]

Claims

1. A method for improving the oil yield from germ in a wet milling process, the method comprising mixing a process stream containing corn germ with an enzyme composition containing an effective amount of one or more hydrolytic enzymes, wherein at least one of the hydrolytic enzymes is at least one xylanase polypeptide selected from the group consisting of: GH5 polypeptide. The xylanase polypeptide has 100% sequence identity with the mature polypeptide of SEQ ID NO:4; wherein the mature polypeptide is amino acid 1 to 551 of SEQ ID NO:

4.

2. The method of claim 1, wherein the amount of oil released from the germ is increased compared to the method without xylanase addition.

3. The method of claim 1, wherein the method comprises the following steps: a) Soak corn kernels in water to produce soaked kernels; b) Grind the soaked seeds to release the germ from the seeds; c) Separate the germ from these soaked and milled grains; as well as d) subject the germ to enzyme treatment with at least one xylanase polypeptide selected from the group consisting of: GH5 xylanase.

4. The method of claim 3, wherein the germ is mixed with one or more xylanases during or after step b), during step c), or after the germ has been separated.

5. The method of claim 4, wherein the germ is mixed with one or more xylanases after step c).

6. The method of any one of claims 1 to 5, wherein the germ is allowed to react with one or more xylanases for at least 45 minutes, at least 1 hour, at least 2 hours, at least 2.5 hours, or at least 3 hours.

7. The method according to any one of claims 1 to 5, wherein the hydrolytic enzyme is present in an amount of 0.0005 to 1.5 mg enzyme protein / gDS grain.

8. The method of claim 7, wherein the hydrolytic enzyme is present in an amount of 0.001 to 1 mg enzyme protein / g DS grain.

9. The method of claim 7, wherein the hydrolytic enzyme is present at an amount of 0.002 to 0.5 mg enzyme protein / g DS grain.

10. The method of claim 7, wherein the hydrolytic enzyme is present at an amount of 0.003 to 0.4 mg enzyme protein / g DS grain.

11. The method of any one of claims 1 to 5, wherein the hydrolytic enzymes comprise at least one xylanase and at least one arabinofuranase.

12. The method of any one of claims 1 to 5, wherein the hydrolases comprise at least one β-glucanase.

13. The method of any one of claims 1 to 5, wherein the hydrolases further comprise cellulase.

14. The method of claim 13, wherein the cellulases are selected from endoglucanase, cellobiase I, cellobiase II, GH61, β-glucosidase, or combinations thereof.

15. The method of claim 14, wherein the cellulases comprise at least an endoglucanase and a cellobiase.

16. The method of claim 15, wherein the cellulases comprise at least cellobiase I, cellobiase II, and endoglucanase I.

17. The method of claim 13, wherein the cellulases are derived from the Trichoderma genus.

18. The method of claim 17, wherein the cellulases are derived from Trichoderma reesei.

19. The method of claim 11, wherein the arabinofuranase is GH62 or GH43 arabinofuranase.

20. The method of claim 12, wherein the β-glucanase is GH5 β-glucanase.

21. The method of claim 20, wherein the β-glucanase is GH5_15 β-glucanase.

22. The method of claim 11, wherein the arabinofuranase is selected from the group consisting of: (a) A polypeptide having at least 75%, at least 80%, at least 85%, 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 with the mature polypeptide of SEQ ID NO:2; (b) A variant of the mature polypeptide of SEQ ID NO: 2, comprising substitution, deletion, and / or insertion at one or more sites; and (c) or (a) or (b) fragments of the polypeptide with arabinofuranyl glycosidase activity.

23. The method of claim 22, wherein the arabinofuranases of (a), (b) and (c) have at least 20%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% arabinofuranase activity of the mature polypeptide of SEQ ID NO:

2.

24. The method of claim 22, wherein the arabinofuranase is GH62-arabinofuranase.

25. The method of claim 24, wherein the arabinofuranosylase is derived from *Pseudomonas spp.* (…). Talaromyces phinophilus ).

26. The method of claim 22, wherein the mature polypeptide is amino acid 17 to 325 of SEQ ID NO:

2.

27. The method of claim 12, wherein the β-glucanase is selected from the group consisting of: (a) A polypeptide having at least 75%, at least 80%, at least 85%, 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 with the mature polypeptide of SEQ ID NO:7; (b) A variant of the mature polypeptide of SEQ ID NO: 7, comprising substitution, deletion, and / or insertion at one or more sites; and (c) or (a) or (b) fragments of the polypeptide with β-glucanase activity.

28. The method of claim 27, wherein the β-glucanases of (a), (b) and (c) have at least 20%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% β-glucanase activity of the mature polypeptide of SEQ ID NO:

7.

29. The method of claim 27, wherein the β-glucanase is GH5_15 endoglucanase-1,6-β-glucanase.

30. The method of claim 29, wherein the beta-glucanase is from Trichoderma atroviride (T. harzianum). Trichoderma astroviride ).

31. The method of claim 27, wherein the mature polypeptide is amino acids 17 to 408 of SEQ ID NO:

7.

32. The method of any one of claims 1 to 5, wherein the germ is dehydrated / dried.

33. The method of any one of claims 1 to 5, further comprising the step of extracting oil from the germ.

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