WO2018053220A1 - Fiber washing method and system - Google Patents

Fiber washing method and system Download PDF

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Publication number
WO2018053220A1
WO2018053220A1 PCT/US2017/051701 US2017051701W WO2018053220A1 WO 2018053220 A1 WO2018053220 A1 WO 2018053220A1 US 2017051701 W US2017051701 W US 2017051701W WO 2018053220 A1 WO2018053220 A1 WO 2018053220A1
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minutes
fraction
fiber
hours
system
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PCT/US2017/051701
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French (fr)
Inventor
Bernardo VIDAL
Oscar Pastor FERRER
Scott R. Mclaughlin
Randall Deinhammer
Thomas Patrick GIBBONS
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Novozymes A/S
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Priority to US201662395545P priority Critical
Priority to US62/395,545 priority
Priority to US201662426711P priority
Priority to US62/426,711 priority
Priority to EP17151610 priority
Priority to EP17151610.7 priority
Priority to US201762468704P priority
Priority to US62/468,704 priority
Application filed by Novozymes A/S filed Critical Novozymes A/S
Publication of WO2018053220A1 publication Critical patent/WO2018053220A1/en

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    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A23B - A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L7/00Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/10Cereal-derived products
    • A23L7/197Treatment of whole grains not provided for in groups A23L7/117 - A23L7/196
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A23B - A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/206Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
    • A23L29/212Starch; Modified starch; Starch derivatives, e.g. esters or ethers
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A23B - A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/206Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
    • A23L29/212Starch; Modified starch; Starch derivatives, e.g. esters or ethers
    • A23L29/225Farinaceous thickening agents other than isolated starch or derivatives
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B02CRUSHING, PULVERISING, OR DISINTEGRATING; PREPARATORY TREATMENT OF GRAIN FOR MILLING
    • B02BPREPARING GRAIN FOR MILLING; REFINING GRANULAR FRUIT TO COMMERCIAL PRODUCTS BY WORKING THE SURFACE
    • B02B1/00Preparing grain for milling or like processes
    • B02B1/04Wet treatment, e.g. washing, wetting, softening
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B30/00Preparation of starch, degraded or non-chemically modified starch, amylose, or amylopectin
    • C08B30/02Preparatory treatment, e.g. crushing of raw materials or steeping process
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B30/00Preparation of starch, degraded or non-chemically modified starch, amylose, or amylopectin
    • C08B30/04Extraction or purification
    • C08B30/042Extraction or purification from cereals or grains
    • C08B30/044Extraction or purification from cereals or grains from corn or maize
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B30/00Preparation of starch, degraded or non-chemically modified starch, amylose, or amylopectin
    • C08B30/08Concentration of starch suspensions
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B30/00Preparation of starch, degraded or non-chemically modified starch, amylose, or amylopectin
    • C08B30/12Degraded, destructured or non-chemically modified starch, e.g. mechanically, enzymatically or by irradiation; Bleaching of starch
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B30/00Preparation of starch, degraded or non-chemically modified starch, amylose, or amylopectin
    • C08B30/12Degraded, destructured or non-chemically modified starch, e.g. mechanically, enzymatically or by irradiation; Bleaching of starch
    • C08B30/16Apparatus therefor

Abstract

The present invention provides to a fiber washing system, optimized for the use of hydrolytic enzymes in the system. Furthermore, the present invention provides to a method for improving starch and gluten yield in a wet milling process, preferably comprising the optimized fiber washing system.

Description

FIBER WASHING METHOD AND SYSTEM

FIELD OF THE INVENTION

The present invention relates to a fiber washing system, optimized for the use of hydrolytic enzymes in the system. Furthermore, the present invention relates to a method for improving starch and gluten yield in a wet milling process, preferably comprising the optimized fiber washing system.

BACKGROUND OF THE INVENTION

Conventional wet milling of corn is a process designed for the recovery and purification of starch and several coproducts including germ, gluten and fiber.

Fiber is the least valuable coproduct, so the industry has put substantial effort into increasing the yield of the more valuable products, such as starch and gluten, while decreasing the fiber fraction. High quality starch is valuable as it can be used for a variety of commercial purposes after further processing to products such as dried starch, modified starch, dextrins, sweeteners and alcohol. Gluten is usually used for animal feed, as corn gluten meal (Around 60% protein) or corn gluten feed (Around 20% protein).

The wet milling process can vary significantly dependent on the specific mill equipment used, but usually the process include: grain cleaning, steeping, grinding, germ separation, a second grinding, 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 conditions of time and temperature. Then, the kernels are grinded to break down the pericarp and the germ is separated from the rest of the kernel. The remaining slurry, mainly consisting of fiber, starch and gluten is finely ground and screened in a fiber washing process, to separate the fiber from starch and gluten, before the gluten and starch is separated and the starch can be purified in a washing/filtration process.

The use of enzymes in several steps of the wet milling process has been suggested, such as the use of enzymes for the steeping step of wet milling processes. The commercial enzyme product Steepzyme® (available from Novozymes A/S) has been shown suitable for the first step in wet milling processes, i.e., the steeping step where corn kernels are soaked in water.

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

US 6,566, 125 discloses a method for obtaining starch from maize involving soaking maize kernels in water to produce soaked maize kernels, grinding the soaked maize kernels to produce a ground maize slurry, and incubating the ground maize slurry with enzyme (e.g., protease).

US 5,066,218 discloses a method of milling grain, especially corn, comprising cleaning the grain, steeping the grain in water to soften it, and then milling the grain with a cellulase enzyme.

WO 2002/000731 discloses a process of treating crop kernels, comprising soaking the kernels in water for 1-12 hours, wet milling the soaked kernels and treating the kernels with one or more enzymes including an acidic protease.

WO 2002/000911 discloses a process of starch gluten separation, comprising subjecting mill starch to an acidic protease.

WO 2002/002644 discloses a process of washing a starch slurry obtained from the starch gluten separation step of a milling process, comprising washing the starch slurry with an aqueous solution comprising an effective amount of acidic protease.

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

While the art has investigated the effect of using enzymes in corn wet milling, during steeping/soaking of corn kernels, during grinding of the corn kernels and in starch gluten separation, there is still a need for improved enzyme technology that may lower the energy expenditure and costs associated with corn wet milling and provide increased yield of starch and gluten.

SUMMARY OF THE INVENTION

The present invention relates to a fiber washing system which can be used in corn wet milling and is optimized for use of hydrolytic enzymes in the system.

Furthermore, the present invention relates to a method for improving starch yield and/or gluten yield from corn kernels in a wet milling process, by contacting one or more fractions of the corn kernel mass with an effective amount of one or more hydrolytic enzymes for at least 35 minutes during fiber washing. In a first aspect, the invention relates to a fiber washing system comprising a plurality of screen units being fluidly connected in a counter current washing configuration; each screen unit is configured for separating a stream of corn kernel mass and liquid into two fractions: a first fraction and a second fraction, said second fraction containing a higher amount measured in wt% fiber than the first fraction;

a space arranged in the system and being fluidly connected to receive one of said first fraction, one of said second fraction, or a mixed first and second fraction, preferably only a second fraction, and configured to provide an incubation time for one or both fractions received in the space; and outletting the thereby incubated one or both fractions to a downstream screen unit,

wherein the system is configured for

inletting corn kernel mass and liquid to the most upstream screen unit,

outletting the first fraction from the most upstream screen unit as a product stream containing starch,

- inletting process water, preferably arranged for inletting process water to a most downstream screen unit,

outletting the second fraction from most downstream screen unit as a washed corn kernel mass containing a lower amount of starch and gluten than the original corn kernel mass.

- introducing hydrolytic enzymes into the system.

In a second aspect, the invention relates to an incubator comprising a number of fluidic serially connected chambers by an opening forming a passage between two neighbouring chambers through which opening material may flow from an upstream to a downstream chamber), wherein

a most upstream chamber being connected to receive said first fraction, said second fraction or a combination of a first and a second fraction, and

a most downstream chamber having an outlet connection adapted to deliver incubated material out from the incubator;

the incubator further comprising one or more agitators configured for preventing solid decantation in the incubator.

An incubator according the second aspect may advantageously be used in a fiber washing system according the first aspect but is not considered limited to this use. In a third aspect, the invention relates to a method to improve starch yield and/or gluten yield from corn kernels in a wet milling process, comprising the steps of:

a) soaking the kernels in water to produce soaked kernels;

b) grinding the soaked kernels;

c) separating germs from the ground and soaked kernels to produce a corn kernel mass comprising fiber, starch and gluten; and

d) subjecting the resultant corn kernel mass, to a fiber washing procedure;

wherein during step d) one or more fractions of the corn kernel mass is contacted with an effective amount of one or more hydrolytic enzymes, and step d) has a total retention time of at least 45 minutes.

BRIEF DESCRIPTION OF THE FIGURES

The present invention and in particular preferred embodiments according to the invention will be described in more detail with reference to the accompanying figures. The figures show ways of implementing the present invention and are not to be construed as being limiting to other possible embodiments falling within the scope of the attached claim set.

Figure 1 schematically illustrates a first embodiment of a counter current fiber wash system according to present invention,

Figure 2 schematically illustrates a further embodiment of a system according to the present invention.

Figure 3 schematically illustrates a screen unit with an build-in incubator

Figure 4 schematically illustrates a screen unit in the form of a hydro-cyclone

Figure 5 A flow diagram of a wet milling process segment, showing different dosing locations used for the simulations.

Figure 6 shows enzyme recovery in filtrate, during laboratory fiber incubation

Figure 7 schematically illustrates a preferred embodiment of an incubator according to the present invention; figure 7 illustrates the incubator in a vertical cross sectional view (left side of figure 7), details on agitators arranged inside the incubator (upper right corner of figure 7) and in a horizontal cross sectional view along A-A (lower right corner of figure 7). Figure 8 provides an outline of a process for determining total starch, total protein, dry fiber weight and amount of bound starch. Figure 9 shows mean(% insolubles released from starting fiber) & % insoluble released from starting fiber vs. % dry solids (DS).

DETAILED DESCRIPTION

It is an object of the present invention to provide a fiber washing system optimized for the use of hydrolytic enzymes. Furthermore, it is an object of the present invention to provide a method that improves starch and gluten yield from corn kernels in a wet milling process. Other benefits of the invention include improved fiber dewatering and an anti-foaming effect.

The wet milling process:

Corn kernels are wet milled in order to open up the kernels and separate the kernels into its four main constituents: starch, germ, fiber and gluten.

The wet milling process can vary significantly from mill to mill, however conventional wet milling usually comprises the following steps:

1. Steeping and germ separation,

2. Fiber washing, pressing and drying,

3. Starch/gluten separation, and

4. Starch washing.

1. Steeping, grinding and germ separation

Corn kernels are softened by soaking in water for between about 30 minutes to about 48 hours, preferably 30 minutes to about 15 hours, such as about 1 hour to about 6 hours at a temperature of about 50°C, such as between about 45°C to 60°C. During steeping, the kernels absorb water, increasing their moisture levels from 15 percent to 45 percent and more than doubling in size. The optional addition of e.g. 0.1 percent sulfur dioxide (SO2) and/or NaHSOs to the water prevents excessive bacteria growth in the warm environment. As the corn swells and softens, the mild acidity of the steepwater begins to loosen the gluten bonds within the corn and release the starch. After the corn kernels are steeped they are cracked open to release the germ. The germ contains corn oil. The germ is separated from the heavier density mixture of starch, gluten and fiber essentially by "floating" the germ segment free of the other substances under closely controlled conditions. This method serves to eliminate any adverse effect of traces of corn oil in later processing steps. 2. Fiber washing, pressing and drying

To get maximum starch and gluten recovery, while keeping any fiber in the final product to an absolute minimum, it is necessary to wash the free starch and gluten from the fiber during processing. The free starch and gluten is separated from fiber during screening (washing) and collected as mill starch. The remaining fiber is then pressed to decrease the water content and dried.

3. Starch gluten separation

The starch-gluten suspension from the fiber-washing step, called mill starch, is separated into starch and gluten. Gluten has a low density compared to starch. By passing mill starch through a centrifuge, the gluten is readily spun out.

4. Starch washing

The starch slurry from the starch separation step contains some insoluble protein and much of solubles. They have to be removed before a top quality starch (high purity starch) can be made. The starch, with just one or two percent protein remaining, is diluted, washed 8 to 14 times, re-diluted and washed again in hydroclones to remove the last trace of protein and produce high quality starch, typically more than 99.5% pure.

Products of wet milling: Wet milling can be used to produce, without limitation, 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.

Definition of enzymes:

Arabinofuranosidases/polypeptide with arabinofuranosidase activity: The term "arabinofuranosidase" means an alpha L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzyme acts on alpha-L-arabinofuranosides, alpha- L-arabinans containing (1 ,3)- and/or (1 ,2)- and/or (1 ,5)-linkages, arabinoxylans, and arabinogalactans. Alpha-L arabinofuranosidase is also known as arabinosidase, alpha- arabinosidase, alpha-L-arabinosidase, alphaarabinofuranosidase, polysaccharide alpha-L- arabinofuranosidase, alpha-L-arabinofuranoside hydrolase, L-arabinosidase, or alpha-L- arabinanase. Arabinofuranosidase activity can be determined using 5 mg of medium viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5 in a total volume of 200 μΙ for 30 minutes at 40°C followed by arabinose analysis by AM IN EX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Beta-glucosidase/polypeptide with beta-glucosidase activity: The term "beta- glucosidase" means a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminal non-reducing beta-D-glucose residues with the release of beta-D- glucose. Beta-glucosidase activity can be determined using pnitrophenyl-beta-D- glucopyranoside as substrate according to the procedure of Venturi et al., 2002, J. Basic Microbiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0 μηιοΐβ of pnitrophenolate anion produced per minute at 25°C, pH 4.8 from 1 mM p-nitrophenyl-beta- Dglucopyranoside as substrate in 50 mM sodium citrate containing 0.01 % TWEEN® 20.

Beta-xylosidase/polypeptide with beta-xylosidase activity: The term "beta-xylosidase" means a beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of short beta (1→4)-xylooligosaccharides to remove successive D-xylose residues from non- reducing termini. Beta-xylosidase activity can be determined using 1 mM p-nitrophenyl- beta-D-xyloside as substrate in 100 mM sodium citrate containing 0.01 % TWEEN® 20 at pH 5, 40°C. One unit of beta-xylosidase is defined as 1.0 μηιοΐβ of p15 nitrophenolate anion produced per minute at 40°C, pH 5 from 1 mM p-nitrophenyl-beta-Dxylosidein 100 mM sodium citrate containing 0.01 % TWEEN® 20.

Cellobiohydrolase/polypeptide with cellobiohydrolase activity: The term "cellobiohydrolase" means a 1 ,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176) that catalyzes the hydrolysis of 1 ,4-beta-D15 glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1 ,4-linked glucose containing polymer, releasing cellobiose from the reducing end (cellobiohydrolase I) or non-reducing end (cellobiohydrolase II) 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 ai, 1988, Eur. J. Biochem. 170: 575-581.

Cellulolytic enzyme or cellulase/polypeptide with cellulase activity or cellulolytic activity: The term "cellulolytic enzyme" or "cellulase" means one or more (e.g., several) enzymes that hydrolyze a cellulosic material, which comprise any material comprising cellulose, such as fiber. Cellulytic enzymes include endoglucanase(s) (E.C 3.2.1.4), cellobiohydrolase(s) (E.C 3.2.1.91 and E.C 3.2.1.150), beta-glucosidase(s) (E.C. 3.2.1.21), or combinations thereof. The two basic approaches for measuring cellulolytic enzyme activity include: (1) measuring the total cellulolytic enzyme activity, and (2) measuring the individual cellulolytic enzyme activities (endoglucanases, cellobiohydrolases, and beta- glucosidases) as reviewed in Zhang et al. , 2006, Biotechnology Advances 24: 452-481. 5 Total cellulolytic enzyme activity can be measured using insoluble substrates, including Whatman N°1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc. The most common total cellulolytic activity assay is the filter paper assay using Whatman N°1 filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (lUPAC) (Ghose, 10 1987, Pure Appl. Chem. 59: 257-68).

Cellulolytic enzyme activity can be determined by measuring the increase in production/release of sugars during hydrolysis of a cellulosic material by cellulolytic enzyme(s) under the following conditions: 1-50 mg of cellulolytic enzyme protein/g of cellulose in pretreated corn stover (PCS) (or other pretreated cellulosic material) for 3-7

15 days 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 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, compared to a control hydrolysis without addition of cellulolytic enzyme protein. Typical conditions are 1 ml reactions, washed or unwashed PCS, 5% insoluble solids (dry weight), 50 mM sodium acetate 5 pH 5, 1 mM MnS04, 50°C, 55°C, or

20 60°C, 72 hours, sugar analysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Hydrolytic enzymes or hydrolase/polypeptide with hydrolase activity: "Hydrolytic enzymes" refers to any catalytic protein that use water to break down substrates. Hydrolytic enzymes include cellulases (EC 3.2.1.4), xylanases (EC 3.2.1.8) arabinofuranosidases 25 (EC 3.2.1.55 (Non-reducing end alpha-L-arabinofuranosidases); EC 3.2.1.185 (Non- reducing end beta-L-arabinofuranosidases) cellobiohydrolase I (EC 3.2.1.150), cellobiohydrolase II (E.C. 3.2.1.91), cellobiosidase (E.C. 3.2.1.176), beta-glucosidase (E.C. 3.2.1.21), beta-xylosidases (EC 3.2.1.37).

Xylanases/polypeptide with xylanase activity: The term "xylanase" means a 1 ,4-beta- 30 D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1 ,4-beta-D- xylosidic linkages in xylans. Xylanase activity can be determined with 0.2% AZCL- arabinoxylan as substrate in 0.01 % TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C. One unit of xylanase activity is defined as 1.0 μηιοΐβ of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6.

Other definitions:

In the present context, terms are used in manner being ordinary to a skilled person. Some of these terms are elucidated below:

Anti-foaming effect: Foaming is a widely observed phenomenon in wet milling. An "anti- foaming effect" refers to means of reducing foaming.

Contact time: For one or more enzymes to react with a substrate, the one or more enzymes have to be in contact with the substrate. "Contact time" refers to the time period in which an effective amount of one or more enzymes is in contact with at least a fraction of a substrate mass. The enzymes may not be in contact with all of the substrate mass during the contact time, however mixing the one or more enzymes with a substrate mass allows the potential of enzymatically catalyzed hydrolysis of a fraction of the substrate mass during the contact time.

Corn kernel: A variety of corn kernels are known, including, e.g., dent corn, flint corn, pod corn, striped maize, sweet corn, waxy corn and the like.

Some corn kernels has an outer covering referred to as the "Pericarp" that protects the germ in the kernels. It resists water and water vapour and is undesirable to insects and microorganisms. The only area of the kernels not covered by the "Pericarp" is the "Tip Cap", which is the attachment point of the kernel to the cob.

Corn kernel mass: is preferably used to reference a mass comprising fiber, gluten and starch, preferably achieved by steaming and grinding crop kernels and separating a mass comprising fiber, gluten and starch from germs. As the corn kernel mass move through the fiber washing, it is separated into several fractions, including first (s) and second fractions (f). Hence, "fractions of corn kernel mass" and "one or more fractions of corn kernel mass" refer to these first (s) and second fractions (f).

Dewatering: "Dewatering" refers to any process in which excess water is removed from corn fiber.

Expression: The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Germ: The "Germ" is the only living part of the corn kernel. It contains the essential genetic information, enzymes, vitamins, and minerals for the kernel to grow into a corn plant. In yellow dent corn, about 25 percent of the germ is corn oil. The endosperm covered or surrounded by the germ comprises about 82 percent of the kernel dry weight and is the source of energy (starch) and protein for the germinating seed. There are two types of endosperm, soft and hard. In the hard endosperm, starch is packed tightly together. In the soft endosperm, the starch is loose.

GH10 polypeptide: refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 10 in the database of Carbohydrate- Active enZYmes (CAZymes) available at http:/'/www. cazy . org/. (Lombard, V.; Golaconda Ramulu, H.; Drula, E.; Coutinho, P. M.; 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).

GH11 polypeptide refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 1 1 in the database of Carbohydrate- Active enZYmes (CAZymes).

GH62 polypeptide: refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 62 in the database of Carbohydrate- Active enZYmes (CAZymes).

Gluten: Gluten is a protein, made up from two smaller proteins, glutenin and gliadin. Herein "gluten" refers to the majority of proteins found in corn kernels. The major products of gluten from corn wet milling is corn gluten meal (Approximately 60% protein) and corn gluten feed (Approximately 20% protein).

Grind or grinding: The term "grinding" refers to breaking down the corn kernels into smaller components.

Incubation time: Time in which the one or more fractions of the corn kernel mass is/are in contact with hydrolytic enzyme during fiber washing, without being screened. In many preferred embodiments, a system and method according to the present invention utilises an incubator inside which the material is "left to be affected" by the enzymes and in such situations, the incubation time may be determined by: volume of incubator [m3] * density of inflow to incubator tit =

mass inflow per time unit to the incubator

Alternatively, if the inflow to the incubator is expressed in terms of volume per time unit:

volume of incubator [m3]

vo lume inflow per time unit to the incubator

Insolubles: In the present context, "insolubles" is used interchangeably with "insoluble solids"; it is defined as materials that is able to pass through a 75μηι sieve and cannot be dissolved in water.

Mill equipment: "Mill equipment" refers to all equipment used on a mill. The wet milling process will vary dependent on the available mill equipment. Examples of mill equipment can be steeping tanks, evaporator, screw press, rotatory dryer, dewatering screen, centrifuge, hydrocyclone, ect. The size, and number of each mill equipment/milling lines can vary on different mills, which will affect the milling process. For example, the number of fiber washing screen units can vary and so can the size of a centrifuge.

Retention time: The total retention time, is the time period in which the corn kernel mass, received in the first screen unit (S1) and one or more fractions thereof, 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 corn kernel mass is incubated with one or more hydrolytic enzymes in a space (V), before it leaves the fiber washing system, as part of a first fraction (s1) from the most upstream screen unit (S1) or as part of a second fraction (f4) from the most downstream screen unit (S4). Retention time may preferably be estimated as the average duration of time solid mater spends in a system according to the present invention. This may be estimated by the following relation:

Figure imgf000012_0001

Alternatively, if the inflow to the system is expressed in terms of volume per time unit: volume of system [m3]

lume inflow per time unit to the system

The volume of the system is typically set equal to the sum of the volumes of all voids in the system; however, as the tubing in the system typically is made small, and it may thus be preferred to discard the volume of the tubing in the determination of the retention time.

Screened: The term "screened" refers to the process of separating corn kernel mass into a first fraction s and a second fraction f and movement of these fractions from one screen unit to another. A screen unit may for example be a pressure-fed screen/feed pressure screen wherein material is fed through a nozzle or a rotary screen, wherein material is forced through the screen by gravity. Examples of such screens could be DSM screen and ICM screens respectively.

A non-screening period is a non-separating period provided for incubation of corn kernel mass or fractions thereof with enzymes.

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

For 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 Needle program of the EMBOSS 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 was used.

The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labelled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment).

Starch: The term "starch" means any material comprised of complex polysaccharides of plants, composed of glucose units that occurs widely in plant tissues in the form of storage granules, consisting of amylose and amylopectin, and represented as (C6HioOs)n, where n is any number. Steeping or soaking: The term "steeping" means soaking the crop kernel with water and optionally SO2.

Viscosity: The viscosity of a fluid is a measure of its resistance to gradual deformation by shear stress or tensile stress. In the present application "viscosity" also refers to the informal concept of "thickness".

Description of the invention:

In processes for conventional corn wet milling fiber washing and drying procedures have been optimized to achieve maximum throughput of fiber slurry. Hence, although the fiber washing process varies from mill to mill, it typically takes less than 30 minutes, which makes it little attractive to dose enzymes during fiber washing.

The present inventors have observed that dosing enzymes in fiber washing has a surprisingly great potential: When dosed correctly and with the appropriate retention time, the enzymes provide not only dewatering of the fiber fraction; the enzymes also release considerable amounts of valuable starch and gluten bound in the fiber. Further, the inventors have observed that with correct application of enzymes in the fiber washing process the enzymes also reduce foam formation, which reduces or may even obviate the need for anti-foaming chemicals. Hence, the present invention provides means for appropriate dosing of enzymes and means of increasing contact time between enzymes and corn kernel mass in the fiber washing process

In one aspect, the present invention related to a fiber washing system comprising:

a plurality of screen units (S1... S4) being fluidly connected in a counter current washing configuration; each screen unit is configured for separating a stream of corn kernel mass and liquid into two fractions: a first fraction (s) and a second fraction (f), said second fraction (f) containing a higher amount measured in wt% fiber than the first fraction (s);

a space (V) arranged in the system and being fluidly connected to receive said first fraction (s), said second fraction (f), or a mixed first and second fraction (s,f), preferably only a second fraction (f), and configured to provide an incubation time for one or both fractions received in the space; and outletting the thereby incubated one or both fractions to a downstream screen unit (S4),

wherein the system is configured for

inletting corn kernel mass and liquid to the most upstream screen unit (S1) outletting the first fraction (s1) from the most upstream screen unit (S1) as a product stream containing starch,

inletting process water, preferably arranged for inletting process water to a most downstream screen unit (S4),

- outletting the second fraction (f4) from most downstream screen unit (S4) as a washed corn kernel mass containing a lower amount of starch and gluten than the original corn kernel mass,

introducing hydrolytic enzymes into the system.

Figure 1 schematically illustrates an embodiment of a fiber washing system according to the present invention. As illustrated in figure 1 , the fiber washing system comprises a plurality of screen units S1 , S2, S3, S4 being fluidly connected in a counter current washing configuration. By fluidly connected typically means that the screen units are connected by use of flow lines, such as pipes for transporting matter between the screen units. Each of the screen units S1-S4 is configured for separating a stream of corn kernel mass and liquid into two fractions: a first fraction s (s1 , s2, s3, s4) and a second fraction f (f1 , f2, f3, f4). As the skilled person will understand, the number of first fractions produced in the fiber washing system depends on the number screen units included in the system. The number of screen units in the system is preferably between 2-8, and in such embodiments the number of firsts and second fractions will also be between 2-8. The screen units are typically configured so that the solid matter is separated out in a separate stream whereby the second fraction f contains a higher amount measured in wt% fiber than the first fraction s. In the figure, notation "s" preferably refers to a fibreless stream (containing starch) and notation "f preferably refers to a fiber containing stream. Index on f and s refers to the origin of the stream. It is noted that although it is preferred that the first fractions s does not contain any fiber, this may in a practical set-up be difficult to achieve.

The flow in the system has a downstream direction and an upstream direction: each screen unit; e.g. screen unit S3, receives a stream; e.g. f2, from an upstream screen unit, e.g. S2 and delivers a stream; e.g. s3, to the upstream screen unit; e.g. S2. Similarly, the screen unit S3 receives a stream s4 from a downstream screen unit S4 and delivers a stream f3 to the downstream screen unit S4.

As illustrated in figure 1 , process water, that is typically water that is used as washing water in the system, is provided to the most downstream screen unit S4, and the process water is typically water not containing fiber. Corn kernel mass is typically a liquid suspension (typically a suspension in water), provided at the most upstream screen unit S1. This is in fig. 1 indicated by the arrow labelled "From milling". Thereby, and by the fluid connection between the screen units, the corn kernel mass and fractions f thereof flow downstream in the system and the process water moves upstream in the system. Thus, the fluid configuration in the system can be seen as the cornel kernel mass is washed in the most upstream screen unit S1 by a fluid containing high amount of starch and in the most downstream screen unit S4 washed by a fluid containing low amount of starch. Further, the corn kernel mass in the most upstream screen unit S1 contains a higher amount of starch than the fraction f of the corn kernel mass in the most downstream screen unit S4.

One of the aims of the invention is to provide a contact time between corn kernel mass or fractions thereof and enzymes in the system, in order to increase the efficiency of the removal of the starch from fiber. The contact time between enzymes and corn kernel mass or fractions thereof in the system is also referred to as retention time. By adding the enzymes at an optimal point in the fiber washing system, the retention time can be prolonged, which may increase the efficiency of the removal or separation of starch from fiber. In order to provide a longer retention time than that provided by a typical mill, a space V (not shown in fig. 1) may be arranged in the system and being fluidly connected to receive one of said first fractions s, one of said second fractions f, or a mixed first and second fraction s, f, preferably only a second fraction f, and configured to provide an incubation time for one or both fractions received in the space; and outletting the thereby incubated fraction or fractions to a downstream screen unit S4. It is noted that while it may be preferred to have a separate incubator unit arranged in the system, the flow lines connecting the screen units may also be used to provide the space.

Further and as presented herein, it has been found advantageous to add enzymes at a position being downstream of a most upstream screen unit S1 and upstream of a most downstream screen unit S4; in the embodiment of fig. 1 , the addition of enzymes is illustrated as being at the fluid position of the screen unit S3 (illustrated by the arrow in fig. 1 labelled "Enzymes".

According to embodiments wherein the fiber washing system comprises 2 screen units, dosing is preferred between the first and second screen unit or in a space configured between screen unit 1 and screen unit 2.

According to embodiments wherein the fiber washing system comprises 3 screen units, dosing is preferred in the second screen unit or in a space configured between screen unit 1 and screen unit 3, most preferred in screen unit 2, or a space configured between screen unit 2 and 3. According to embodiments wherein the fiber washing system comprise 4 screen units, dosing is preferred in the second or third screen unit or in a space configured between screen unit 1 and screen unit 4, most preferred in screen unit 2, or a space configured between screen unit 2 and 3.

According to embodiments wherein the fiber washing system comprise 5 screen units, dosing is preferred in the second, third or fourth screen unit, or in a space configured between screen unit 1 and screen unit 5, most preferred in screen unit 3 or a space configured between screen unit 3 and 4.

According to embodiments wherein the fiber washing system comprise 6 screen units, dosing is preferred in the second, third, fourth or fifth screen unit, or in a space configured between screen unit 1 and screen unit 6, most preferred in screen unit 4, or a space configured between screen unit 4 and 5.

According to embodiments wherein the fiber washing system comprise 7 screen units, dosing is preferred in the second, third, fourth, fifth or sixth screen unit, or in a space configured between screen unit 1 and screen unit 7, most preferred in screen unit 4 or a space configured between screen unit 4 and 5.

According to embodiments wherein the fiber washing system comprise 8 screen units, dosing is preferred in the second, third, fourth, fifth, sixth and seventh screen unit, or in a space configured between screen unit 1 and screen unit 8, most preferred in screen unit 5 or a space configured between screen unit 5 and 6.

Thus, a system according to preferred embodiments of the invention is configured for inletting corn kernel mass and liquid to the most upstream screen unit S1 , preferably by comprising an inlet into system feeding the matter to the most upstream screen unit SI ;

- outletting the first fraction s1 from the most upstream screen unit S1 as a product stream containing starch, preferably by comprising an outlet from the most upstream screen unit feeding a fibreless stream out from the system;

inletting process water, preferably arranged for inletting process water to a most downstream screen unit S4; the inlet of process water is preferably an inlet to the most downstream screen unit S4;

outletting the second fraction f4 from most downstream screen unit S4 as a washed corn kernel mass containing a lower amount of starch and gluten than the original corn kernel mass; preferably by comprising an outlet from the most downstream screen unit. The system is also configured for introducing hydrolytic enzymes into the system, which may be an inlet arranged at a preferred position to allow contact between the corn kernel mass or fractions thereof and the one or more hydrolytic enzymes.

Reference is made to fig. 2 schematically illustrating a further embodiment of a system according to the present invention. The same notation as used in fig. 1 is used in fig. 2. As presented in fig. 2, the screen units S1 to S4 all comprises a screening element (screen) indicated by a slanted, dotted line inside the screen units. This slanted dotted line illustrate a device configured for separating out a fraction f containing fiber and a fraction preferably not containing any fiber; this could for instance be provided by a band filter or a filter in general arranged inside wall parts defining an interior void of a screen unit.

In the embodiment shown in fig. 2, the various fraction to be mixed are illustrated as being mixed outside the screen units S1-S4. However, they may be mixed inside the screen units.

As also illustrated in fig. 2, the space V is a separate container being fluidly connected to the screen unit S3 so that the screen unit S3 receives fluid with fiber and enzymes after the fluid with fiber and enzymes has had an incubation time in the space V. As schematically illustrated in fig. 2, the space V may have baffle plates for assuring the fluid does not flow in a straight line from inlet to outlet of the container, which could otherwise short-cut the flow to provide an incubation time.

Fig. 2 also illustrates that enzymes are applied to the streams f2 and s4 going into the space V. In the embodiment shown, the enzymes are dosed by a dosing pump 10 illustrated schematically be a piston pump driven by a crank shaft where the amount of enzymes dosed is controlled by the rotation of the crank shaft (one-way inlet and outlet valves are present in the cylinder or cylinder head but not illustrated).

Thus, a system according to the present invention is preferably configured for introducing hydrolytic enzymes into said first fraction (s), and/or into said second fraction (f), and/or into a mixed first and second fraction and/or into the stream of process water supplied to the system.

The number of screen units S may be selected according to e.g. the volumetric capacity to separate into two stream and/or the other design aims. However, a system according to present invention will in general have a most upstream screen unit, a most downstream screen unit and preferably one or more intermediate screen units fluidic arranged in between the most upstream and most downstream screen units. That is, with reference to figs. 1 and 2, a preferred system will comprise a most upstream screen unit S1 and a most downstream screen unit S4 and a number of screen units (e.g. 2) arranged in between, where arranged in between refers to being fluidly connected as illustrated in the figs. 1 and 2.

In detail, the fluidly connected counter current washing configuration, as disclosed in fig.s 1 and 2, typically comprising the plurality of screen units S1 ... S4 being arranged in a manner so:

a second fraction f1 produced by an upstream screen unit S1 is mixed with a first fraction s3 produced by a downstream screen unit S3, and said mixed fractions being separated by a screen unit S2, being intermediate between said upstream and said downstream screen units S1 , S3, into a first fraction s2 and a second fraction f2.

While this disclosure is made with reference to screen unit S3, the same description may apply for any intermediate screen units, such as screen S2, or other intermediate screen units where intermediate screen unit refers to a screen unit being arranged downstream of the most upstream screen unit and upstream of a most downstream screen unit.

As illustrated in fig. 2, it is in some embodiments preferred that mixing of a second fraction f 1 and a first fraction s3 occurs prior to being inlet into an intermediate screen unit S2. Such a mixing may be provided by inletting the two fractions into a mixing chamber comprising stirring means providing typically a vigorous agitation of the fluid or the mixing may be provide by a manifold having an inlet for each stream and an outlet for the mixed stream.

As an alternative to mixing prior to be inlet into a screen unit, mixing of a second fraction f1 and a first fractions 3 may occur inside an intermediate screen unit S2. This may for instance be accomplished by the interior of the screen unit being equipped with a stirring means providing typically a vigorous agitation of the fluid inside the screen unit.

Although the embodiments disclosed in figs. 1 and 2 are shown to comprise more than two screen units, a system is considered to be fully operational with as little as two screen units. Thus, it is generally preferred that the system comprising 2-8 screen units typically arranged as illustrated in fig. 1 or 2.

A system according to the present invention may preferably be configured to provide a total retention time in said fiber washing system of at least 35 minutes, such as at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, at least 65 minutes, at least 70 minutes, at least 75 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 105 minutes, at least 110 minutes, at least 1 15 minutes, at least 120 minutes, during which the one or more hydrolytic enzymes is/are in contact with said corn kernel mass and/or one or more fractions thereof, where one or more fractions of corn kernel mass preferably refers to any fraction of corn kernel mass containing fiber and/or starch and/or gluten including any of fractions s and f. Here, retention time refers to the time a fluid element is present in the system calculated from the time where it enters into screen unit S1 and to the time where it leaves screen unit S4. This is typically determined on the basis of e.g. the total volume in m3 of the system (volume of screen units, flow lines and space (V) divided by the total volume flow of fibreless product out from screen unit S1 in m3/h. In a practical approach, the total volume flow of fibreless product is a predetermined design parameter, and the total volume of the system is then selected on the basis of this design parameter.

The total retention time in the fiber washing system may be between 35 minutes and 5 hours, such as between 35 minutes and 4 hours, 35 minutes and 3 hours, 35 minutes and 2,5 hours, 35 minutes and 2 hours, 35 minutes and 1 ,5 hour, 45 minutes and 5 hours, 45 minutes and 4 hours, 45 minutes and 3 hours, 45 minutes and 2,5 hours, 45 minutes and 2 hours, 1 hour and 5 hours, 1 hour and 4 hours, 1 hour and 3 hours, 1 hour and 2,5 hours, 1 hour and 2 hours, 70 minutes and 5 hours, 70 minutes and 4 hours, 70 minutes and 3 hours, 70 minutes and 2,5 hours, 70 minutes and 2 hours, 75 minutes and 5 hours, 75 minutes and 4 hours, 75 minutes and 3 hours, 75 minutes and 2,5 hours, 75 minutes and 2 hours, 80 minutes and 5 hours, 80 minutes and 4 hours, 80 minutes and 3 hours, 80 minutes and 2,5 hours, 80 minutes and 2 hours, 85 minutes and 5 hours, 85 minutes and 4 hours, 85 minutes and 3 hours, 85 minutes and 2,5 hours, 85 minutes and 2 hours, 90 minutes and 5 hours, 90 minutes and 4 hours, 90 minutes and 3 hours, 90 minutes and 2,5 hours, 90 minutes and 2 hours.

Further, the dimension of the space (in m3) is preferably configured to provide an incubation time of at least at least 5 minutes, such as 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. The incubation time in the space (V) configured into the fiber washing procedure may be less than 24 hours, such as less than 12 hours, less than 8 hours, less than 5 hours, less than 3 hours, less than 2,5 hours, less than 2 hours, less than 1 ,5 hours, less than 1 hour.

Preferably, the incubation time in said space (V) is in the range of 0.5-3 hours, such as 1-3 hours, 1-2 hours or such as 85-95 minutes. Again, the flow of fibreless product may be the design parameter and the layout of the system together with a desired incubation time can be used to determine the volume of the space V.

The space V designated for incubation preferably has a volume of at least 30 m3, at least 5 40 m3, at least 50 m3, at least 60 m3, at least 70, m3, at least 80, m3, at least 90, m3, at least 100 m3, at least 110 m3, at least 120 m3, at least 130 m3, at least 140 m3, at least 150 m3, at least 160 m3, at least 170 m3, at least 180 m3, at least 190 m3, at least 200 m3, at least 210 m3, at least 220 m3, at least 230 m3, at least 240 m3, at least 250 m3, at least 260 m3, at least 270 m3, at least 280 m3, at least 290 m3, at least 300 m3, at least 400 m3, or at least

10 500 m3. The incubation time may also be in more than one space V with a total or combined volume of at least 100 m3, at least 110 m3, at least 120 m3, at least 130 m3, at least 140 m3, at least 150 m3, at least 160 m3, at least 170 m3, at least 180 m3, at least 190 m3, at least 200 m3, at least 210 m3, at least 220 m3, at least 230 m3, at least 240 m3, at least 250 m3, at least 260 m3, at least 270 m3, at least 280 m3, at least 290 m3, at least 300 m3, at least

15 400 m3, at least 500 m3.

During the incubation time, it is preferred that the fluid received in the space V is not screened. Thus, the fluid leaving the space V has the same composition, e.g. of starch and fiber, as the fluid received in the space V, although it preferably contains a higher proportion of starch that has been released from the fibers.

20 To assure intimate contact between the enzymes and the fiber, it may be preferred to configure the space V for agitation of matter contained in said space V, such as by comprising a rotor or impeller.

As illustrated in fig. 2, it is preferred to arrange the space V downstream of the most upstream screen unit S1 and upstream of said most downstream screen unit S4; in 25 particular, the embodiment of fig. 2 illustrates that the space V is arranged to feed fluid into the second most downstream screen unit S3.

As disclosed herein, the space may be provided in different manner and as illustrated in fig. 2 the space V may preferably be provided as a separate incubator unit. The incubator unit may be configured by suitable fluid lines to receive a first fraction s, a second fraction f or a 30 combination of a first and a second fraction s,f, preferably only a second fraction f, and deliver the thereby incubated material to a downstream screen unit S3.

Reference is made to figure 7 which schematically illustrates a preferred embodiment of an incubator according to the present invention; figure 7 illustrates the incubator in a vertical cross sectional view (left side of fig. 7), details on agitators arranged inside the incubator (upper right corner of fig. 7) and in a horizontal cross sectional view along A-A (lower right corner of fig. 7).

As illustrated in fig. 7, the incubator 14 comprising a number of fluidic serially connected chambers 30 (four is shown). The chambers 30 are connected serially by an opening 32 forming a passage between two neighbouring chambers 30 through which opening 32 material may flow from an upstream to a downstream chamber 30. It is noted that the flow direction "from upstream to downstream" is in the figure vertically downwardly and this flow direction is typically dictated by a pump (not shown) and/or as elucidated below, by use of impellers arranged inside the incubator 14.

As illustrated in fig. 7, the most upstream chamber 30 is connected to receive a first fraction s, a second fraction f or a combination of a first and a second fraction s,f. In fig. 7 the first and the second fraction s, f are mixed outside the incubator 14 and fed into the most upstream chamber 30.

The most downstream chamber 30 of the incubator has an outlet connection 26 adapted to deliver incubated material out from the incubator 14. Further, the incubator of fig. 7 comprises one or more agitators 20 (four are shown in fig. 7) configured for preventing solid decantation in the incubator 14. Decantation may be an issue to consider, since the material to be incubated in the incubator comprises solid material contained in a liquid (typically water) and the material typically has a density being different from the liquid whereby if the mixture of solid matter and liquid is left with no agitation, solid matter may (depending on the density) either settle on the bottom of a chamber or float at the surface of the liquid. To avoid this, the incubator comprises the agitators 20 to provide agitation.

The incubator 14 preferably has a cylindrical shape so as to provide circular cross sections as illustrated in the cross sectional view along line A-A.

As illustrated in fig. 7, the incubator 14 may have a number of walls, preferably funnel shaped walls 28 (in fig. 7, three such funnel shaped walls are shown), disposed horizontally distanced inside the incubator 14 to divide the interior of the incubator 14 into chambers 30. The funnel shaped walls 28 are arranged so as to funnel material downwardly from one chamber to a downstream chamber 30. That is the angle a in fig. 7 is larger than 90 degrees. Typically the angle a is between 90 and 110, such as between 90 and 100, preferably between 90 and 95 degrees. As illustrated in the cross sectional view along line A-A in fig. 7, the funnel shaped walls 28 each has its opening 32 geometrical centred at the smallest cross section of each of the funnel shaped wall.

Since the most downstream chamber 30 of the incubator 14 is not fluidic serially connected to a downstream chamber 30, the bottom wall of such a most downstream chamber 30 is a sloping bottom wall 36 sloping towards an inlet of said outlet connection 26. The angle of the slope is typically selected as for the angle a of the funnel shaped walls 28. Such a one- side-sloping wall has the advantage that material contained in the chamber will be guided by the wall towards the outlet connection 26.

The outlet connection 26 is arranged so as to outlet material from the incubator 14 at the bottom thereof. This may be embodied as a horizontal extending tube extending through the wall of the incubator 14 at position close to the bottom wall 36. In the embodiment shown in figure 7, the outlet connection 26 is in a form of a tube extending internally inside the incubator 14 from a position at the top and outside of the incubator 14 and to the bottom of the most downstream positioned chamber 30.

As illustrated in fig. 7 - see in particular upper right corner of the fig. 7 - the agitators 20 are preferably impellers comprising a plurality of impeller blades 34. Further, the incubator 14 further comprising a shaft 18 and a motor 22 configured for rotating the shaft 14; and as the impellers are arranged on said shaft 14 rotation of the shaft 14 provides a rotation of the impellers. Since impellers beside prevents solid decantation (by agitation) also may provide a pumping action, the impellers may be configured to provide a pumping action pumping material through the incubator 14.

The combination of pumping and decantation may be designed by considering the vertical position of the impellers in the openings 32. This is illustrated in fig. 7 by the magnified view (indicated by the dotted lined circle) illustrating that each of the impellers arranged in one of said openings 32 are arranged in a position where a lower end of the impeller being disposed a distance h below an edge 32 of the opening 32. The distance h being preferably smaller than a height of the impeller blade 34 measured at the opening 32. If for instance, the impellers are arranged so that the height h equal zero, substantially no pumping action (towards a downstream chamber) occurs and the impeller substantially only provides a agitation action.

It is also noted that in the exemplified embodiment of fig. 7, the funnel shaped wall 28 is illustrated as not having a substantial thickness (compared to the height of the impeller blade 34); however, if the wall 28 is designed with a substantial thickness thereby defining an upper and lower edge, the height h is considered to be the height of the impeller blade 34 at the lower edge of the wall 28. It is noted that although it is preferred that all such impellers are arranged identical in each of the openings 32, the impellers may be arranged at different vertical heights h in each opening 32.

It is noted that fig. 7 in the magnified view also indicates a clearance c. This clearance is typically selected so as to allow the impeller to avoid mechanical contact with the funnel shaped wall 28.

Thus, one or more of the agitators 20 / impellers are preferably each arranged in one of said openings 32, so as to provide an agitation of material flowing from one chamber 30 to a downstream chamber 30 through the opening 32. Typically, an incubator according to preferred embodiments of the invention, has a total volume larger than 50 m3, such as larger than 100m3, preferably larger than 150m3, such as larger than 200m3.

The incubator may preferably be made, partly or fully, from stainless steel, but other materials, such as coated steel, coated stainless steel, polymers and fibre-reinforced polymers may also be used in the construction of the incubator.

If the incubator is made partly or fully from coated metal, the coating may include fused glass, fused flourpolymers or a thermoset coating system based on epoxy, polyurethane, vinylesther or polyester. If the incubator is made from a polymer, the polymer is preferably a polyolefine preferably chosen between polypropylene or polyethylene.

If the incubator is made partly or fully from a fibre-reinforced polymer, the reinforcing fibres 5 are preferably glass or basalt, and the matrix material is preferably chosen from the group of epoxies, vinylesters or polyesters.

Preferably, an incubator according to the present invention may comprise a number of fluidic serially connected chambers 30 being two, such as three, preferably four, such as 10 five, and even preferably six fluidic serially connected chambers 30.

Preferably, all of the fluidic serially connected chambers 30 have the same volume.

Reference is made to fig. 3 schematically illustrating a screen unit with an build-in incubator/space V. As illustrated the screen unit/incubator comprising at the lower end, a 15 screening element 14 and above that a space V. Inside the space baffle plates are arranged to avoid short-cut in the fluid flow from the upper end (receiving in the disclosed embodiment of fig. 3 fractions f1 and s3) towards the screening element 14. As also illustrated, the fiberless stream s2 is screened out providing a fiber containing fraction f2.

The enzyme used in releasing the starch from the corn kernel mass typically has a thermal 20 window inside which the release of enzyme is most efficient and it may therefore be advantageous to be able to control the temperature at selected positions in the system, such as in the space V. To this, a system according to the present invention may preferably comprising thermo elements for providing an incubation temperature of the fluid inside said space (V), preferably in the range 35-70°C, such of 40-60°C, such as in the range of 46- 25 48°C, such as in the range of 45-49°C, such as in the range of 45-48°C, such as 47C. In the embodiment where the space is provided as a separate incubation unit (as in fig. 2) the thermo elements may be arranged inside the incubation unit and/or on a shell defining the enclosure of the incubation unit.

The thermo elements are preferably thermostat-able heating/cooling elements being 30 adapted to measure the temperature and change the heat flux into/out from the space to control the temperature of material contained in the space to be within a predefined range.

In some preferred embodiments, the thermostat-able heating elements comprising electrical heating/cooling elements or liquid heating/cooling elements and temperature sensors. As presented herein, the screen unit provides a separation of fluid into two fractions s and f and the screen unit typically screens in a mechanical manner where one or more, such as all the screen units, comprises one or more screening element having openings (as illustrated e.g. in fig. 2 with a slanted, dotted lines) configured for allowing passage of solid matter below a predefined size. The predefined size may be defined according to a number of design criteria. However, it typically preferred than no fiber is allowed to pass through the opening. On the other hand to small opening may have a tendency to become blocked and in many instances the actual size of openings is selected by taking the blocking aspect and the screening aspect into consideration, which may result in that smaller amounts of fibers are allowed to pass through.

In some preferred embodiments, one or more such as all screen units comprises rotor blade and/or sieves configured for providing said two fractions s, f. As an alternative to screening elements made up by openings one or more such as all screen units may be hydro-cyclones 16 as illustrated schematically in fig. 4.

As disclosed above, the system is configured to introduce hydrolytic enzymes into said first fraction s and/or into said second fraction f and/or into a mixed first and second fraction and/or into the process water, by means of a dosing device 10 - see fig. 2.

Such a dosing device 10 is typically adapted to provide a controllable dosing quantity of enzymes, preferably according to a predetermined specific ratio between amount of enzymes and infeed of corn kernel mass to the system. To accomplish this, the dosing device 10 could be a metering pump as illustrated by a piston pump in fig. 2.

Alternatively, the dosing device 10 may be a gravity flow dispenser having a controllable outflow valve configured for controlling the amount of enzyme flowing through the flow valve.

In one aspect the present invention relates to a method to improve starch yield and/or gluten yield from corn kernels in a wet milling process, comprising the steps of:

a) soaking the kernels in water to produce soaked kernels;

b) grinding the soaked kernels;

c) separating the germs from the ground and soaked kernels to produce a corn kernel mass comprising fiber, starch and gluten; and

d) subjecting the resultant corn kernel mass, to a fiber washing procedure;

wherein during step d) one or more fractions of the corn kernel mass is/are contacted with an effective amount of one or more hydrolytic enzymes, and step d) has a total retention time of at least 35 minutes, such as at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, at least 65 minutes, at least 70 minutes, at least 75 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 105 minutes, at least 1 10 minutes, at least 115 minutes, at least 120 minutes.

The total retention time in the fiber washing procedure may be between 35 minutes and 5 hours, such as between 35 minutes and 4 hours, 35 minutes and 3 hours, 35 minutes and 2,5 hours, 35 minutes and 2 hours, 35 minutes and 1 ,5 hour, 45 minutes and 5 hours, 45 minutes and 4 hours, 45 minutes and 3 hours, 45 minutes and 2,5 hours, 45 minutes and 2 hours, 1 hour and 5 hours, 1 hour and 4 hours, 1 hour and 3 hours, 1 hour and 2,5 hours, 1 hour and 2 hours, 70 minutes and 5 hours, 70 minutes and 4 hours, 70 minutes and 3 hours, 70 minutes and 2,5 hours, 70 minutes and 2 hours, 75 minutes and 5 hours, 75 minutes and 4 hours, 75 minutes and 3 hours, 75 minutes and 2,5 hours, 75 minutes and 2 hours, 80 minutes and 5 hours, 80 minutes and 4 hours, 80 minutes and 3 hours, 80 minutes and 2,5 hours, 80 minutes and 2 hours, 85 minutes and 5 hours, 85 minutes and 4 hours, 85 minutes and 3 hours, 85 minutes and 2,5 hours, 85 minutes and 2 hours, 90 minutes and 5 hours, 90 minutes and 4 hours, 90 minutes and 3 hours, 90 minutes and 2,5 hours, 90 minutes and 2 hours.

The one or more fractions of the corn kernel mass may be a first fraction (s), a second fraction (f), or a mixed first and second fraction as defined above.

In the fiber washing procedure, it can be difficult to obtain a retention time that allows the one or more hydrolytic enzymes to work optimally. Hence, in one embodiment, a space (V) is configured into the fiber washing procedure, to provide said total retention time of at least 45 minutes, such as at least 50 minutes, at least 55 minutes, at least 60 minutes, at least 65 minutes, at least 70 minutes, at least 75 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 105 minutes, at least 1 10 minutes, at least 115 minutes, at least 120 minutes, in which said one or more hydrolytic enzymes is/are in contact with one or more fractions of said corn kernel mass.

The total retention time in the fiber washing procedure may be between 35 minutes and 5 hours, such as between 35 minutes and 4 hours, 35 minutes and 3 hours, 35 minutes and 2,5 hours, 35 minutes and 2 hours, 35 minutes and 1 ,5 hour, 45 minutes and 5 hours, 45 minutes and 4 hours, 45 minutes and 3 hours, 45 minutes and 2,5 hours, 45 minutes and 2 hours, 1 hour and 5 hours, 1 hour and 4 hours, 1 hour and 3 hours, 1 hour and 2,5 hours, 1 hour and 2 hours, 70 minutes and 5 hours, 70 minutes and 4 hours, 70 minutes and 3 hours, 70 minutes and 2,5 hours, 70 minutes and 2 hours, 75 minutes and 5 hours, 75 minutes and 4 hours, 75 minutes and 3 hours, 75 minutes and 2,5 hours, 75 minutes and 2 hours, 80 minutes and 5 hours, 80 minutes and 4 hours, 80 minutes and 3 hours, 80 minutes and 2,5 hours, 80 minutes and 2 hours, 85 minutes and 5 hours, 85 minutes and 4 hours, 85 minutes and 3 hours, 85 minutes and 2,5 hours, 85 minutes and 2 hours, 90 minutes and 5 hours, 90 minutes and 4 hours, 90 minutes and 3 hours, 90 minutes and 2,5 hours, 90 minutes and 2 hours.

The total retention time, is the time period in which the corn kernel mass, received in the first screen unit (S1) and one or more fractions thereof, 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 corn kernel mass is incubated with one or more hydrolytic enzymes in a space (V), before it leaves the fiber washing system, as part of a first fraction (s1) from the most upstream screen unit (S1) or as part of a second fraction (f4) from the most downstream screen unit (S4).

Preferably, all of the second fractions will be in contact with the one or more hydrolytic enzymes through the whole fiber washing procedure, but the enzyme concentration will vary in the different fractions (f).

In one embodiment, the incubation time in said space configured into the fiber washing procedure is at least 5 minutes, such as 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.

The incubation time in the space (V) configured into the fiber washing procedure may be less than 24 hours, such as less than 12 hours, less than 8 hours, less than 5 hours, less than 3 hours, less than 2,5 hours, less than 2 hours, less than 1 ,5 hours, less than 1 hour.

Preferably, the incubation time in said space (V) is in the range of 0.5-3 hours, such as 1-3 hours, 1-2 hours or such as 85-95 minutes.

The enzymes can be dosed into a screen unit, in a connection point between two screen units, in a space (V) configured into the fiber washing system or in the process water. Preferably, the one or more hydrolytic enzymes is dosed continuous, upstream of the most downstream screen unit and downstream of the most upstream screen unit, using a dosing device with a controllable flow rate.

According to preferred embodiments of the invention, the incubation time in said space V and/or total retention time in the fiber washing procedure provides reduced foam formation in the fiber washing process. Preferably, the formation of foam is reduced by at least 90% (v/v), such as by at least 80% (v/v), such as by at least 70%(v/v), such as by at least 60% (v/v), such as by at least 50% (v/v), such as by at least 30%(v/v), or such as by at least 20% (v/v).

In one embodiment, the fiber washing procedure comprises 2-8 fiber washing steps.

According to embodiments wherein the fiber washing system comprise 2 screen units, dosing is preferred between the first and second screen unit or in a space configured between screen unit 1 and screen unit 2.

According to embodiments wherein the fiber washing system comprise 3 screen units, dosing is preferred in the second screen unit or in a space configured between screen unit 1 and screen unit 3, most preferred in screen unit 2, or a space configured between screen unit 2 and 3.

According to embodiments wherein the fiber washing system comprise 4 screen units, dosing is preferred in the second or third screen unit or in a space configured between screen unit 1 and screen unit 4, most preferred in screen unit 2, or a space configured between screen unit 2 and 3.

According to embodiments wherein the fiber washing system comprise 5 screen units, dosing is preferred in the second, third or fourth screen unit, or in a space configured between screen unit 1 and screen unit 5, most preferred in screen unit 3 or a space configured between screen unit 3 and 4.

According to embodiments wherein the fiber washing system comprise 6 screen units, dosing is preferred in the second, third, fourth or fifth screen unit, or in a space configured between screen unit 1 and screen unit 6, most preferred in screen unit 4, or a space configured between screen unit 4 and 5.

According to embodiments wherein the fiber washing system comprise 7 screen units, dosing is preferred in the second, third, fourth, fifth or sixth screen unit, or in a space configured between screen unit 1 and screen unit 7, most preferred in screen unit 4 or a space configured between screen unit 4 and 5.

According to embodiments wherein the fiber washing system comprise 8 screen units, dosing is preferred in the second, third, fourth, fifth, sixth and seventh screen unit, or in a space configured between screen unit 1 and screen unit 8, most preferred in screen unit 5 or a space configured between screen unit 5 and 6.

In one embodiment, a fiber washing step comprises passing a stream of corn kernel mass and liquid/a liquid suspension, such an aqueous suspension, of corn kernel mass through a screen unit configured for separating said stream of corn kernel mass and liquid into two fractions: a first fraction s and a second fraction f, said second fraction f containing a higher amount of wt% fiber than the first fraction.

Some material from the corn kernel mass received in the first screening unit (S1) will go through all screening units in the fiber washing system, the number of screen units depending on the specific mill, and may thus be part of more than one second fraction and/or become part of more than one first fraction, while other material from the corn kernel mass will not go through all the screen units. These fractions will become part of one or more of a first fraction and/or one or more of a second fraction, but not all second fractions. Preferably, the material from the corn kernel mass going through all screen units in the fiber washing procedure, comprise mostly fiber.

Preferably, the material from the corn kernel mass that are not going through all of the screen units in the fiber washing procedure, comprise mostly starch and/or gluten.

In one embodiment, the wt% of starch and/or gluten is reduced in a downstream second fraction (f4) compared to an upstream second fraction (f1).

The wt% of starch and/or gluten may be higher in an upstream first fraction (s1) compared to a downstream first fraction (s4).

In addition to observing the benefits of dosed enzymes correctly in the fiber washing procedure and with the appropriate retention time, the inventors also observed that the release of insolubles from the fibers: As shown in Figure 9 herein, he most efficient release insoluble was achieved when the relative amount of fiber in the reaction corresponded to approximately 7,5-10% dry solids (DS).

Hence, the invention provides in particular embodiments a method as described above, wherein a fraction of the corn kernel mass is contacted with an effective amount of said one or more hydrolytic enzymes, said fraction containing an amount of fiber, which corresponds to 2-15% (w/w) dry solids (DS), such as to 5-15% (w/w) DS, to 5-12% (w/w) dry solids (DS), to 5-10% (w/w) dry solids (DS), to 7.5-12.5% (w/w) DS, to 8-12% (w/w) DS or to 9-11 % dry solids (DS).

The amount of dry solids may in particular be determined in a sample of said one or more fractions of the corn kernel mass, taken from a space (V) or incubator unit as defined above, or from an inlet into said space (V) or incubator unit, such as through an opening in space (V) or incubator unit or in said inlet.

The inlet may be an inlet that connects space (V) or said incubator unit with a upstream screening unit (S), which is the closest upstream screening unit to space (V) or to said incubator. Hence, it is understood that, preferably, the sample is preferable taken from the fraction or fractions that are delivered to the space (V) or incubator unit disclosed above in such a way that the content of dry solids determined in the sample is a direct measure of the amount of dry solids present in the fraction or fractions which enter the space (V) or incubator and are contacted with the one or more hydrolytic enzymes.

In particular, the amount of dry solids may be determined by:

i) Providing a sample as defined as defined above, having a wet weight of 100 g; ii) Washing the sample in 5 L distilled water and passing it through a 250 μηι sieve; iii) Drying the solids, which are retained by the sieve, overnight at 50°C;

iv) Determining the weight of the dried solids and calculating the % dry solids (DS) according to the formula:

Figure imgf000031_0001

In one embodiment, the one or more of said hydrolytic enzymes is/are 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 alpha-L-arabinofuranosidases); cellobiohydrolase I (EC 3.2.1.150), cellobiohydrolase II (E.C. 3.2.1.91), cellobiosidase (E.C. 3.2.1.176), beta- glucosidase (E.C. 3.2.1.21), and beta-xylosidases (EC 3.2.1.37).

In one embodiment, the one or more of said hydrolytic enzymes is expressed in an organism with a cellulase background, such as Trichoderma reesei.

An organism with a cellulase background, should be understood as an organism naturally expressing one or more cellulytic enzymes.

In one embodiment, the one or more of said hydrolytic enzymes comprise a xylanase, which is a GH10 xylanase.

In one embodiment, the one or more of said hydrolytic enzymes comprise a xylanase, which is a GH1 1 xylanase. In one embodiment, the one or more of said hydrolytic enzymes comprise an arabinofuranosidase, which is a GH62 arabinofuranosidase.

In one embodiment, the one or more of said hydrolytic enzymes comprise a GH10 polypeptide with xylanase activity, which is selected from the group consisting of

i) An amino acid sequence as set forth in any one of SEQ ID NOs: 22-26 ii) An amino acid sequence which has at least 60%, such as 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% identity to any one of SEQ ID NOs: 22-26; and iii) A subsequence of any one of the amino acid sequences in i) and ii).

In one embodiment, the one or more of said hydrolytic enzymes comprise a GH1 1 polypeptide with xylanase activity, which is selected from the group consisting of

i) An amino acid sequence as set forth in any one of SEQ ID NOs: 27-35 ii) An amino acid sequence which has at least 60%, such as 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% identity to any one of SEQ ID NOs: 27-35; and iii) A subsequence of any one of the amino acid sequences in i) and ii).

In one embodiment, the one or more of said hydrolytic enzymes comprise a GH62 polypeptide with arabinofuranosidase activity, which is selected from the group consisting of:

iv) An amino acid sequence as set forth in any one of SEQ ID NOs: 1-21 v) An amino acid sequence which has at least 60%, such as 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% identity to any one of the amino acid sequences set forth in SEQ ID NO: 1-21 ; and

vi) A subsequence of any one of the amino acid sequences in iv) and v).

The subsequences defined above, may be subsequences in which 30 amino acid residues or less, such as 25 amino acid residues or less, 20 amino acid residues or less, 15 amino acid residues or less, 10 amino acid residues or less, or 5 amino acid residues or less have been deleted.

In other embodiments, the amino acid sequences in items ii) and/or v) above has a length, which in 75% or more, such as 80% or more, 90% or more, 95% or more, or 98% or more of the length of the sequence set forth under the respective SEQ ID NO.

In one embodiment, the one or more of said hydrolytic enzymes is expressed in Trichoderma reesei and comprise a xylanase, which is a GH10 xylanase or a GH1 1 xylanase and a arabinofuranosidase, which is a GH62 arabinofuranosidase. In one embodiment, the effective amount of one or more hydrolytic enzymes contacted with one or more fractions of the corn kernel mass does not exceed 0.5, 0.4, 0.3, 0.2 kg enzyme protein/metric tonne (MT) corn kernel mass.

In one embodiment, the effective amount of one or more hydrolytic enzymes contacted with one or more fractions of said corn kernel mass is between 0.010-0.5 kg/MT corn kernel mass, such as between 0.05-0.5 kg/MT corn kernel mass or 0.075-0.5 kg/MT or 0.1-0.5 kg/MT corn kernel mass or 0.01-0.4 kg/MT corn kernel mass or 0.05-0.4 kg/MT corn kernel mass or 0.075-0.4 kg/MT corn kernel mass or 0.1-0.4 kg/MT corn kernel mass or 0.01-0.3 kg/MT corn kernel mass or 0.05-0.3 kg/MT corn kernel mass or 0.075-0.3 kg/MT or 0.1-0.3 kg/MT corn kernel mass or 0.010-0.2 kg/MT corn kernel mass or 0.05-0.2 kg/MT corn kernel mass or 0.075-0.2 kg/MT or 0.1-0.2 kg/MT corn kernel mass or such as 0.075-0.10 kg/MT corn kernel mass or 0.075-0.1 1 kg/MT corn kernel mass..

In one embodiment said fiber washing procedure is performed with the use of a fiber washing system as defined above in aspects and embodiments of the invention. Hence, the fiber washing procedure may be performed with the use of a fiber washing system comprising:

a plurality of screen units (S1... S4) being fluidly connected in a counter current washing configuration; each screen unit is configured for separating a stream of corn kernel mass and liquid into two fractions: a first fraction (s) and a second fraction (f), said second fraction (f) containing a higher amount measured in wt% fiber than the first fraction (s);

a space (V) arranged in the system and being fluidly connected to receive said first fraction (s), said second fraction (f), or a mixed first and second fraction (s,f), preferably only a second fraction (f), and configured to provide an incubation time for one or both fractions received in the space; and outletting the thereby incubated one or both fractions to a downstream screen unit (S4),

wherein the system is configured for

inletting corn kernel mass and liquid to the most upstream screen unit (S1) outletting the first fraction (s1) from the most upstream screen unit (S1) as a product stream containing starch,

inletting process water, preferably arranged for inletting process water to a most downstream screen unit (S4),

outletting the second fraction (f4) from most downstream screen unit (S4) as a washed corn kernel mass containing a lower amount of starch and gluten than the original corn kernel mass. introducing hydrolytic enzymes into the system.

In one aspect, the invention is a wet milling process, which comprises the use of a fiber washing procedure as defined above in aspects and embodiments of the invention. Hence, the milling process may comprise subjecting corn kernel mass, to a fiber washing procedure 5 wherein one or more fractions of the corn kernel mass is contacted with an effective amount of one or more hydrolytic enzymes, and step d) has a total retention time of at least 35 minutes, such as at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, at least 65 minutes, at least 70 minutes, at least 75 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 105 minutes, at least 10 1 10 minutes, at least 1 15 minutes, at least 120 minutes.

In particular, the total retention time in the fiber washing procedure may be between 35 minutes and 5 hours, such as between 35 minutes and 4 hours, 35 minutes and 3 hours, 35 minutes and 2,5 hours, 35 minutes and 2 hours, 35 minutes and 1 ,5 hour, 45 minutes and 5 hours, 45 minutes and 4 hours, 45 minutes and 3 hours, 45 minutes and 2,5 hours,

15 45 minutes and 2 hours, 1 hour and 5 hours, 1 hour and 4 hours, 1 hour and 3 hours, 1 hour and 2,5 hours, 1 hour and 2 hours, 70 minutes and 5 hours, 70 minutes and 4 hours, 70 minutes and 3 hours, 70 minutes and 2,5 hours, 70 minutes and 2 hours, 75 minutes and 5 hours, 75 minutes and 4 hours, 75 minutes and 3 hours, 75 minutes and 2,5 hours, 75 minutes and 2 hours, 80 minutes and 5 hours, 80 minutes and 4 hours, 80 minutes and

20 3 hours, 80 minutes and 2,5 hours, 80 minutes and 2 hours, 85 minutes and 5 hours, 85 minutes and 4 hours, 85 minutes and 3 hours, 85 minutes and 2,5 hours, 85 minutes and 2 hours, 90 minutes and 5 hours, 90 minutes and 4 hours, 90 minutes and 3 hours, 90 minutes and 2,5 hours, 90 minutes and 2 hours.

The invention further comprises a composition comprising corn starch, said composition 25 being obtainable by the wet milling process defined above.

Finally, the invention provides a composition comprising corn gluten, said composition being obtainable by the wet milling process defined above.

The invention is further summarized in the following paragraphs:

30 1. A fiber washing system comprising a plurality of screen units (S1 ... S4) being fluidly connected in a counter current washing configuration; each screen unit is configured for separating a stream of corn kernel mass and liquid into two fractions: a first fraction (s) and a second fraction (f), said second fraction (f) containing a higher amount measured in wt% fiber than the first fraction (s);

a space (V) arranged in the system and being fluidly connected to receive one of said first fraction (s), one of said second fraction (f), or a mixed first and second fraction (s,f), preferably only a second fraction (f), and configured to provide an incubation time for one or both fractions received in the space; and outletting the thereby incubated one or both fractions to a downstream screen unit (S4), wherein the system is configured for

inletting corn kernel mass and liquid to the most upstream screen unit (S1) - outletting the first fraction (s1) from the most upstream screen unit (S1) as a product stream containing starch,

inletting process water, preferably arranged for inletting process water to a most downstream screen unit (S4),

outletting the second fraction (f4) from most downstream screen unit (S4) as a washed corn kernel mass containing a lower amount of starch and gluten than the original corn kernel mass,

introducing hydrolytic enzymes into the system.

2. The system according to paragraph 1 , wherein the system is configured for introducing said hydrolytic enzymes into said first fraction (s), and/or into said second fraction (f), and/or into a mixed first and second fraction and/or into the stream of process water supplied to the system.

3. The system according to paragraphs 1 or 2, wherein the system comprise a most upstream screen unit (S1... S4), a most downstream screen unit and preferably one or more intermediate screen units fluidic arranged inbetween the most upstream and most downstream screen units (S1 , S4).

4. A fiber washing system according to any of paragraphs 1-3, wherein said fluidly connected counter current washing configuration, comprising the plurality of screen units

(S1... S4), is arranged in a manner so:

a second fraction (f1) produced by an upstream screen unit (S1) is mixed with a first fraction (s3) produced by a downstream screen unit (S3), and said mixed fractions being separated by a screen unit (S2), being intermediate between said upstream and said downstream screen units (S1 , S3), into a first fraction (s2) and a second fraction (f2).

5. A fiber washing system according to any of claims 1-4, wherein mixing of said second fraction (f1) and said first fraction (s3) occurs prior to being inlet into said intermediate screen unit (S2).

6 A fiber washing system according to any of paragraphs 1-4, wherein mixing of said second fraction (f1) and said first fraction(s3) occurs inside said intermediate screen unit (S2).

7. The system according to any of paragraphs 1-3, comprising 2-8 screen units.

8 The system according to any of the preceding paragraphs, wherein the system is configured to provide a total retention time in said fiber washing system of at least 35 minutes, 45 minutes, 1.5 hours, such as at least 3 hours during which the one or more hydrolytic enzymes is/are in contact with said corn kernel mass and/or one or more fractions thereof. 9. The system according to any of the preceding paragraphs, wherein said space (V) is configured to provide an incubation time of at least 5 minutes, preferably at least 15 minutes, such as at least 30 minutes, preferably at least 45 minutes, such as at least 1 hours, and preferably less than 24 hours, such as less than 12 hours. 10. The system according to any of the preceding paragraphs, wherein the fluid received in said space (V) is not screened during said incubation time.

11. The system according to any of the preceding paragraphs, wherein said space (V) is configured for agitating matter contained in said space (V), such as by comprising a rotor or impeller.

12. The system according to any of the preceding paragraphs, wherein said space (V) is arranged downstream of said most upstream screen unit (S1) and upstream of said most downstream screen unit (S4). 13. The system according to any of the preceding paragraphs, wherein said space is provided as a separate incubator unit (14) configured to receive said first fraction (s), said second fraction (f) or a combination of a first and a second fraction (s,f), preferably only a second fraction (f), and deliver the incubated material to a downstream screen unit (S3).

14. The system according to paragraph 13, wherein the incubator (14) comprise a number of fluidic serially connected chambers (30) by an opening (32) forming a passage between two neighbouring chambers (30) through which opening (32) material may flow from an upstream to a downstream chamber (30), wherein

- a most upstream chamber (30) being connected to receive said first fraction (s), said second fraction (f) or a combination of a first and a second fraction (s,f), and a most downstream chamber (30) having an outlet connection (26) adapted to deliver incubated material out from the incubator (14);

the incubator (14) further comprising one or more agitators (20) configured for preventing solid decantation in the incubator (14).

15. The system according to paragraph 14, wherein the incubator (14) comprise a number of funnel shaped walls (28) disposed horizontally distanced inside the incubator (14) to divide the interior of the incubator (14) into chambers (30) and funnelling material downwardly from one chamber to a downstream chamber (30).

16. The system according to paragraph 15, wherein the funnel shaped walls (28) each comprise said opening (32) geometrical centred at the smallest cross section of each of the funnel shaped wall.

17. The system according to any of the preceding paragraphs 14-16, wherein the incubator comprise a sloping bottom wall (36) sloping towards an inlet of said outlet connection (26).

18. The system according to any of the preceding paragraphs 14-17, wherein said outlet connection (26) is arranged so as to outlet material from the incubator (14) at the bottom thereof.

19. The system according to any of the preceding paragraphs 14-18, wherein said outlet connection (26) being in a form of a tube extending internally inside the incubator (14) from a position at the top and outside of the incubator (14) and to the bottom of the most downstream positioned chamber (30).

20. The system according to any of the preceding paragraphs 14-19, wherein one or more, such as all, of the agitators (20) are impellers comprising a plurality of impeller blades (34), the incubator (14) further comprising a shaft (18) and a motor (22) configured for rotating the shaft (14) and wherein the impellers are arranged on said shaft (14) so that rotation of the shaft (14) provides a rotation of the impellers. 21. The system according to paragraph 20, wherein the impellers are configured to provide a pumping action pumping material through the incubator (14).

22. The system according to paragraphs 20 or 21 , wherein each of the impellers arranged in one of said openings (32) are arranged in a position where a lower end of the impeller being disposed a distance (h) below an edge (32) of the opening (32), said distance being preferably smaller than a height of the impeller blade (34) measured at the opening (32).

23. The system according to any of the preceding paragraphs 14-22, wherein one of said agitators (20) each are arranged in one of said openings (32), so as to provide an agitation of material flowing from one chamber (30) to a downstream chamber (30) through the opening (32).

24. The system according to any of the preceding paragraphs 14-23, wherein the incubator has a total volume larger than 50 m3, such as larger than 100m3, preferably larger than 150m3, such as larger than 200m3.

25. The system according to any of the preceding paragraphs 14-24, wherein the number of fluidic serially connected chambers (30) are two, such as three, preferably four, such as five, and even preferably six.

26. The system according to any of the preceding paragraphs 14-25, wherein all of the fluidic serially connected chambers (30) have the same volume.

27. The system according to any paragraph 1-1 1 , wherein said space is provided internally in a screen unit. 28. The system according to any of the preceding paragraphs, further comprising thermo elements for providing an incubation temperature of the fluid inside said space (V), preferably in the range 35-70C, such of 40-60°C, such as in the range of 46-48°C, such as in the range of 45-49°C, such as in the range of 45-48°C, such as 47C.

29. The system according to paragraph 28, wherein the thermo elements are thermostat- able heating/cooling elements being adapted to measure the temperature and change the heat flux into/out from the space to control the temperature of material contained in the space to be within a predefined range.

30. The system according to paragraph 28 or 29, wherein the thermostat-able heating elements comprising electrical heating/cooling elements or liquid heating/cooling elements and temperature sensors.

31. The system according to any of the preceding paragraphs, wherein one or more such as each of the screen units (S1...S4) comprising one or more screening element (22) having openings configured for allowing passage of solid matter below a predefined size. 32. The system according to any of the preceding paragraphs where one or more such as all screen unit comprising rotor blade and/or sieves configured for providing said two fractions (s, f).

33. The system according to any of the preceding paragraphs, wherein one or more such as all screen units are hydro-cyclones (16).

34. The system according to any of the preceding paragraphs, wherein the system is configured to introduce hydrolytic enzymes into said first fraction (s) and/or into said second fraction (f) and/or into a mixed first and second fraction and/or into the process water, by means of a dosing device (10).

35. The system according to any of the preceding paragraphs, wherein said dosing device (10) is adapted to provide a controllable dosing quantity of enzymes, preferably according to a predetermined specific ratio between amount of enzymes and infeed of corn kernel mass to the system. 36. The system according to paragraphs 34 or 35, wherein the dosing device (10) is a metering pump. 37. The system according to paragraphs 34 or 35, wherein the dosing device (10) is a gravity flow dispenser having a controllable outflow valve configured for controlling the amount of enzyme flowing through the flow valve.

38. A crop kernel wet milling system comprising a fiber washing system as defined in any of the preceding claims.

39. A method to improve starch yield and/or gluten yield from corn kernels in a wet milling process, comprising the steps of:

a) soaking the kernels in water to produce soaked kernels;

b) grinding the soaked kernels;

c) separating germs from the ground and soaked kernels to produce a corn kernel mass comprising fiber, starch and gluten; and

d) subjecting the resultant corn kernel mass, to a fiber washing procedure;

wherein during step d) one or more fractions of the corn kernel mass is contacted with an effective amount of one or more hydrolytic enzymes, and step d) has a total retention time of at least 45 minutes.

40. The method according to paragraph 39, wherein said one or more fractions of the corn kernel mass is a first fraction (s), a second fraction (f), or a mixed first and second fraction as defined in any of claims 1- 37.

41. The method according to paragraph 39 or 40, wherein a space (V) is configured into the fiber washing procedure, to provide said total retention time of at least 45 minutes in which said one or more hydrolytic enzymes is/are in contact with one or more fractions of said corn kernel mass.

42. The method according to any of paragraphs 39-41 , wherein the incubation time in said space (V) configured into the fiber washing procedure is at least 5 minutes and less than 24 hours. 43. The method according to any of the preceding paragraphs 39-42, wherein said fiber washing procedure comprises 2-8 fiber washing steps.

44. The method according to any of paragraphs 39-43, wherein a fiber washing step 5 comprises passing a stream of corn kernel mass and liquid through a screen unit configured for separating a stream of corn kernel mass and liquid into two fractions: a first fraction (s) and a second fraction (f), said second fraction (f) containg a higher amount of wt% fiber than the first fraction.

45. The method according to any of paragraphs 39-44, wherein the wt% of starch and/or gluten is reduced in a downstream second fraction (f4) compared to an upstream second fraction (f1).

46. The method according to any of paragraphs 39-45, wherein a fraction of the corn kernel mass is contacted with an effective amount of said one or more hydrolytic enzymes, said fraction containing an amount of fiber, which corresponds to 2-15% (w/w) dry solids (DS), such as to 5-15% (w/w) DS, to 5-12% (w/w) dry solids (DS), to 5-10% (w/w) dry solids (DS), to 7.5-12.5% (w/w) DS, to 8-12% (w/w) DS or to 9-1 1 % dry solids (DS).

20 47. The method according to paragraph 46, wherein the amount of dry solids is determined in a sample of said one or more fractions of the corn kernel mass, taken from a space (V) or incubator unit as defined in any of claims 1-37 or from an inlet into said space (V) or incubator unit, such as through an opening in space (V) or incubator unit or in said inlet.

25 48. The method according to paragraph 47, wherein said inlet connects space (V) or said incubator unit with a upstream screening unit (S), which is the closest upstream screening unit to space (V) or to said incubator.

49. The method according to any of paragraphs 46-48, wherein the amount of dry solids 30 are determined by:

i) Providing a sample as defined in claim 46 or 47, having a wet weight of 100 g; ii) Washing the sample in 5 L distilled water and passing it through a 250 μηι sieve; iii) Drying the solids, which are retained by the sieve, overnight at 50°C;

iv) Determining the weight of the dried solids and calculating the % dry solids (DS) 35 according to the formula: weiqht of dried solids\

%DS = -— — xlOO

\ wet weight of sample J

50. The method according to any of paragraphs 39-49 , wherein one or more of said hydrolytic enzymes is/are 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 alpha-L- arabinofuranosidases); EC 3.2.1.185 (Non-reducing end beta-L-arabinofuranosidases) cellobiohydrolase I (EC 3.2.1.150), cellobiohydrolase II (E.C. 3.2.1.91), cellobiosidase (E.C. 3.2.1.176), beta-glucosidase (E.C. 3.2.1.21), beta-xylosidases (EC 3.2.1.37).

51. The method according to any of paragraphs 39-50, wherein the one or more of said hydrolytic enzymes is expressed in an organism with a cellulase background, such as Trichoderma reesei.

52. The method according to any of paragraphs 39-52, wherein the one or more of said hydrolytic enzymes comprise a xylanase, which is a GH 10 xylanase

53. The method according to any of paragraphs 39-52, wherein the one or more of said hydrolytic enzymes comprise an arabinofuranosidase, which is a GH62 arabinofuranosidase.

54. The method according to any of paragraphs 39-53, wherein the one or more of said hydrolytic enzymes comprise a GH10 polypeptide with xylanase activity, which is selected from the group consisting of

i) An amino acid sequence as set forth in any one of SEQ ID NOs: 22-26;

ii) An amino acid sequence which has at least 80% identity to any one of SEQ ID NOs: 22-26; and

iii) A subsequence of any one of the amino acid sequences in i) and ii).

55. The method according to any of paragraphs 39-54, wherein the one or more of said hydrolytic enzymes comprise a GH11 polypeptide with xylanase activity, which is selected from the group consisting of

i) An amino acid sequence as set forth in any one of SEQ ID NOs: 27-35 ii) An amino acid sequence which has at least 80% identity to any one of SEQ ID NOs: 27-35; and iii) A subsequence of any one of the amino acid sequences in i) and ii).

56. The method according to any of paragraphs 39-55, wherein the one or more of said hydrolytic enzymes comprise a GH62 polypeptide with arabinofuranosidase activity, which

5 is selected from the group consisting of:

i) An amino acid sequence as set forth in any one of SEQ ID NOs: 1-21 ii) An amino acid sequence which has at least 80% identity to any one of the amino acid sequences set forth in SEQ ID NO: 1-21 ; and

iii) A subsequence of any one of the amino acid sequences in iv) and v).

10

57. The method according to any of paragraphs 39-56, wherein the one or more of said hydrolytic enzymes is expressed in Trichoderma reesei and comprise a xylanase, which is a GH 10 xylanase and a arabinofuranosidase, which is a GH62 arabinofuranosidase.

15 58. The method according to any of paragraphs 39-57, wherein the effective amount of one or more hydrolytic enzymes contacted with one or more fractions of the corn kernel mass does not exceed 0,5 kg enzyme protein/metric tonne corn kernel mass.

59. The method according to any of paragraphs 39-58, wherein the effective amount of one 20 or more hydrolytic enzymes contacted with one or more fractions of said corn kernel mass, is between 0.010-0.5 kg/metric tonne corn kernel mass.

60. The method according to any of preceding paragraphs 39-59, wherein said fiber washing procedure is performed with the use of a system as defined in any one of

25 paragraphs 1-38.

61. A wet milling process, which comprises the use of a fiber washing procedure as defined in any of paragraphs 39-60.

30 62. A composition comprising corn starch, said composition being obtainable by the wet milling process defined in paragraph 61.

63. A composition comprising corn gluten, said composition being obtainable by the wet milling process defined in paragraph 61.

35 Although the present invention has been described in connection with the specified embodiments, it should not be construed as being in any way limited to the presented examples. The scope of the present invention is set out by the accompanying claim set. In the context of the claims, the terms "comprising" or "comprises" do not exclude other possible elements or steps. Also, the mentioning of references such as "a" or "an" etc. should not be construed as excluding a plurality. The use of reference signs in the claims with respect to elements indicated in the figures shall also not be construed as limiting the scope of the invention. Furthermore, individual features mentioned in different claims, may possibly be advantageously combined, and the mentioning of these features in different claims does not exclude that a combination of features is not possible and advantageous.

EXAMPLES

Example 1 : The optimal enzyme dosing point in wet milling

Because of short residence times in the wet milling process, selecting the optimum location to dose enzymes is critical for maximizing contact between enzyme and substrate. This means achieving the highest enzyme concentration at a given dose rate within the process sections where the substrate is most available. However, this determination is not easily done because of numerous splitting and recycling of streams in the process. To help provide an answer for the optimal dosing point, a process simulation was conducted.

Method:

1. The model simulation was done in SuperPro Designer V.9 (evaluation version). The USDA model represents a 100,000 bu/day wet mill plant, including all unit operations. No change was made to the model, except to introduce the enzyme at four different points in the process, illustrated in figure 5. These dose points, referred to as cases, are (1) after 2nd grind, (2) after 3rd grind, (3) after 4th fiber washing step, and (4) after 6th fiber washing step (last wash stage before fiber press). The enzyme feed rate was pegged at a dose of 0.20 kg/MT corn.

2. A determining parameter for the simulation is the enzyme's partitioning characteristic. In this case, the simple assumption was taken that the enzyme behaves just like a soluble solid. This assumption is supported by data in figure 6 showing close to 100% enzyme recovery in the filtrate, during laboratory fiber incubation. The partitioning of soluble components in the model was mainly based on empirical data on solubles and moisture of some of the streams coming out of separation units, such as centrifuges and screens (Reference: Ramirez et al. 2008; Industrial Crops and Products 27; 91-97), with the rest approximated using the model.

Taking the above parameters as-is from the USDA model, the mass balance was solved using Superpro (energy balance was ignored in this case). The mass balance results were checked for accuracy after convergence, as shown in table 1 for one of the cases simulated. Once the mass balance was solved, the mass flowrates (at steady state) of the different intermediate streams are obtained as output. These mass flowrates are the needed data to calculate an effective dose of enzyme (or ratio of enzyme to fiber solids) at the different stages of the fiber wash segment.

Figure imgf000045_0001

Table 1 : Overall component balance (kg/h) Since fiber is considered the target substrate, the effective dose was taken as the ratio of enzyme to fiber within the relevant segment of the process. For simplicity, this segment is taken to begin at the second grind and end at the last fiber washing step just before fiber pressing. This would include every step of the process where relevant substrate-enzyme contact is expected to take place. A weighted averaged dose is determined for the whole segment by factoring in the residence times of the receiving tanks, placed after each screen to the individual effective dose at every process step included. The whole segment in this case, had a total residence time of 1.28 hr. However, it should be noted that the residence time is due to the use of receiving tanks, which some plants may or may not have. Furthermore, the tank size may vary from plant to plant. However, regardless of tank size, the conclusions arrived here shouldn't change.

Results:

Table 2 below depicts weighted average effective enzyme doses (factoring in residence times) at various stages of the wet milling process, for each of the dosing cases simulated. From this it appears that dosing in the fiber washing result in higher enzyme levels compared to 2nd grind or 3rd grind. The best case suggests dosing towards the end of the fiber wash steps, but not at the last wash step.

Figure imgf000046_0001

Table 2: Weighted average enzyme cone, for the different cases (weights based residence times of the operations included).

The residual enzyme concentrations {ppm based on total product weight) under the different dosing cases are given in Table 3 below. In general, the levels are very similar for the different cases. The highest levels are found in the dry gluten feed (DGF), since most of the enzymes (>85%) end up with the steep liquor that is added back to the DGF. The starch slurry has the least, less than a tenth of a ppm, because of the extensive starch washing that takes place. This is important, since the starch stream is the likeliest place for the enzyme to retain any activity; all others will go through high temperature drying that would potentially deactivate the enzymes.

Enzyme Residual (ppm)

End Products CASE CASE CASE CASE

1 2 3 4

Starch slurry <0.1 <0.1 <0.1 <0.1

Dry Gluten Meal 218 218 207 170

Dry Gluten Feed 814 814 821 844

Dry Germ 169 168 160 131

Table 3: Residual enzyme concentrations (total weight basis) in the final product streams. Example 2. The effect of enzyme on fiber slurry viscosity

In this example, we measured the viscosity profile of fiber slurry with time using an RVA (Rapid Visco Analyzer). The fiber slurry was taken from the overflow of the 5th fiber washing stage containing 12% of dry solids. In one treatment, hydrolytic enzymes were added at a dose of 1 mg BCA proteins/ g dry solids. In another, only water of equivalent volume was added, to serve as a negative control. The total time of the RVA run was 90 minutes, and the temperature was set to 48°C. The absolute viscosity numbers are very sensitive to the consistency of the samples, and due to the inhomogeneity of the slurry, it is difficult to ensure the starting values are similar. We set the rpm of the RVA impeller at different values in order to keep the starting viscosity as close as possible between the two treatments. However, it should be noted that more important than the absolute value of the results is the change in viscosity with time.

The change in viscosity is a convenient way to follow the change in water concentration of the slurry due to enzyme treatment. In an ideal case of a Newtonian suspension, the effective viscosity is a positive function of the volume fraction of solids (Bailey, J.E. and Ollis, D.F., Biochemical Engineering Fundamentals 2nd ed, 1986, McGraw-Hill, Inc. page 502). While most likely the slurry behaves as non-Newtonian, it does not change the generality that as the solid volume fraction increases (or equivalently, the aqueous volume fraction decreases), the viscosity increases, and vice versa. So if dewatering occurs during enzyme reaction, this should cause the solid volume fraction to go down (or equivalently, the aqueous volume fraction to go up), and consequently the viscosity to go down with it.

The RVA measurements were taken continuously, and in order to summarize the results, a running average was calculated for each 10-minute duration. Each average and standard deviation calculated consist of >300 data points. Tables 4 and 5 below shows the results for the enzyme treated and control samples, respectively.

Figure imgf000047_0001
Figure imgf000048_0001

Table 4: Average viscosity (in centipoise) measured within 10-min time durations over the 90-min RVA run. With addition of hydrolytic enzymes

Figure imgf000048_0002

Table 5. Average viscosity (in centipoise) measured within 10-min time durations over the 90-min RVA run. Without addition of hydrolytic enzymes.

Very clear differences in viscosity profile emerged between enzyme treated and untreated fiber slurry. Significant reduction (by more than 50%) of viscosity during the 90 min reaction time was observed when enzyme was added. In contrast, no viscosity change was observed without enzyme addition in the fiber slurry during the same period. In addition, majority of the viscosity change in the enzyme-treated slurry occurred in the first 40 min, and then a more gradual decrease takes place from there on. (It is not as clear in the discretized averages above, but in the continuous viscosity data, the inflection point occurs close to the 40 min time point). Another striking feature is the more erratic viscosity values obtained in the enzyme-treated slurry, especially in the first 10 to 20 min, as described by the high standard deviations observed during this period, and which gradually subsided after 40 min. This was not observed in the case of the negative control. This surprising observation is likely due to a phasing-out taking place during the early time period, wherein water that is newly released from being bound by fiber has not yet equilibrated with the aqueous phase, thus resulting to a transient non-homogeneity that adds to the viscosity variation. These observations point to the notion of a rapid dewatering phase occurring during the enzyme reaction. Example 3. The effect of incubation time and enzyme dose on starch and gluten yield.

In this example, we measured the starch and gluten separated from the fiber after incubation with and without hydrolytic enzyme (mature polypeptides of SEQ ID NO: 7 and 22, expressed in Trichoderma reesei), while varying incubation time and enzyme dose. The fiber sample was obtained from a wet-mill plant after fiber pressing with a total dry matter content of 41 %. The sample was re-suspended in buffer (pH 4, 0.02M Na Acetate) to 100-g slurry containing 5% dry solids. To this slurry was added enzyme at a final ratio of 0, 2, 6, and 10-mg enzyme concentrate per g dry-solids substrate (one gram enzyme concentrate contains about 280 mg proteins). The incubation was done at 50C in an air- heated incubator with constant shaking, varying the incubation time to 10, 50 and 90 min. After incubation, the samples were cooled quickly in ice-water (5°C) before processing. The slurry was transferred on to a 100-micron sieve, while collecting the filtrate that passed through. The fiber that was retained over the sieve was pressed using a spatula to recover as much filtrate as possible. The pressed fiber was then transferred to a beaker containing 200-ml of water and stirred. This slurry was again passed through the 100-micron sieve, and the collected filtrate was combined with the first. The pressing, washing and filtering steps above were repeated once more, so that a final filtrate was recovered and combined with the first two. The combined filtrate was then vacuum filtered, this time through a glass micro filter paper (Whatman) which retained the insoluble solids that were released from the fiber and passed through the 100-micron screen. After passing 200 ml water over the filter paper to remove any trace of solubles, the total insoluble solids retained on the filter paper was dried and weighed. The dry weight was reported as Starch+Gluten released as percentage of fiber dry matter of starting substrate. The results are shown in Table 6.

Starch+Gluten Released (% of

Incubation Time (min) Dose (mg/g fiber) fiber)

0 4.98%

2 7.49%

10

6 8.54%

10 8.13%

0 6.16%

2 9.04%

50

6 12.29%

10 13.29%

0 5.04%

90 2 10.44%

6 14.02% I U I 15.1 1 % I

Table 6. Yield of starch and gluten released from fiber under different enzyme dose and incubation time.

The effect of enzyme addition is apparent from the increase in the starch and gluten yields for all incubation time. However, the response to dose is flat for an incubation time of 10 minutes, whereas there is a steep response to dose in the 50 min, and especially in the 90 min incubation. With these longer residence times, a lower dose is able to achieve the yields that only a much higher dose is capable for a shorter residence time. For example, a 6 mg dose at 90 min was able to achieve a higher yield than a 10 mg dose at 50 min. This also underlines the importance of having a minimum sufficient incubation time for the enzyme to perform, as for example an incubation of 10 min, regardless of the dose, could not attain yields of even the lowest dose (i.e., 2 mg) with longer incubation.

Example 4: Effect of increased retention time on starch and gluten yields in wet milling

Fiber washing system:

The fiber washing lay-out of the test facility consists of four screen units. An incubation tank (250m3) was installed between the second and third screen unit to allow additional retention time of the corn kernel mass or fractions thereof. The enzymes (mature polypeptides of SEQ ID NO: 7 and 22, expressed in Trichoderma reesei) were dosed in the second screen, before the holding tank. Without the additional incubation tank, the retention time (RT) in the fiber washing system was estimated to be around 10 min (based on the total capacity of the pre-existing receiving tanks). The effect of retention time was evaluated by increasing the utilization capacity of the additional tank from 0%, to 40% (100m3, adding 40 min RT), and then finally to 80% (200m3, adding 80 min RT).

Method:

The fiber was washed in a fiber washing system as described above. The trial without enzymes and incubation tank was run for 3 months. The trial without incubation tank (10 min RT), but with enzymes, was run over 4 weeks at an enzyme dose of 0,5 kg/MT corn. The experiment with incubation tank (100m3), 50 min RT (40 min incubation time) and enzymes was run over 1 week at an enzyme dose of 0,5 kg/MT corn. The experiment with incubation tank (200m3), 90 min RT (80 min incubation time) and enzymes was run over 2 weeks at an enzyme dose of 0,5 kg/MT corn.

After fiber washing, the fiber was pressed to decrease the water content before drying.

One sample of wet fiber was weighted before total starch (%) and total protein (%) in wet fiber was determined. The total starch (%) was determined by an enzymatic method established for measuring total starch in cereal products (Method 76-13, AACC International 2000). The total protein (%) was determined by the micro-Kjeldahl method of measuring crude protein (AACCI Method 46-13.01).

One sample of wet fiber was then dried and the dry fiber weight was determined, to allow calculation of total starch and total protein (%) in dry fiber.

The total starch can be divided into extractable starch (starch that can be washed off), and bound starch (starch that is stuck with the fiber even washing). The amount of starch remaining in a sample after an additional washing step in clean water (bound starch) was used as a control, to make sure that starch and protein were released from the fiber. The sample was washed with 10 L water and filtered through a 50-micron sieve to wash away extractable starch before measuring the bound starch. The process is illustrated in a flow diagram in figure 8.

The total starch (wt%) and protein (wt%) of dry fiber reflects the amount of starch and protein still present in the fiber after fiber wash, pressing and drying. This amount of starch and protein should in theory decrease as the retention time increase, as the hypothesis is that enzymes catalyse the release of starch and protein into the process stream. The effect on total starch (wt%) and total protein (wt%) of dry fiber, by change in retention time is given in table 7 below. As shown, there is a significant effect on the removal of starch and protein by the addition of the enzyme, even at the original retention time of 10 min. With increased retention time (+80 min RT) a decrease in both total starch and protein can be seen, reflecting that increased retention time in fiber washing can indeed increase the total starch and protein yields in corn wet milling.

The total moisture (wt%) of the pressed wet fiber was calculated as: the wet fiber weight minus the dry fiber weight. The moisture content decreases in the wet fiber (dewatering) with addition of enzymes, but does not seem to be effected by retention time.

Wet fiber Baseline3 Enzyme Enzyme Enzyme

(After fiber pressing) (10 min RT)b (50 min RT)C (90 min RT)d Dry fiber (%) of wet fiber 38.5 42.0 43.7 33.05

Moisture (%) of wet fiber 61.5 58.0 56.3 56.95

Total starch (%) of dry 25 85 21.85 21.2 19.25

fiber

Protein (%) of dry fiber 11.44 10.68 11.16 10.35

Bound starch (%) of dry 20.37 17.54 17.3 15.35

fiber

Table 7:

a Baseline average over 3 months without enzyme" Average over 4 weeks at 0.5 kg/MT dose (<1 week @ 1.2 kg/MT)

c Average over 1 week at 0.5 kg/MT dose

d Average over 2 weeks at 0.5 kg/MT dose

Example 5: Effect of the amount of dry solids on insolubles released form starting fiber in enzyme reaction. In this example, we measured the insoluble solids that are separated from the fiber after incubation with enzyme (Frontia® Fiberwash, commercially available from Novozymes A/S) at varying % dry solids.

The fiber sample was obtained from a wet-mill plant after the vetter press with a total dry matter content of 42.80%. The sample was re-suspended in buffer (pH 4, 0.02M Na Acetate) to a slurry containing various dry solids. To this slurry the enzyme was added at 0.9kg/MT wet corn.

The %DS was adjusted with various amounts of water as shown in the table below to achieve a range of %DS.

(Fiber dry solids / % dried solids) - Fiber weight = Water added

Figure imgf000052_0001
8,00% 2 4 13,54 5,80 123 1 ,00 59

8,00% 2 5 13,21 5,65 120 1 ,00 57

8,00% 2 6 13,34 5,71 121 1 ,00 58

12,00% 2 7 13, 14 5,62 119 1 ,00 34

12,00% 2 8 13,23 5,66 120 1 ,00 34

12,00% 2 9 13,06 5,59 118 1 ,00 34

15,00% 2 10 13,01 5,57 118 1 ,00 24

15,00% 2 11 13,06 5,59 118 1 ,00 24

15,00% 2 12 13,08 5,60 119 1 ,00 24

18,00% 2 13 13,20 5,65 120 1 ,00 18

18,00% 2 14 13, 10 5,61 119 1 ,00 18

18,00% 2 15 13,03 5,58 118 1 ,00 18

The incubation was done at 48C in a Werner Mathis AG Labomat reactor with constant mixing for 120 minutes. After incubation, the samples were cooled quickly in ice-water (5°C) before processing.

The slurry was then poured into an Omega 1000 Juicer and the variable autotransformer was set to 30% power for 1 minute. The filtrate was caught with a 75μηι screen and catch pan. The insoluble solids in the catch pan were transferred into a 500ml Nalgene bottle. The fiber was scraped out of the Omega 1000 juicer basket and washed with 200 mis of Dl water in a beaker with a spatula. The fiber slurry was then poured back into the juicer and run for 1 minutes at 30% power. Again the filtrate was poured over a 75μηι sieve and catch pan the insoluble solids were transferred to the same Nalgene bottle. The juice basket was taken out of the juicer and the juicer body was rinsed with a water spray bottle to rinse any and all insoluble over the 75μηι sieve and catch pan and transferred to the 500ml Nalgene bottle.

The bottle was then capped and the insoluble solids were separated using vacuum filtration. The vacuum filtration set up utilized a funnel with filter paper (Whatman) the insoluble solids slurry was poured over the filter paper under vacuum. A weight was taken of the filter paper before filtration and filter paper was put in a 50C oven to dry and a weight was taken after 24 hours in the oven.

The insoluble solids are reported in the table below and are also shown in Figure 9.

%DS % insoluble solids released from starting Fiber

2.00% 11.74%

2.00% 16.65% 2.00% 13.25%

8.00% 23.94%

8.00% 21.47%

8.00% 21.91 %

12.00% 18.57%

12.00% 20.52%

12.00% 19.10%

15.00% 17.55%

15.00% 15.14%

15.00% 16.53%

18.00% 9.44%

18.00% 10.68%

List of reference symbols used:

S Screen

5 f Fraction produced by a screen

V Void/space

c Clearance

h height

10 dosing device

10 12 screening element

14 Incubator

16 Hydro-cyclone

18 Shaft

20 Stirring means; agitator

15 22 Motor

26 Outlet connection

28 Funnel shaped wall

30 Chamber

32 Opening

20 34 Impeller blades

36 Sloping bottom wall

32 Edge of opening

Claims

1. A fiber washing system comprising a plurality of screen units (S1 ... S4) being fluidly connected in a counter current washing configuration; each screen unit is configured for separating a stream of corn kernel mass and liquid into two fractions:
a first fraction (s) and a second fraction (f), said second fraction (f) containing a higher amount measured in wt% fiber than the first fraction (s);
a space (V) arranged in the system and being fluidly connected to receive one of said first fraction (s), one of said second fraction (f), or a mixed first and second fraction (s,f), preferably only a second fraction (f), and configured to provide an incubation time for one or both fractions received in the space; and outletting the thereby incubated one or both fractions to a downstream screen unit (S4), wherein the system is configured for
inletting corn kernel mass and liquid to the most upstream screen unit (S1) - outletting the first fraction (s1) from the most upstream screen unit (S1) as a product stream containing starch,
inletting process water, preferably arranged for inletting process water to a most downstream screen unit (S4),
outletting the second fraction (f4) from most downstream screen unit (S4) as a washed corn kernel mass containing a lower amount of starch and gluten than the original corn kernel mass,
introducing hydrolytic enzymes into the system.
2. The system according to claim 1 , wherein one or more such as all screen units are hydro- cyclones (16).
3. The system according to any of the preceding claims, wherein the system is configured to introduce hydrolytic enzymes into said first fraction (s) and/or into said second fraction (f) and/or into a mixed first and second fraction and/or into the process water, by means of a dosing device (10).
4. The system according to any of the preceding claims, wherein said dosing device (10) is adapted to provide a controllable dosing quantity of enzymes, preferably according to a predetermined specific ratio between amount of enzymes and infeed of corn kernel mass to the system.
5. The system according to claims 3 or 4, wherein the dosing device (10) is a metering pump.
6. The system according to claims 3 or 4, wherein the dosing device (10) is a gravity flow dispenser having a controllable outflow valve configured for controlling the amount of enzyme flowing through the flow valve.
7. A crop kernel wet milling system comprising a fiber washing system as defined in any of the preceding claims.
8. A method to improve starch yield and/or gluten yield from corn kernels in a wet milling process, comprising the steps of:
a) soaking the kernels in water to produce soaked kernels;
b) grinding the soaked kernels;
c) separating germs from the ground and soaked kernels to produce a corn kernel mass comprising fiber, starch and gluten; and
d) subjecting the resultant corn kernel mass, to a fiber washing procedure;
wherein during step d) one or more fractions of the corn kernel mass is contacted with an effective amount of one or more hydrolytic enzymes, and step d) has a total retention time of at least 45 minutes.
9. The method according to claim 8, wherein one or more of said hydrolytic enzymes is/are 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 alpha-L-arabinofuranosidases); EC 3.2.1.185 (Non-reducing end beta-L-arabinofuranosidases) cellobiohydrolase I (EC 3.2.1.150), cellobiohydrolase II (E.C. 3.2.1.91), cellobiosidase (E.C. 3.2.1.176), beta- glucosidase (E.C. 3.2.1.21), beta-xylosidases (EC 3.2.1.37).
10. The method according to any of claims 8-9, wherein the one or more of said hydrolytic enzymes is expressed in an organism with a cellulase background, such as Trichoderma reesei.
1 1. The method according to any of claims 8-10, wherein the one or more of said hydrolytic enzymes comprise a xylanase, which is a GH10 xylanase
12. The method according to any of claims 8-1 1 , wherein the one or more of said hydrolytic enzymes comprise an arabinofuranosidase, which is a GH62 arabinofuranosidase.
13. The method according to any of claims 8-12, wherein the one or more of said hydrolytic enzymes comprise a GH10 polypeptide with xylanase activity, which is selected from the group consisting of
iv) An amino acid sequence as set forth in any one of SEQ ID NOs: 22-26;
v) An amino acid sequence which has at least 80% identity to any one of SEQ ID NOs: 22-26; and
vi) A subsequence of any one of the amino acid sequences in i) and ii).
14. The method according to any of claims 8-13, wherein the one or more of said hydrolytic enzymes comprise a GH11 polypeptide with xylanase activity, which is selected from the group consisting of
iv) An amino acid sequence as set forth in any one of SEQ ID NOs: 27-35 v) An amino acid sequence which has at least 80% identity to any one of SEQ ID NOs: 27-35; and
vi) A subsequence of any one of the amino acid sequences in i) and ii).
15. The method according to any of claims 8-14, wherein the one or more of said hydrolytic enzymes comprise a GH62 polypeptide with arabinofuranosidase activity, which is selected from the group consisting of:
iv) An amino acid sequence as set forth in any one of SEQ ID NOs: 1-21 v) An amino acid sequence which has at least 80% identity to any one of the amino acid sequences set forth in SEQ ID NO: 1-21 ; and
vi) A subsequence of any one of the amino acid sequences in iv) and v).
16. The method according to any of claims 8-15, wherein the one or more of said hydrolytic enzymes is expressed in Trichoderma reesei and comprise a xylanase, which is a GH10 xylanase and a arabinofuranosidase, which is a GH62 arabinofuranosidase.
17. The method according to any of preceding claims 8-16 wherein said fiber washi procedure is performed with the use of a system as defined in any one of claims 1-7.
18. A wet milling process, which comprises the use of a fiber washing procedure as defined in any of claims 8-17.
19. A composition comprising corn starch, said composition being obtainable by the wet milling process defined in claim 18.
20. A composition comprising corn gluten, said composition being obtainable by the wet milling process defined in claim 18.
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