PHYTASE IN ENZYMATIC WET MILLING PROCESS

PRIORITY

The present application claims priority to U.S. Provisional Patent Application 61/091,975, which was filed on August 26, 2008, and which is incorporated by reference it' s entirety.

FIELD OF THE INVENTION

The present invention relates to utilization of phytase in enzymatic wet milling processes to obtain starch.

BACKGROUND OF THE INVENTION

The preparation of grain for alcohol fermentation is generally accomplished by two methods which are known in the industry as dry milling and wet milling. Dry milling methods involve the grinding of whole grains. In contrast, wet milling methods involve soaking the grain with water and SO ₂ to soften the grain kernel (steeping) and loosen the hull. In a conventional wet milling process corn kernels are initially steeped in an aqueous solution of sulfur dioxide for a period of generally 20 to 60 hours ((at about 48°- 54° C) sulfurous (0.1-0.2%) water). Steeping times shorter than about 24 hours generally result in poor starch yields and loss of starch to the fiber and protein fractions of the kernel. After the kernels are soaked they are passed though a series of mills which frees the intact germ from the rest of the kernel. The germ which contains a high concentration of oil (~ 45%), generally is separated, for example by density difference using hydrocyclones. The remaining slurry is comprised of starch, protein and fiber. In a series of steps, the starch is separated from the protein, resulting in a starch fraction containing less than 0.30% total protein and less than 0.01% soluble protein. Starch is then further converted into low molecular weight fermentable sugars (e.g., glucose and maltose) and processed for different products such as alcohols (e.g., ethanol) or corn syrups. Numerous references specifically detail the conventional wet milling process such as The Alcohol Textbook 3 ^rd Ed: A Reference for the Beverage, Fuel and Industrial Alcohol Industries, Eds. KA Jacques, TP Lyons and DR Kelsall, 1999 Nottingham University Press, Chapter 4; Watson, S. A., et al., Cereal Chem., 38:22-23 (1961) and Eckhoff, S. R., Wet milling short course, Course Notes, American Association of Cereal Chemists, St. Paul, Minn., (1999).

One of the disadvantages of the conventional wet milling process outlined above is the use of SO ₂ in the steeping process. However, SO ₂ helps break down the protein matrix that surrounds the starch particles and further increases starch yield during the milling process. One improvement on the conventional wet milling process has been development of an enzymatic wet milling process. The enzymatic wet milling process comprises a) size reduction of corn after a short water soaking of intact kernels and b) controlled incubation of the coarsely ground slurry with protease. Following the enzyme incubation step, the corn slurry is fractionated using conventional wet milling processes.

The enzymatic wet milling process reduces and potentially eliminates the need for SO ₂ during steeping. Further the steeping time may be reduced by about 70% to about 80% and higher starch yields may be produced when compared to the conventional wet milling process. For example, comparison of the enzymatic corn wet milling process with the conventional corn wet milling process showed that significantly higher amounts of starch (approximately 1.0%) could be obtained with the enzymatic wet milling process. (Johnston and Singh (2001) Cereal Chem. 78:405 - 411; Singh and Johnston (2002) Cereal Chem. 79:523 - 527; Singh and Johnston (2004) Adv. Food Nutr. Rs. 48: 151 - 171; Johnston and Singh (2004) Cereal Chem. 81:626 - 632; Johnston and Singh (2005) Cereal Chem. 82:523 - 527 and United States Patent 6,566,125 (herein incorporated by reference in its entirety).

It would be desirable to improve the enzymatic wet milling process by a) reducing steeping time, b) decreasing or eliminating SO ₂ utilization, c) improving starch yield, d) improving germ floatation, e) decreasing the dose of protease utilized during incubation of steeped (soaked) grain, and/or f) improving fiber separation.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to enzymatic wet milling processes which comprise addition of phytase during an incubation step.

In one aspect, the invention provides an incubation step of steeped grain with a phytase in addition to a protease. In a further aspect, the invention relates to methods for obtaining starch from grain, which comprises a) steeping grain in water and optionally SO ₂ , for a sufficient period of time to produce soaked grain; b) grinding the steeped grain to produce a slurry; c) incubating the slurry with a protease and a phytase; and obtaining a slurry comprising starch.

In another aspect, the invention relates to methods for recovering starch, protein and fiber from the incubated slurry.

In yet another aspect, the invention relates to a method for increasing the starch yield from an enzymatic wet milling process, the method comprising adding to the slurry of an enzymatic wet milling incubation step a phytase in addition to a protease to obtain a starch yield which is greater than the starch yield obtained under essentially the same conditions absent addition of phytase during the incubation.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates starch yield (% dry weight basis, db) under different soaking and incubation conditions as further described in example 1.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise indicated, the practice of the invention involves conventional techniques commonly used in molecular biology, protein engineering, recombinant DNA techniques, microbiology, cell biology, cell culture, transgenic biology, immunology, and protein purification. Such techniques are known to those of skill in the art and are described in numerous texts and reference works. All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference in their entirety.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described as these may vary, depending upon the context they are used by those of skill in the art.

Accordingly the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular "a", "an" and "the" includes the plural reference unless the context clearly indicates otherwise. Thus, for example, reference to a composition containing "a compound" includes a mixture of two or more compounds. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise. Unless otherwise indicated amino acids are written left to right in amino to carboxy orientation, respectively.

Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole.

In order to facilitate understanding of the invention, a number of terms are defined herein below.

Definitions

As used herein the term "starch" refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin with the formula (C ₆ H ₁₀ O ₅ ) _x , wherein x can be any number. The term "steeping" means soaking the grain with water and optionally SO ₂ to soften the grain kernel in a wet milling process.

The term "incubating" as used with reference to the enzymatic wet milling process means contacting a slurry comprising ground grain with an enzyme composition. The term "granular starch" means raw starch, that is, starch which has not been subject to temperatures of gelatinization.

The term "oligosaccharides" refers to any compound having 2 to 10 monosaccharide units joined in glycosidic linkages. These short chain polymers of simple sugars include dextrins. "Dextrins" are short chain polymers of glucose (e.g., 2 to 10 units). The term "fermentable sugars" refers to any sugars that are capable of being fermented by a fermenting organism. Fermentable sugars include oligosaccharides and dextrins.

The term "DE" or "dextrose equivalent" is an industry standard for measuring the concentration of total reducing sugars, calculated as D-glucose on a dry weight basis. Unhydrolyzed granular starch has a DE that is essentially 0 and D-glucose has a DE of 100.

The term "glucose syrup" refers to an aqueous composition containing glucose solids. Glucose syrup will have a DE of at least 20. In some embodiments, glucose syrup will not contain more than 21% water and will not contain less than 25% reducing sugar calculated as dextrose. In one embodiment, glucose syrup will include at least about 90% D-glucose and in another embodiment glucose syrup will include at least about 95% D-glucose. In some embodiments the terms glucose and glucose syrup are used interchangeably. The term "total sugar content" refers to the total sugar content present in a starch composition.

The term "dry solids (ds)" refers to the total solids of a slurry in % on a dry weight basis.

The term "milled" is used herein to refer to plant material that has been reduced in size, such as by grinding, crushing, fractionating or any other means of particle size reduction. Milling includes dry or wet milling. "Dry milling" refers to the milling of whole dry grain. "Wet milling" refers to a process whereby grain is first soaked in water to soften the grain (steeping).

The term "gelatinization" means solubilization of a starch molecule, generally by cooking, to form a viscous suspension.

The term "slurry" refers to an aqueous mixture comprising insoluble solids.

The term "fermentation" refers to the enzymatic and anaerobic breakdown of organic substances by microorganisms to produce simpler organic compounds. While fermentation occurs under anaerobic conditions it is not intended that the term be solely limited to strict anaerobic conditions, as fermentation also occurs in the presence of oxygen. The term "end-product" refers to any carbon-source derived product which is enzymatically converted from a fermentable substrate. In some embodiments, the end- product is an alcohol (e.g., ethanol).

The term "derived" encompasses the terms "originated from", "obtained" or "obtainable from", and "isolated from" and in some embodiments as used herein means that a polypeptide encoded by the nucleotide sequence is produced from a cell in which the nucleotide is naturally present or in which the nucleotide has been inserted.

As used herein the term "fermenting organism" refers to any microorganism or cell, which is suitable for use in fermentation for directly or indirectly producing an end-product.

As used herein the term "ethanol producer" or ethanol producing microorganism" refers to a fermenting organism that is capable of producing ethanol from a mono- or oligosaccharide. The terms "recovered", "isolated", and "separated" as used herein refer to a protein, cell, nucleic acid, amino acid, molecule or compound that is removed from at least one component with which it is naturally associated.

As used herein, the term "phytase" refers to an enzyme which is capable of catalyzing the hydrolysis of esters of phosphoric acid inositol, including phytate and releasing inorganic phosphate and inositol. In some embodiments, in addition to phytate, the phytase can be capable of hydrolyzing at least one of the inositol- phosphates of intermediate degrees of phosphorylation. Phytases can be grouped according to their preference for a specific position of the phosphate ester group on the phytate molecule at which hydrolysis is initiated, (e.g., as 3-phytases (EC 3.1.3.8) or as 6-phytases (EC 3.1.3.26)). A typical example of phytase is myo-inositol- hexakiphosphate-3-phosphohydrolase.

The term "wild-type" as used herein refers to an enzyme naturally occurring (native) in a host cell. In some embodiments, the term "parent" or "parent sequence" is used interchangeably with the term wild-type. As used herein, the term "contacting" refers to the placing of at least one enzyme in sufficiently close proximity to its respective substrate to enable the enzyme(s) to convert the substrate to at least one end-product. In some embodiments, the end-product is a "product of interest" (i.e., an end-product that is the desired outcome of the fermentation reaction). Those skilled in the art will recognize that mixing at least one solution comprising the at least one enzyme with the respective enzyme substrate(s) results in "contacting." Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Detailed Embodiments

The methods of the invention include the use of phytase during enzymatic wet milling.

In some embodiments, the first step of the process involves hydrating grain, such as corn, in an aqueous solution (e.g., water) for a period of time to hydrate the grain such that the grain becomes pliable but does not break when the grain is ground. While a preferred grain for use in the process is corn, other grains such as wheat and sorghum may be used in the enzymatic wet milling process of the invention.

This step is referred to as the soaking or steeping step. Steeping may take place for about 30 minutes (mins) to about 15 hours (hrs), also for about 30 mins to about 12 hrs, also for about 30 mins to about 10.0 hrs, also for about 1.0 hr to about 8.0 hrs, also for about 1.0 hr to about 6.0 hrs, also for about 30 mins to 3.0 hrs, and further for about 2.0 hrs to about 4.0 hrs. Steeping temperature is generally between about 45°C to 60 ⁰ C, also between about 45°C and 55°C, also between about 45°C to 50 ⁰ C, and also about between 50 ⁰ C to 55°C.

The addition of sulfur dioxide may be used during this step or during the incubation step discussed below. The amount of sulfur dioxide in either step will range from about 0 to about 2000 ppm, from about 0 to 1000 ppm, from about 0 to about 750 ppm, from about 0 to about 500 ppm, from about 0 to 250 ppm, and from about 0 to about 100 ppm. In some embodiments, the amount of sulfur dioxide will be less than about 100 ppm, less than about 50 ppm, less than about 25 ppm and less than about 5 ppm. In some embodiments, no sulfur dioxide will be added. In some embodiments, sulfur dioxide will be added to the steeping step but not the incubation step, and in other embodiments, sulfur dioxide will be added to the incubation step but not the steeping step.

After the steeping step the hydrated grain is ground, to form a slurry. One of skill in the art is familiar with various grinding (milling) processes, and generally wet milling involves the coarse milling of grain. Reference is made to USP 6,566,125. This step is also referred to as the first ground.

The slurry is combined in an incubation step with enzymes. The incubation time is between about 0.5 hrs to about 12 hrs, also about 0.5 hrs to about 10.0 hrs, also about 1.0 hr to about 8.0 hrs, also about 1.0 hr to about 6.0 hrs, also about 1.0 hr to about 4.0 hrs, and further about 2.0 hrs to 4.0 hrs. Incubation time may be increased or decreased depending on the amount of enzyme that is used. Incubation temperature is generally between about 25 ⁰ C to 75 ⁰ C, between about 30 ⁰ C to 70 ⁰ C, between about 40 ⁰ C to 65 ⁰ C, between about 45 ⁰ C to 60 ⁰ C, between about 45 ⁰ C and 55 ⁰ C, between about 45°C to 50 ⁰ C, and also between about 50 ⁰ C to 55°C. The temperature may change depending on the specific enzymes used in the incubation step. However, the temperature will generally be below the gelatinization temperature of the starch in the grain.

Additional chemicals may be added to the slurry including for example sodium metabisulfite (e.g., 0 to 2000 ppm), lactic acid (about 0 to 0.7% w/w, e.g., about 0.5%), and buffers to maintain pH 4.0 ± 0.2. In addition, lactic acid (about 0 to 0.7% w/w) may be added to the steeping step. In the incubation step, the slurry is combined with a protease enzyme and a phytase enzyme. It is within the skill of one in the art to determine which proteases or phytases can be successfully utilized in the present invention.

The proteases used in the methods are not critical, and generally any protease that can hydrolyze proteins in grain (e.g., corn) may be used. The protease can be an acid, an alkaline, or a neutral protease. In some embodiments, the protease is an acid protease. Proteases can be obtained from bacteria, fungi and/or plants. Proteases obtained from bacteria include proteases from Bacillus such as B. amyloliquefaciens, B. lentus, B. licheniformis, and B. subtilis. These sources include subtilisin such as a subtilisin obtainable from B. amyloliquefaciens and mutants thereof (USP 4,760,025).

Proteases useful in the invention may also be derived from fungal sources such as Trichoderma (e.g., T. reesei), Aspergillus (e.g., A. niger, A. awamori, and A. oryzae), Humicola and Penicillium, Mucor (e.g., M. miehei) and Rhizopus. In some embodiments, the protease is an acid fungal protease (AFP). AFPs can be obtained from the heterologous or endogenous protein expression of bacteria, plants and fungi sources. In some embodiments, proteases secreted from strains of Trichoderma and Aspergillus are suitable. AFPs include naturally occurring wild-type AFP as well as variant and genetically engineered mutant AFP. Reference is made to United States patent application 11/312,290, filed December 20, 2005 for an AFP useful in the invention.

Suitable commercial proteases include PROSTEEP™ (Danisco, Genencor), MULTIFECT P 3000 (Danisco Genencor) and SUMIZYME FP (Shin Nihon). Examples of plant protease include for example, bromelain (Sigma). In addition, pepsin from porcine and Aspergillus acid proteinase, Type XIII from A. saitoi may be purchased from Sigma.

In some embodiments, the slurry is incubated with protease enzyme in an amount about 0.25 kg to about 3.0 kg/metric ton (MT) dry solids basis (dsb) of substrate, about 0.5 kg to about 2.5 kg/MT dsb, about 0.7 kg to about 2.0 kg/MT dsb, and about 1.0 kg to about 1.5 kg/MT dsb. In some embodiments, the substrate is grain and in some embodiments the grain is corn. In other embodiments, the concentration of protease used in the incubation is in the range of about 1500 mg per lOOg of grain, about 1000 mg per lOOg of grain, about 500 mg per 100 g of grain, and about 250 mg per lOOg of grain, about 100 mg per 100 grain and about 50 mg per 100 g of grain.

In some embodiments, the phytase useful in the invention is one isolated from fungal or yeast sources, such as Aspergillus spp. (e.g., A. fumigatus and A. nidulans), Penicillium spp. (e.g., P. chrysogenum), Fusarium spp. (e.g., F. javanicum),

Humicola spp. (e.g., H. grisea), Talaromyces spp. (e.g., T. thermophilus), Peniophora (e.g. P. lycii) and Trichoderma spp. (e.g., T. reeseϊ). Reference is also made to WO 1998/44125; WO 1998/28408; and Folio Microbiol. 43 (4) 323-338 (1998) - Phytase: Sources, Preparation and Exploitation, J. Dvorakova. Yeast sources include Saccharomyces and Schwanniomyces.

In other embodiments, the phytase useful in the invention is one isolated from a bacterial source, such as Escherichia ( E.coli), Citrobacter spp., and Buttiauxiella spp. Buttiauxiella spp. includes B. agrestis, B. brennerae, B. ferragutiase, B. gaviniae, B. izardii, B. noackiae, and B. wαrmboldiαe. Strains of B uttiαuxellα species are available from DSMZ, the German National Resource Center for Biological Material (Inhoffenstrabe 7B, 38124 Braunschweig, Germany). Buttiαuxellα sp. strain Pl-29 deposited under accession number NCIMB 41248 is an example of a useful strain from which a phytase can be obtained and used according to the invention. Variant phytases derived from wildtype phytase enzymes are also useful according to the invention.

Examples of variant phytases derived from Buttiαuxellα include those disclosed in WO 2006/043178 and WO 2008/092901. Additionally phytases useful according to the invention include the phytase comprising the amino acid sequence of

NDTPASGYQVEKVVILSRHGVRAPTKMTQTMRDVTPNTWPEWPVKLGYITPRGEH LISLMGGFYRQKFQQQGILSQGSCPTPNS IYVWTDVDQRTLKTGEAFLAGLAPQC GLTIHHQQNLEKADPLFHPVKAGICSMDKTQVQQAVEKEAQTPIDNLNQHYIPSL ALMNTTLNFSKSPWCQKHSADKSCDLGLSMPSKLSIKDNGNEVSLDGAIGLSSTL AEIFLLEYAQGMPQAAWGNIHSEQEWALLLKLHNVYFDLMERTPYIARHKGTPLL QAISNALNPNATESKLPDISPDNKILFIAGHDTNIANIAGMLNMRWTLPGQPDNT PPGGALVFERLADKSGKQYVSVSMVYQTLEQLRSQTPLSLNQPAGSVQLKIPGCN DQTAEGYCPLSTFTRVVSQSVEPGCQLQ (SEQ ID NO: 1) and variants thereof. In one embodiment, the phytase is the phytase comprising the amino acid sequence of

NDTPASGYQVEKVVILSRHGVRAPTKMTQTMRDVTPNTWPEWPVKLGYITPRGEHLI SLMGGFYRQKFQQQGILSQGSCPTPNSIYVWTDVAQRTLKTGEAFLAGLAPQCGLTI HHQQNLEKADPLFHPVKAGICSMDKTQVQQAVEKEAQTPIDNLNQHYIPSLALMNTT LNFSKSPWCQKHSADKSCDLGLSMPSKLSIKDNGNEVSLDGAIGLSSTLAEIFLLEY AQGMPQAAWGNIHSEQEWALLLKLHNVYFDLMERTPYIARHKGTPLLQAISNALNPN ATESKLPDISPDNKILFIAGHDTNIANIAGMLNMRWTLPGQPDNTPPGGALVFERLA DKSGKQYVSVSMVYQTLEQLRSQTPLSLNQPAGSVQLKIPGCNDQTAEGYCPLSTFT RVVSQSVEPGCQLQ (SEQ ID NO: 2) and variants thereof.

In general, bacterial phytases are disclosed in WO 1998/06856, WO 1997/33976, and WO 1997/48812. Variant phytases are also obtainable by methods known in the art, and for example by methods disclosed in WO 1999/49022.

In some embodiments, the slurry is incubated with a phytase enzyme in an amount of about 0.01 FTU/g to 25.0 FTU/g dry solid basis (dsb) of substrate, about 0.01 FTU/g to 20.0 FTU/g dsb of substrate, about 0.01 FTU/g to 15.0 FTU/g dsb of substrate, 0.01 FTU/g to 10.0 FTU/g dsb of substrate, about 0.01 FTU/g to 5.0 FTU/g dsb of substrate, about 0.05 FTU/g to 20.0 FTU/g dsb of substrate, about 0.05 FTU/g to 15.0 FTU/g dsb of substrate, about 0.05 FTU/g to 10.0 FTU/g dsb of substrate, about 0.1 FTU/g to 15.0 FTU/g dsb of substrate, about 0.1 FTU/g to 10.0 FTU/g dsb of substrate, about 0.1 FTU/g to 5.0 FTU/g dsb of substrate, about 0.25 FTU/g to 10.0 FTU/g dsb of substrate, about 0.5 FTU/g to 20.0 FTU/g dsb of substrate, about 0.5 FTU/g to 10.0 FTU/g dsb of substrate, about 0.5 FTU/g to 5.0 FTU/g dsb of substrate, 1.0 FTU/g to 10.0 FTU/g dsb of substrate, 1.0 FTU/g to 5.0 FTU/g dsb of substrate and about 2.0 FTU/g to 8.0 FTU/g dsb of substrate. In some embodiments, the substrate is grain (e.g., corn).

In other embodiments, the concentration of phytase used in the incubation step is in the range of about 10 mg to 3000 mg per 100 g grain, about 10 mg to 1500 mg per 100 g grain, about 10 mg to 1000 mg per 100 g grain and about 10 mg per 100 mg per 100 g grain. In some embodiments, the phytase concentration used in the incubation step will range from about 0.1 to about 20 FTU/gds starch, about 0.5 to about 15 FTU/gds starch, also from about 1.0 to about 15 FTU/gds starch, also from about 1.0 FTU to 10 FTU/gds starch, and also from about 2.0 to about 8.0 FTU/gds starch.

While the invention includes an incubation step comprising a protease and a phytase, the invention may include the addition of a phytase in the steeping step and further other enzymes may be used in either the steeping step or incubating step. For example, the additional enzymes include without limitation: alpha amylases, other proteases (including acid fungal proteases), glucoamylases, other phytases, cellulases, hemicellulases, xylanases, pullulanases, beta amylases, lipases, cutinases, pectinases, beta-glucosidases, galactosidases, esterases, cyclodextrin transglycosyltransferases (CGTases), oxido-reductases, esterases, beta-amylases and combinations thereof. In some embodiments, the methods of the invention will include the addition of xylanases, cellulases, hemicellulases and pectinases.

In some embodiments, the additional enzyme is an alpha amylase. Alpha amylases useful according to the invention can be fungal alpha amylases or bacterial alpha amylases. In some embodiments, the one or more alpha amylases include those obtained from filamentous fungal strains including but not limited to strains of Aspergillus (e.g., A. niger, A. kawachi, and A. oryzae); Trichoderma sp., Rhizopus sp., Mucor sp., and Penicillium sp. In some embodiments, the alpha amylase is obtained from a strain of Aspergillus kawachi or a strain of Trichoderma reesei. In some embodiments, the alpha amylase will be a hybrid enzyme, for example, the hybrid enzyme may comprise fragments that are derived from a strain of Aspergillus kawachi and a strain of A. niger. In other embodiments, the alpha amylase is derived from a bacterial source, such as Bacillus spp (e.g., B. licheniformis, B. lentus, B. coagulans, B. amyloliquefaciens, B. stearothermophilus, and B subtilis). Hybrid, mutants and variants alpha amylase may also be useful. Reference is made to USP 5,763,385;

USP 5,824,532; USP 5,958,739; USP 6,008,026 and USP 6,361,809 for enumeration of various alpha amylases. Commercially available alpha amylase include e.g., TERMAMYL™ SC and SUPRA available from Novo Nordisk A/S, ULTRATHIN from Diversa, LIQUEZYME™ SC from Novo Nordisk A/S and SPEZYME FRED, SPEZYME XTRA, GZYME G997, CLARASE available from Danisco US Inc, Genencor Division.

Cellulases may also be used in the methods of the invention. Cellulases are enzymes that hydrolyze cellulose (β-1, 4-D-glucan linkages) and/or derivatives thereof, such as phosphoric acid swollen cellulose. Cellulases include the classification of exo-cellobiohydrolases (CBH), endoglucanases (EG) and β- glucosidases (BG) (EC3.2.191, EC3.2.1.4 and EC3.2.1.21). Examples of cellulases include cellulases from Penicillium, Trichoderma, Humicola, Fusarium, Thermomonospora, Cellulomonas, Clostridium and Aspergillus. Commercially available cellulases sold for feed applications are beta-glucanases such as ROVABIO (Adisseo), NATUGRAIN (BASF), MULTIFECT BGL (Danisco Genencor) and ECONASE (AB Enzymes).

Xylanases can also be used in the methods of the invention. Xylanases (e.g. endo-β-xylanases (E.C. 3.2.1.8)), which hydrolyze the xylan backbone chain can be obtained from bacterial sources, such as Bacillus, Streptomyces, Clostridium, Acidothermus, Microtetrapsora or Thermonospora. In addition, xylanases can be obtained from fungal sources, such as Aspergillus, Trichoderma, Neurospora, Humicola, Penicillium or Fusarium. (See, for example, EP473 545; USP 5,612,055; WO 92/06209; and WO 97/20920). Commercial preparations of xylanases include MULTIFECT and FEEDTREAT Y5 (Danisco Genencor), RONOZYME WX (Novozymes A/S) and NATUGRAIN WHEAT (BASF).

The above mentioned enzymes may be added in effective amounts well known to the person skilled in the art. After the incubation step, the slurry is processed by conventional wet milling methods. These steps include a second milling wherein the material is then separated and sieved to separate the germ, fiber, protein and starch. Reference is made to Eckhoff, R.S. et al., Cereal Chem. 73:54-57 (1996).

When germ, fiber, gluten and starch are separated from the incubated milled slurry, in some embodiments, the yield of starch obtained is between about 60 and 75%; the yield of fiber obtained is between 7.0 and 20%; the yield of germ obtained in between 6.0 and 7.5%, and the yield of protein (gluten) obtained is between 7.0 and 15%. The % is based on a weight basis. In some embodiments, the yield of starch obtained according to the invention will be in the range of 62% to 75%. In some embodiments, the yield of starch will be greater than 65%, greater than 66%, greater than 67%, greater that 68%, greater than 69%, greater than 70%, greater than 71%, greater than 72%, greater than 73%, and greater than 74%. In some embodiments, the amount of starch will be less 80%.

In some embodiments, the yield of starch according to the invention will be increased relative to the yield of starch obtained under essentially the same process conditions but without the addition of phytase during the incubation step. In some embodiments, the increase in starch yield on a dry weight basis will be at least 0.2%, at least 0.25%, at least 0.5%, at least 0.75%, at least 1.0%, at least 1.25%, at least 1.5%, at least 1.75%, at least 2.0%, at least 2.25%, at least 2.5%, at least 3.0%, at least 3.5%, at least 4.0%, at least 4.5%, and at least 5.0% greater. In further embodiments, the addition of phytase in the incubation step can decrease the amount of protease used in same incubation step while the yield of starch is held constant as compared to essentially the same method under essentially the same conditions absent the phytase in the incubation step. In some embodiments, the method will include addition of sulfur dioxide of between 200 ppm and 1400 ppm in the steeping step but no sulfur dioxide addition in the incubation step.

The components obtained from the methods of the invention (e.g., starch, fiber, germ, gluten and other materials) may then be further processed. The impure starch product obtained according to the invention may be further washed and dried to obtain a pure starch product (e.g., 97 - 99% pure starch). Starch obtained from the enzymatic wet milling process may be further processed in the presence of enzymes (e.g., amylases) which convert starch to oligosaccharides. Glucose and maltose obtained from the starch maybe used as syrups. The syrups may be crystallized into pure dextrose or further processed to create high fructose corn syrup. Following the conversion of starch to oligosaccharides and fermentable sugars such as glucose and maltose, the fermentable sugars may be converted to end- products such as alcohol (e.g., ethanol and butanol) by a fermenting organism. In some embodiments, the fermentable sugars will be converted to ethanol by ethanol fermenting organisms, such as yeast. In further embodiments, the end product is a biological fermentation product. The fermentation end product can include without limitation glycerol, 1,3-propanediol, gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate, 2-keto-L-gulonic acid, succinic acid, lactic acid, amino acids and derivatives thereof. More specifically when lactic acid is the desired end product, a Lactobacillus sp. (L. caseϊ) can be used; when glycerol or 1,3 -propanediol are the desired end-products E.coli can be used; and when 2-keto-D-gluconate, 2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid are the desired end products, Pantoea citrea can be used as the fermenting microorganism. The above enumerated list are only examples and one skilled in the art will be aware of a number of fermenting microorganisms that can be appropriately used to obtain a desired end product.

Methods for separation and purification are known, for example by subjecting the media to extraction, distillation and column chromatography. In some embodiments, the end product is identified directly by submitting the media to high-pressure liquid chromatography (HPLC) analysis.

EXPERIMENTAL The present invention is described in further detail in the following examples which are not in any way intended to limit the scope of the invention. The attached Figure is meant to be considered an integral part of the specification and description of the invention.

In the disclosure and experimental section which follows, the following abbreviations apply: wt% (weight percent); ⁰ C (degrees Centigrade); H ₂ O (water); dH ₂ O (deionized water); dIH ₂ O (deionized water, Milli-Q filtration); g or gm (grams); μg (micrograms); mg (milligrams); kg (kilograms); μL (microliters); ml and mL (milliliters); mm (millimeters); μm (micrometer); M (molar); mM (millimolar); μM (micromolar); U (units); MW (molecular weight); sec (seconds); min(s) (minute/minutes); hr(s) (hour/hours); ds (dry solids); W/V (weight to volume); WAV (weight to weight); V/V (volume to volume); Genencor (Danisco US, Inc., Genencor Division, Palo Alto, CA); MT (Metric ton); and ETOH (ethanol).

Yellow dent corn grown at Agricultural Engineering experiment station at the University of Illinois at Urbana-Champaign was used for the study. Corn samples were hand cleaned to remove broken corn and foreign material, packaged in plastic bags and stored at 4°C until processing. Whole kernel moisture content was measured using the 103 ⁰ C convection oven method (AACC 2000). Whole kernel moisture content of 9.2 and 11.5% was observed for amylase and dent corn, respectively. Experimental phytase (BP- 17) and acid fugal proteases (GC 106 or PROSTEEP) samples was obtained from Danisco US Inc, Genencor Division (Palo Alto, CA).

EXAMPLES

The following example shows the usefulness of the addition of a phytase in combination with a protease in a wet milling process. The protease used in the example was GC106.

Phytic Acid content of wet milled samples (soak/steep water and starch samples) was analyzed by phytic acid and free phosphate content using HPLC (Kwanyuen and Burton 2005). For further verification phytic acid derivatives (Ins P5 to Ins Pl) from HPLC were analyzed using a mass spectrophotometer. Phytase Activity (FTU) was measured by the release of inorganic phosphate.

The inorganic phosphate forms a yellow complex with acidic molybdate/vandate reagent and the yellow complex was measured at a wavelength of 415 nm in a spectrophometer and the released inorganic phosphate was quantified with a phosphate standard curve. One unit of phytase (FTU) is the amount of enzyme that releases 1 micromole of inorganic phosphate from phytate per minute under the reaction conditions given in the European Standard (CEN/TC 327, 2005-TC327WI 003270XX).

EXAMPLE 1 Phytase (BP- 17) and protease (GC106) in an enzymatic wet milling process

Corn kernels samples (1 kg) were fractionated into starch, protein, germ, fiber and steepwater solubles using an enzymatic wet milling procedure. Control treatments consisted of soaking kernels for 12 hrs in water with 0, 200, 400 or 1200 ppm of SO ₂ and 0 or 0.55% lactic acid (LA) at 52°C. After soaking samples were coarsely ground in a Waring blender (with blunt blades) and incubated with Aspergillus niger acid fungal protease, GC 106 (1 ml/kg corn kernels) at pH 4 and 48°C for 4 hr with 0 or 1200 ppm SO ₂ . Except for the addition of phytase, phytase treatments involved the same process conditions as control treatments. Phytase concentration ranged from 1.0 FTU to 10 FTU/g ds starch. In a typical experiment 4 ml of B P- 17 was added during the incubation step and the amount of phytase was equivalent to 5.6 FTU/g ds starch. All treatments were enzymatic wet milled in triplicate. The experimental design with process conditions for addition of phytase is given below. Starch, protein, germ, fiber and soluble yields are reported as a percentage of the original sample dry weight. Moisture contents of fractions were determined using a two stage convection oven method (AACC 2000: Method 44-18).

Table 1 Process conditions for addition of phytase in an enzymatic wet milling process.

Control Phytase Control Phytase Control Phytase Control Phytase Control Phytase

1 1 2 2 3 3 4 4 5 5

Steep SO ₂

(ppm) 0 0 0 0 200 200 400 400 1200 1200

Steep LA

(%) 0 0 0 0 0.55 0.55 0.55 0.55 0.55 0.55

Incub

Phytase

(ml) 0 4 0 4 0 4 0 4 0 4

Incub

GC106

(ml) 1 1 1 1 1 1 1 1 1 1

Incub SO ₂

(ppm) 0 0 1200 1200 0 0 0 0 0 0

Incub LA

(%) 0 0 0.55 0.55 0 0 0 0 0 0

Germ

(%) 6.63 6.74 6.54 6.44 6.74 6.60 6.61 6.49 6.48 6.49

Fiber

(%) 13.56 17.51 8.37 8.49 10.65 9.70 9.19 8.49 7.78 7.59

Gluten

(%) 9.37 8.13 13.05 12.60 10.63 10.95 10.77 10.61 11.06 11.25

Total

(%) 96.73 98.36 99.93 99.18 99.26 98.95 99.15 100.18 100.03 100.80

As observed in Figure 1, addition of phytase during the incubation step affected starch yield for some of the enzymatic wet milling treatments. With no SO ₂ addition during soaking, the effect of phytase addition (during incubation) on starch yields was lower or negligible compared to control treatments. However, when SO ₂ was present during soaking, addition of phytase consistently resulted in comparable or higher starch yields compared to Control treatment (Figure 1). Starch yield for Phytase 3 treatment (200 ppm of SO ₂ during soaking) was 0.5% higher than its corresponding Control treatment. Starch yield for Phytase 4 treatment (400 ppm of SO ₂ during soaking) was 1.6% higher than Control 4 treatment. Similarly starch yield for Phytase 5 treatment (1200 ppm of SO ₂ during soaking) was 0.7% higher than Control 5 treatment. Addition of phytase for all treatments improved the germ recovery step for the enzymatic wet milling process. Qualitatively (visual observation) germ was cleaner (no broken germ or no attached endosperm particles with germ) and floated better when phytase was added during incubation compared to treatments in which phytase was not added. For phytase treatments, fiber was also cleaner (less adhering starch) and its separation was better compared to treatments with no phytase addition. Quantitatively, lower germ and fiber yields were observed for phytase treatments (especially when SO ₂ was used in soaking) compared to treatments in which no phytase was added. Lower germ and fiber yield were due to lighter germ and less loss of starch in germ and fiber fractions.

Specific Embodiments Include:

1. A method for obtaining starch from maize, comprising: a) soaking maize kernels in water and SO ₂ produce soaked maize kernels; b) grinding said soaked maize kernels to produce a ground maize slurry; c) incubating the ground maize slurry with at least one protease and at least one phytase and SO ₂ .

2. The method of embodiment 1, wherein said method utilizes less than about 600 ppm SO ₂ , wherein said soaking further comprises the addition of phytase.

3. The method of embodiment 1, wherein said soaking is for about 1 to about 6 hours. 4. The method of embodiment 1, wherein said soaking is for about 2 to about 4 hours.

5. The method of embodiment 1, wherein said soaking is for about 3 hours.

6. The method of embodiment 1, wherein said soaking is at about 45 to about 6O ⁰ C.

7. The method according to embodiment 1, wherein said incubating is for about 0.5 to about 6 hours. 8. The method of embodiment 1, wherein said incubating is for about 1 to about 4 hours.

9. The method of embodiment 1 wherein said incubating is at a temperature of about 20 to about 55°C. 10. The method of embodiment 1 wherein said incubating is at a temperature of about 40 to about 70 ⁰ C.

11. The method of embodiment 1, wherein said soaking utilizes less than about 100

12. The method of embodiment 1, wherein said incubating is in the presence of from about 0 to about 2000 ppm SO ₂ .

13. The method of embodiment 1, wherein the phytase is a wild type or variant phytase isolated from a fungal source, a yeast source or a bacterial source.

14. The method of embodiment 1, wherein the concentration of protease is from about 45-100 mg/lOOg of maize.

15. A method for reducing the amount of sulfur used in a wet milling process, comprising: a) soaking maize kernels in water and SO ₂ for about 1 to about 6 hours to produce soaked maize kernels; b) grinding said soaked maize kernels to produce a ground maize slurry; c) incubating the ground maize slurry with at least one protease and at least one phytase, wherein said method utilizes less than about 600 ppm SO ₂ , wherein said soaking further comprises the addition of phytase.

16. The method of embodiment 15, wherein said soaking is for about 1 to about 6 hours.

17. The method of embodiment 15, wherein said soaking is for about 2 to about 4 hours.

18. The method of embodiment 15, wherein said soaking is for about 3 hours.