Methods, compositions, and systems for co-producing rice protein and rice starch

Abstract

Provided herein are methods, compositions, and systems that allow for the co-production of rice starch and rice protein from a rice substrate in a single process. The methods, compositions, and systems facilitate the extraction of rice starch and rice protein from a rice substrate into separate protein and starch streams for processing, thus allowing such co-production in a single process.

Description

IFF10169-WO-PCTMETHODS, COMPOSITIONS, AND SYSTEMS FOR CO-PRODUCING RICE PROTEIN AND RICE STARCHCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of Indian Patent Application No. 202411097146, filed December 9, 2024, which is incorporated by reference in its entirety.INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING

[0002] The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled IFF10169WOPCT_SequenceListing.xml, created on December 4, 2025, which is 11,088 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.FIELD OF THE INVENTION

[0003] Provided herein are methods, compositions, and systems that allow for the co-production of rice starch and rice protein from a rice substrate in a single process. The methods, compositions, and systems facilitate the extraction of rice starch and rice protein from a rice substrate into separate protein and starch streams for processing, thus allowing such co-production in a single process.BACKGROUND

[0004] Starch and protein are the two main components of rice at roughly 80% and 8% by dry weight, respectively. The starch and protein components of rice have found valuable uses as ingredients in a variety of applications and industries, including food and feed products, pharmaceuticals, cosmetics, and textiles, among various others.

[0005] Extracting both rice starch and rice protein during a single process, however, has remained a challenge. For example, the processes needed to extract rice starch can reduce the availability of rice proteins, rendering the efforts needed for protein recovery cost prohibitive. Furthermore, the alkaline methods for rice starch milling and existing common rice protein recovery methods tend to result in protein with poor functional qualities. Thus, there is a need for new methods that allow for the co-production of rice starch and rice protein.

[0006] The methods, compositions, and systems described herein address these and other needs in the art.IFF10169-WO-PCTSUMMARY OF THE INVENTION

[0007] In an aspect is provided a method for co-producing rice starch and rice protein, including contacting a rice slurry with an enzyme composition to produce a treated slurry, wherein the enzyme composition includes a protease. In some embodiments, the enzyme composition consists essentially of a protease. In some embodiments, the enzyme composition further includes a cellulase. In some embodiments, the cellulase includes a cellobiohydrolase, an endoglucanase, a beta-glucosidase, or any combination thereof. In some embodiments, the cellulase is derived from a fungus. In some embodiments, the cellulase is derived from a strain of Trichoderma or a strain of Penicillium. In some embodiments, the protease is an alkaline protease. In some embodiments, the protease is a non-alkaline protease. In some embodiments, the protease includes an amino acid sequence set forth by SEQ ID NOs: 1, 2, or 3, or an amino acid sequence having at least 70, 80, 90, 95, 98, or 100% sequence identity to the sequence set forth by SEQ ID NOs: 1, 2, or 3. In some embodiments, the enzyme composition includes a hemicellulase. In some embodiments, the hemicellulase is a xylanase, an esterase, an arabinofuranosidase, or any combination thereof. In some embodiments, the enzyme composition further includes a xylanase. In some embodiments, the xylanase includes an amino acid sequence set forth by SEQ ID NOs: 4 or 5, or an amino acid sequence having at least 70, 80, 90, 95, 98, or 100% sequence identity to the sequence set forth by SEQ ID NOs: 4 or 5. In some embodiments, the rice slurry is produced by milling a rice substrate. In some embodiments, the rice substrate is polished rice. In some embodiments, the rice substrate is broken rice. In some embodiments, the rice substrate is contacted with the enzyme composition during milling. In some embodiments, the rice slurry is contacted with the enzyme composition after milling. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 8 to about 10. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 5 to about 7. In some embodiments, the method includes adjusting a pH of the treated slurry to a pH in a range of about 8 to about 10. In some embodiments, the adjusting the pH of the treated slurry occurs at least 30 minutes after the rice slurry is contacted with the enzyme composition. In some embodiments, the adjusting the pH of the treated slurry occurs at least 1 hour after the rice slurry is contacted with the enzyme composition. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a temperature in a range of about 30 to 55°C. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a temperature below 30°C. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a temperature below 30°C to produce the treated slurry, and the treated slurry is heated to a temperature in a range of about 30 to 55 °C. In some embodiments,IFF10169-WO-PCT the treated slurry is heated at least 30 minutes after the rice slurry is contacted with the enzyme composition. In some embodiments, the treated slurry is heated at least 1 hour after the rice slurry is contacted with the enzyme composition. In some embodiments, the treated slurry is incubated for about 2 to about 8 hours. In some embodiments, the method includes separating the treated slurry to produce a protein stream and a starch stream. In some embodiments, the treated slurry is diluted before and / or during the separating. In some embodiments, the method includes concentrating and / or purifying the protein stream. In some embodiments, the method includes contacting the protein stream with a second enzyme or second enzyme composition. In some embodiments, the second enzyme or second enzyme composition includes a protease and / or a phytase. In some embodiments, the protease is a glutamine- specific protease and / or a prolinespecific protease. In some embodiments, the treated slurry, the protein stream, and / or the starch stream is heated to at least about 80°C, optionally for at least about 10 min. In some embodiments, the method includes drying the protein stream to produce a dry rice protein. In some embodiments, the dry rice protein is a dry hydrolyzed rice protein isolate. In some embodiments, the method includes concentrating and / or purifying the starch stream. In some embodiments, the method includes drying the starch stream to produce a dry rice starch.

[0008] In an aspect is a rice protein produced according to the methods described herein. In some embodiments, the rice protein has a molecular weight of less than 5 kilodaltons. In some embodiments, the rice protein is at least 70% pure.

[0009] In an aspect is provided a product including a rice protein produced according to the methods described herein, where the product is a food product, a beverage product, a functional nutrition product, a health ingredient product, a cosmetic product, a personal care product, a pharmaceutical product, a pet food product, an animal nutrition product, or a product for industrial application.

[0010] In another aspect is provided a system for producing rice protein, including a concentration unit and a dryer unit, wherein the concentration unit is in operable contact with the dryer unit; and wherein the concentration unit receives a protein stream and produces a concentrated protein, and the dryer unit receives the concentrated protein and produces a dry rice protein. In some embodiments, the concentration unit includes one or more of a strainer, a cross flow membrane filtration system, a dynamic cross flow membrane filtration system, a disk stack centrifuge, a decanter bowl centrifuge, a vortex separator, a gravity setter, a pressure filter, a vacuum filter, a mechanical pressure filter, an evaporator, or any combinations thereof. In some embodiments, the system includes a purification unit in operable contact with the concentration unit and the dryer unit, and wherein the purification unit receives the concentrated protein from the concentrationIFF10169-WO-PCT unit, purifies the concentrated protein stream to produce a purified protein, and the dryer unit receives the purified protein from the purification unit and produces a dry rice protein. In some embodiments, the purification unit includes one or more of a cross flow membrane filtration system, a dynamic cross flow membrane filtration system, an ion exchange resin system, or an electrodialysis system. In some embodiments, the cross flow membrane filtration system and / or the dynamic cross flow membrane filtration system includes, independently, a microfiltration membrane, an ultrafiltration membrane, a nanofiltration membrane, or reverse osmosis membrane. In some embodiments, the system includes one or more pairs of a concentration unit and a purification unit. In some embodiments, the concentration unit is in further operable contact with a separation unit, wherein the separation unit is included in a rice starch extraction system, and wherein the separation unit receives a rice slurry contacted with an enzyme composition and produces the protein stream and a starch stream. In some embodiments, the separation unit includes one or more of a disk stack centrifuge, a decanter bowl centrifuge, a filtering centrifuge, a vortex separator, a gravity settler, a pressure filter, a vacuum filter, a mechanical pressure filter, a dynamic cross flow membrane, or a cross flow membrane filter. In some embodiments, the system further includes a reaction unit in operable contact with the purification unit and the dryer unit, wherein the reaction unit receives a purified protein from the purification unit and produces a reacted protein, and the dryer unit receives the reacted protein from the reaction unit and produces a dry rice protein.

[0011] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIGs: 1A-1C show the amount of protein recovered from a rice slurry using an alkaline method and an exemplary enzymatic method. FIG. 1A shows the total protein in grams per 100 grams in the supernatant and pellet determined by Kjeldahl analysis for each method. FIG. IB shows the total protein in grams in the supernatant and pellet for each method. FIG. 1C shows the percentage of total protein in the supernatant and pellet for each method.

[0013] FIG. 2 shows the average moisture content percentage in wet pellets obtained using the alkaline method (61.5%) and the exemplary enzymatic method (57.9%).

[0014] FIGs. 3A and 3B show the viscosity profiles for slurries obtained using the alkaline method and the exemplary enzymatic method. FIG. 3 A shows the viscosity profile for the slurry over time according to treatment method. FIG. 3B shows the final viscosity of the slurry according to treatment method.IFF10169-WO-PCT

[0015] FIG. 4 shows average starch damage in starch cakes obtained using the alkaline method (8.98) and the exemplary enzymatic method (4.33).

[0016] FIG. 5 shows an exemplary rice protein production system and its connection to a rice starch extraction system.

[0017] FIG. 6 shows an exemplary rice protein production system and its connection to a rice starch extraction system.

[0018] FIG. 7 shows the percentage of rice protein having a particular molecular weight (D, dalton) by method of extraction (Alkaline Method (conventional) or an exemplary Enzyme Method).

[0019] FIG. 8 shows the percentage of solubilized rice protein using an exemplary Enzyme Method performed using two different enzymes (proteases).

[0020] FIG. 9 shows the percentage of rice protein having a particular molecular weight (D, dalton) for two different enzymes (proteases) using an exemplary Enzyme Method.DETAILED DESCRIPTION

[0021] Rice is a major food grain for more than half of the world's population. Proteins (approximately 8%) and starch (approximately 80%) represent the two main components of rice. Compared with other grain starch granules, rice starch has small, uniform granules, a soft mouthfeel, is odorless, and waxy rice starch has excellent freeze-thaw stability, making rice starch useful in a variety of applications, including, but not limited to, in the food and pharmaceutical industries, among others. Rice protein is hypoallergenic and high in essential amino acids, making it useful in various applications, such as, but not limited to, human food.

[0022] Rice protein is known to have bioactive peptides that offer numerous health benefits, such as anti-hypertensive, anti-inflammatory and immunomodulatory effects. Rice protein isolate is known for its innate properties of high purity, hypoallergenicity, and compatibility with cleanlabel formulations. These characteristics make it suitable for applications such as protein-enriched snacks, baked goods, and dairy alternatives, where its nutritional value and allergen-free profile are advantageous. However, rice protein isolate exhibits low solubility under neutral and acidic pH conditions due to its hydrophobic amino acid composition and strong intermolecular bonding, which limits its use in aqueous systems unless modified.

[0023] The main difference between hydrolyzed rice protein isolates and non-hydrolyzed isolates lies in their molecular weight distribution. Hydrolyzed isolates consist of low molecular weight peptides resulting from enzymatic hydrolysis, which significantly enhances solubility, digestibility, and bioavailability. This transformation enables their use in applications requiringIFF10169-WO-PCT rapid absorption and functional efficacy, such as clinical nutrition, hypoallergenic infant formulas, and cosmetic formulations.

[0024] A known challenge associated with hydrolyzed plant proteins, including hydrolyzed rice protein, is their inherent bitterness. This sensory drawback is primarily due to the exposure of hydrophoic amino acids due to hydrolysis. However, the bitterness can be addressed through selective enzymatic processing, activated carbon treatment, or the incorporation of flavor-masking agents, allowing for improved palatability in products that are consumed, for example in food and beverage, functional nutrition and health ingredients, and in pet food and animal nutrition products.

[0025] As a result, hydrolyzed rice protein isolate can be effectively used in specialized applications such as functional beverages, therapeutic nutrition products, and personal care formulations, where its enhanced solubility and bioactive properties offer distinct advantages over conventional rice protein isolate.

[0026] Although both rice starch and rice protein are valuable ingredients for numerous applications and industries, it remains a challenge to extract both components in a single process.

[0027] Rice endosperm has a compact internal structure with protein molecules aggregated by crosslinking through disulfide bonds and hydrophobic groups, resulting in the starch being tightly embedded in the protein network. Methods to release one of the rice starch or rice protein often result in the destruction (e.g., degradation) of or otherwise render the other component unavailable or unusable due to poor quality or low yield. For example, a common starch extraction method uses alkaline leaching, which includes soaking rice in a dilute alkali solution, wet grinding the soaked rice, grading in a precipitation tank, removing the supernatant, and centrifuging the starch suspension to separate starch. Not only is the starch structure obtained under alkaline conditions easily damaged, but the protein residue in the separated starch can be high due to an inability to effectively separate out the protein. Starches with too high a protein content are susceptible to deterioration and non-enzymatic browning of the protein by the glucose converted in the starch. Furthermore, the alkaline method is time intensive, costly, and can be a pollutant to the environment.

[0028] Rice starch plants typically discard rice protein because the protein concentration in the protein containing effluent stream is too low, making it cost prohibitive to recover. Furthermore, the alkaline process for rice starch milling, which typically occurs at a pH above 10.5, e.g., about 11-12, and existing common rice protein recovery methods (e.g. isoelectric precipitation) tend to result in protein with poor functional qualities. Thus, there is a need for new methods, compositions, and systems to facilitate the extraction of rice starch and rice protein from riceIFF10169-WO-PCT substrates in a single process to afford rice processing plants the ability to co-produce rice starch and rice protein. The methods, compositions, and systems provided herein address such needs.

[0029] As described in Section IV, it was surprisingly found that contacting a rice slurry with an enzyme composition including a protease, a combination of protease and cellulase, and optionally including a xylanasc, resulted in improved separation of rice protein and rice starch, allowing for separate protein and starch streams to be independently processed to produce both rice starch and rice protein in a single process. The exemplary enzyme compositions provided herein can be used at lower pHs than the typical alkaline methods, which occur above 10.5, e.g., 1 1-12, thus preserving the rice protein’s functional properties that may be compromised when subjected to higher alkaline conditions. Furthermore, the methods of using an enzyme composition provided herein may increase the solubility of rice protein at lower pH conditions, e.g., less than pH 11. In some embodiments, contacting a rice slurry with an enzyme composition provided herein increases rice starch yield and / or decreases damage to the rice starch. Thus, in some cases, the methods including the use of the enzyme compositions provided herein allow for improved protein extraction while maintaining extracted rice starch quality, increasing rice starch yield, and / or decreasing damage to the rice starch. In some cases, use of the enzyme composition reduces the viscosity of the rice slurry. Decreases in viscosity may improve separation of rice protein from the rice starch and realize cost efficiencies and increased productivity. In addition, in some embodiments, the enzyme composition reduces the water holding capacity of the rice starch. A decrease in water retention can decrease the drying step (e.g., reduce drying time) needed to produce a dry rice starch product, thereby decreasing energy consumption and realizing further cost efficiencies. Thus, in an aspect is provided a method for co-producing rice starch and rice protein, including contacting a rice slurry with an enzyme composition to produce a treated slurry, where the enzyme composition includes a protease, a protease and a cellulase, and, optionally, a xylanase.

[0030] It was also found that the rice protein could be separated from the rice starch and independently processed from the rice starch using a rice protein production system that may interface with a rice starch extraction system. Thus, in an aspect is also provided a system for producing rice protein, including at least one of a concentration unit or a purification unit, and a dryer unit. In some embodiments, the system for producing rice protein includes at least one of a concentration unit or a purification unit, a reaction unit, and a dryer unit. Such a system may be attached to an existing rice starch production system to capture extracted protein separated from the extracted starch and process the extracted protein to produce a dried rice protein.IFF10169-WO-PCT

[0031] Thus, the methods, compositions, and systems provided herein offer a holistic solution for co-producing rice starch and rice protein in a single process. In some embodiments, the rice protein produced from the method and systems provided herein is a hydrolyzed rice protein. In some embodiments, the rice protein produced from the method and systems provided herein is a hydrolyzed rice protein isolate. The methods, compositions, and systems described herein enable improved protein quality, improved separation efficiency, and, therefore, improved protein yield, without negatively impacting starch quality. The methods, compositions, and systems provided herein produce rice protein, e.g., hydrolyzed rice protein or hydrolyzed rice protein isolate, for use in various products.

[0032] The headings provided herein are not limitations of the various aspects or embodiments of this disclosure which can be had by reference to the specification as a whole. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. The reader will appreciate that statements made in one section may apply to other sections. Any terms defined may be more fully defined by reference to the specification as a whole.

[0033] All publications, including patent documents, scientific articles, and databases, referred to in this application are incoiporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications, and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.Definitions

[0034] Definitions of terms may appear throughout the specification. It is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0035] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” include “at least one” and “one or more.”

[0036] The terms “comprising,” “comprises,” and “comprised of” as used herein are synonymous with “including,” “includes,” “containing,” “contains,” “having,” “has,” and grammatical variants thereof, and are inclusive or open-ended and do not exclude additional, non-recited members,IFF10169-WO-PCT elements, or method steps. The terms “comprising.” “comprises,” “comprised of,” “including,” “includes,” “containing,” “contains,” “having,” “has,” and grammatical variants thereof also include the term "consisting of.” For example, it is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of and / or “consisting essentially of’ arc also provided.

[0037] The term “consisting of means “including and limited to.”

[0038] The term “consisting essentially of” means the specified material of a composition, or the specified steps of a method, and those additional materials or steps that do not materially affect the basic characteristics of the material or method. For example, an enzyme composition may include one or more enzymes as well as additives, antioxidants, sugars, preservatives, etc.

[0039] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation.

[0040] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0042] The term “rice” refers to a plant which is classified as a strain of Oryza sativa.

[0043] The term “rice substrate” includes all forms of rice (e.g., polished or unpolished), such as whole grains, broken rice, rice grits and rice flour, and any plant part.

[0044] 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 (CeHioOslx, wherein X can be any number.

[0045] The term “slurry” refers to a mixture of liquid and solid components, where the solid components are insoluble.

[0046] The term “dry solids content (ds)” refers to the total solids of a slurry (in %) on a dry weight basis.

[0047] The term “incubating” refers to admixing a rice slurry with an enzyme, e.g., an enzyme composition as described herein, under given conditions for a defined period of time.

[0048] As used herein, the term “Enzyme Commission” Number, abbreviated “EC," refers to enzyme nomenclature recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), as generally known to one skilledIFF10169-WO-PCT in the art (e.g., see enzyme nomenclature from NC-IUBMB, 1992 (Academic Press, San Diego, California), including supplements 1-5 published in 1994 (Eur. I. Biochem., 223: 1-5), 1995 (Eur. I. Biochem. 232: 1-6); 1996 (Eur. J. Biochem, 237: 1-5), 1997 (Eur. J. Biochem. 250: 1-6) and 1999 (Eur. J. Biochem. 264: 610-650), respectively. Likewise, the nomenclature is regularly supplemented and updated (sec, e.g., chcm.qmul.ac.uk / iubmb / cnzymc / mdcx.html).

[0049] As used herein, the term “cellulase” means enzymes that hydrolyze cellulosic material. Such enzymes include endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. The two basic approaches for measuring cellulolytic activity include: (1) measuring the total cellulolytic activity, and (2) measuring the individual cellulolytic activities (endoglucanases, cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al., Outlook for cellulase improvement: Screening and selection strategies, 2006, Biotechnology Advances 24: 452-481. Total cellulolytic activity is usually measured using insoluble substrates, including Whatman Nol 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 Nol filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (1987, Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

[0050] As used herein, the term “bcta-glucosidasc,” also referred to as BGL, 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.

[0051] As used herein, the term “cellobiohydrolase” includes l,4- / ?-D-glucan glucohydrolases (E.C. 3.2.1.74), 1,4-p-D-glucan cellobiohydrolase (E.C. 3.2.1.91), and enzymes having activity classified according to E.C. 3.2.1.176, e.g., cellulose 1,4-beta-cellobiosidase (reducing end). Cellobiohydrolases typically cleave cellulose strands to produce cellobiose. In some embodiments, the cellobiohydrolase is a cellobiohydrolase I (CBHI). In some embodiments, the cellobiohydrolase is a cellobiohydrolase II (CBHII).

[0052] The term “endoglucanase,” also referred to as EG, refers to an endo-l,4-(l,3;l,4)- beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) that catalyzes endohydrolysis of 1,4-beta-D- glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta- 1,4 bonds in mixed beta- 1,3 glucans such as cereal beta- D-glucans or xyloglucans, and other plant material containing cellulosic components. An endoglucanase also refers to an enzyme classified according to E.C. 3.2.1.6. In some embodiments, the endoglucanase is an endoglucanase I (EGI). In some embodiments, the endoglucanase is an endoglucanase II (EGII).IFF10169-WO-PCT

[0053] The term “hemicellulase” means enzymes that hydrolyze a hemicellulosic material. See, for example, Shallom, D. and Shoham, Y. Microbial hemicellulases. Current Opinion In Microbiology, 2003, 6(3): 219-228). Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to, an acetylmannan esterase, an acctylxylan esterase, an arabinanasc, an arabinofuranosidasc, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a pectate lyase, a xylanase, and a xylosidase. The substrates of these enzymes, the hemicelluloses, are a heterogeneous group of branched and linear polysaccharides that are bound via hydrogen bonds to the cellulose microfibrils in the plant cell wall, crosslinking them into a robust network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a highly complex structure. The variable structure and organization of hemicelluloses require the concerted action of many enzymes for its complete degradation. The catalytic modules of hemicellulases are either glycoside hydrolases (GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze ester linkages of acetate or ferulic acid side groups. These catalytic modules, based on homology of their primary sequence, can be assigned into GH and CE families. Some families, with an overall similar fold, can be further grouped into clans, marked alphabetically (e.g., GH-A). A most informative and updated classification of these and other carbohydrate active enzymes is available in the Carbohydrate- Active Enzymes (CAZy) database. Hemicellulolytic enzyme activities can be measured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a suitable temperature, e.g., 50°C, 55°C, or 60°C, and pH, e.g., 5.0 or 5.5.

[0054] As used herein, the term “protease” includes any enzyme belonging to the EC 3.4 enzyme group (including each of the eighteen subclasses thereof). As described herein, proteins (polypeptides) having protease activity (i.e., proteases), are also known in the art as peptidases, proteinases, peptide hydrolases, and proteolytic enzymes.

[0055] As used herein, protease activity means proteolytic activity (EC 3.4). Protease activity can generally be measured using any assay, in which a substrate is employed, that includes peptide bonds relevant for the specificity of the protease in question. Assays for pH and assays for temperature are likewise to be adapted to the protease in question. Examples of assay pH-values are pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 and examples of assay temperatures are 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 80°C, 90°C, or 95°C. Examples of general protease substrates are casein, bovine serum albumin (BSA), and hemoglobin.IFF10169-WO-PCT

[0056] As used herein, proteases may be of the “exo-type” (i.e., exopeptidases) that hydrolyze proteins (peptide bonds) starting at either N-terminal or C-terminal end of the protein chain, or of the “endo-type” (i.e., endopeptidases) that hydrolyze peptide bonds of the nonterminal ends of the protein chain (i.e., internal peptide bonds).

[0057] As used herein, the terms “contacting," “admixing," “adding," “combining,” and the like, including grammatical variants thereof, refer to the combining of one or more ingredients and / or enzymes, where the one or more ingredients or enzymes are combined in any order and in any combination. In some embodiments, contact may relate to mixing one or more ingredients and / or enzymes. Those skilled in the art will recognize that mixing solutions of the enzymes with the respective rice substrate can affect contacting.

[0058] The terms “recovered,” “isolated,” “extracted,” “separated," and the like, including grammatical variants thereof, refer to a compound, protein (polypeptide), cell, nucleic acid, amino acid, or other specified material or component that is removed from at least one other material or component. In some embodiments, the at least one other material or component is at least one other material or component with which the compound, protein (polypeptide), cell, nucleic acid, amino acid, or other specified material or component is naturally associated as found in nature. In some embodiments, the at least one other material or component is at least one other material or component with which the compound, protein (polypeptide), cell, nucleic acid, amino acid, or other specified material or component is associated with under experimental or production conditions and / or systems. For example, an “isolated” polypeptide includes, but is not limited to, a polypeptide removed from a culture broth containing a heterologous host cell expressing the polypeptide.

[0059] As used herein, “from” encompasses “derived from,” “originated from,” “obtained from," “isolated from," and the like, and grammatical variants thereof.

[0060] As used herein, the terms “wild-type” and “native” are used interchangeably and refer to genes, proteins, strains, or other components found in nature, or that are not intentionally modified.

[0061] The term “amino acid sequence” is synonymous with the terms “polypeptide,” “protein,” and “peptide,” and are used interchangeably. Where such amino acid sequences exhibit activity, they may be referred to as an “enzyme.” The conventional one-letter or three-letter codes for amino acid residues are used, with amino acid sequences being presented in the standard amino-to-carboxy terminal orientation (i.e., N— >C).

[0062] The term “nucleic acid” encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or doubleIFF10169-WO-PCT stranded and may contain chemical modifications. The terms “nucleic acid” and “polynucleotide” are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences arc presented in 5'-to-3' orientation.

[0063] “Hybridization” refers to the process by which one strand of nucleic acid forms a duplex with, i.e., base pairs with, a complementary strand, as occurs during blot hybridization techniques and PCR techniques. Stringent hybridization conditions are exemplified by hybridization under the following conditions: 65°C and 0. IX SSC (where IX SSC = 0.15 M NaCl, 0.015 M Nas citrate, pH 7.0). Hybridized, duplex nucleic acids are characterized by a melting temperature (Tm), where one half of the hybridized nucleic acids are unpaired with the complementary strand. Mismatched nucleotides within the duplex lower the Tm.

[0064] The terms “transformed,” “stably transformed,” and “transgenic,” used with reference to a cell means that the cell contains a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or carried as an episome that is maintained through multiple generations.

[0065] The term “introduced” in the context of inserting a nucleic acid sequence into a cell, encompasses, but is not limited to, “transfection”, “transformation” and “transduction,” as known in the art. Exemplary methods for introducing polynucleotides or polypeptides by transformation into a host cell, include, but are not limited to, microinjection, electroporation, stable transformation methods, transient transformation methods (such as induced competence using chemical (e.g. divalent cations such as CaCh), mechanical (electroporation) means, or methods such as those described in published international applications WO2018 / 114983 and W02010 / 149721, which are incorporated herein by reference in their entireties), ballistic particle acceleration (particle bombardment), direct gene transfer, viral -mediated introduction, cellpenetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery. Introducing a nucleic acid, construct, plasmid, or vector into a host cell may be carried out by conjugation, which is a specific method of natural DNA exchange requiring physical cell-to-cell contact. Introducing a nucleic acid, construct, plasmid, or vector into a host cell may be carried out by transduction, which is the introduction of DNA via a virus (e.g., phage) infection which is also a natural method of DNA exchange. Generally, such methods involve incorporating a polynucleotide within a viral DNA or RNA molecule.

[0066] A “host cell” is an organism into which an expression vector, phage, virus, or other nucleic acid sequence including a polynucleotide encoding a polypeptide of interest (e.g., anIFF10169-WO-PCT epimerase) has been introduced. Exemplary host cells are microorganism cells (e.g., bacteria, filamentous fungi, and yeast), mammalian cells, and plant cells capable of expressing the polypeptide of interest. The term “host cell” includes protoplasts created from cells.

[0067] Those of skill in the art are well aware of suitable methods for introducing polynucleotides into filamentous fungal cells (e.g., Aspergillus sp., Trichoderma sp., etc.), wherein standard techniques for transformation of filamentous fungi and culturing the fungi (which are well known to one skilled in the art) are used to transform a fungal host cell of the disclosure. Thus, the introduction of a DNA construct or vector into a fungal host cell includes techniques such as transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection mediated and DEAE-Dextrin mediated transfection), incubation with calcium phosphate DNA precipitate, high velocity bombardment with DNA-coated microprojectiles, gene gun or biolistic transformation, protoplast fusion and the like. General transformation techniques are known in the art (see, e.g., Ausubel et al., 1987, Sambrook etal., 2001 and 2012, and Campbell et al., 1989). Also of use is the Agrobacterium-mediated transfection method such as the one described in U.S. Patent No. 6,255,115. The expression of heterologous proteins in Trichoderma has been described, for example, in U.S. Patent Nos. 6,022,725; 6,268,328; Harkki et al., 1991 and Harkki et al., 1989. Reference is also made to Cao et al. (2000), for transformation of Aspergillus strains.

[0068] Transformation of Trichoderma sp. cells generally use protoplasts or cells that have been subjected to a permeability treatment, typically at a density of 105to 107 / mL, particularly 2xlO6 / mL. A volume of 100 pL of these protoplasts or cells in an appropriate solution (e.g., 1.2 M sorbitol and 50 mM CaCh) is mixed with the desired DNA. Generally, a high concentration of polyethylene glycol (PEG) is added to the uptake solution. Additives, such as dimethyl sulfoxide, heparin, spermidine, potassium chloride and the like, may also be added to the uptake solution to facilitate transformation. Similar procedures are available for other fungal host cells.

[0069] Nucleic acid sequences (i.e. polynucleotides) or proteins (i.e. polypeptides) may be native or heterologous to the genome of the host cell.

[0070] ‘ ‘Native,” “homologous,” or “endogenous” with respect to a host cell, means that the nucleic acid sequence does naturally occur in the genome of the host cell or that the protein is naturally produced by that cell. The terms “native,” “homologous,” and “endogenous” are used interchangeably herein.

[0071] As used herein, “heterologous” may refer to a nucleic acid sequence or a protein. For example, “heterologous,” with respect to the host cell, may refer to a polynucleotide that does not naturally occur in that way in the genome of the host cell or that a polypeptide or protein is notIFF10169-WO-PCT naturally produced in that manner by that cell. A heterologous nucleic acid sequence is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. For example, a promoter operably linked to a native structural gene is from a species different from that from which the structural gene is derived, or, if from the same species, one or both arc substantially modified from their original form. A heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention. That is, heterologous protein expression includes expression of a protein that is not naturally expressed in that way in the host cell. The term “heterologous expression’’ refers to the expression of heterologous nucleic acids in a host cell. The expression of heterologous proteins in eukaryotic host cell systems such as yeast and fungus are well known to those of skill in the art. A polynucleotide comprising a nucleic acid sequence of a gene encoding a certain protein or enzyme with a specific activity can be expressed in such a eukaryotic system. In some embodiments, transformed / transfected cells may be employed as expression systems for the expression of the enzymes.

[0072] The term “expression” refers to the process by which a polypeptide is produced based on a nucleic acid sequence. The process includes both transcription and translation.

[0073] A “vector” refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.

[0074] An “expression vector” refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

[0075] The terms “operably linked,” “operable contact,” “operably connected,” and the like, mean that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner.

[0076] A “signal sequence” is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal sequence, which is cleaved off during the secretion process.

[0077] As used herein, the terms “recombinant” or “non-natural” refer to an organism, microorganism, cell, nucleic acid molecule, vector, polypeptide and the like that has at least oneIFF10169-WO-PCT engineered genetic alteration, or has been modified by the introduction of a heterologous nucleic acid molecule; or refer to a cell (e.g., a host cell) that has been altered such that the expression of a heterologous nucleic acid molecule or an endogenous nucleic acid molecule or gene can be controlled. Recombinant also refers to a cell that is derived from a non-natural cell or is progeny of a non-natural cell having one or more such modifications. Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins, or other nucleic acid molecule additions, deletions, substitutions, or other functional alteration of a cell’s genetic material. For example, recombinant cells may express genes or other nucleic acid molecules (e.g., polynucleotide constructs) that are not found in identical or homologous form within a native (wild-type) cell or may provide an altered expression pattern of endogenous genes, such as being over-expressed, under-expressed, minimally expressed, or not expressed at all. “Recombination,” “recombining,” or generating a “recombined” nucleic acid is generally the assembly of two or more nucleic acid fragments wherein the assembly gives rise to a chimeric DNA sequence that would not otherwise be found in the genome.

[0078] The term “specific activity” refers to the number of moles of substrate that can be converted to product by an enzyme or enzyme preparation per unit time under specific conditions. Specific activity is generally expressed as units (U) / mg of protein.

[0079] “A cultured cell material” or similar language, refers to a cell lysate or supernatant (including media) that includes a protein of interest as a component. The cell material may be from a cell or host cell that is grown in culture for the purpose of producing the protein. In some embodiments, the enzyme composition, e.g., first enzyme composition, second enzyme composition, is a cultured cell material. For example, the cellulases may be contained in a cultured cell material, such as a whole broth.

[0080] As used herein, “clarified,” when used in reference to cultured cell material, e.g., a whole broth, means a cultured cell material which has been subjected to at least one clarification process to remove cell debris and / or other insoluble components. Clarification processes, as understood in the art include, but are not limited to, centrifugation techniques, cross-flow membrane filtration techniques, solid / liquid filtration techniques, and the like.

[0081] “Percent sequence identity” means that a particular sequence has at least a certain percentage of amino acid residues or nucleotides identical to those in a specified reference sequence, when aligned using e.g., the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. Default parameters for the CLUSTAL W algorithm are:Gap opening penalty: 10.0IFF10169-WO-PCTGap extension penalty: 0.05Protein weight matrix: BLOSUM seriesDNA weight matrix: IUBDelay divergent sequences %: 40Gap separation distance: 8DNA transitions weight: 0.50List hydrophilic residues: GPSNDQEKRUse negative matrix: OFFToggle Residue specific penalties: ONToggle hydrophilic penalties: ONToggle end gap separation penalty OFF.

[0082] Deletions are counted as non-identical residues, compared to a reference sequence.Deletions occurring at either terminus are included.

[0083] The term “increased” as used herein can refer to a quantity or activity that is at least about0.1%, 0.5%, 1%, 2%, 3%. 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%. 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 50%, 100%, or 200% more than the quantity or activity for which the increased quantity or activity is being compared. The terms “increased,” “elevated,” “enhanced,” “improved,” and the like may be used interchangeably herein.

[0084] The temi “decreased” as used herein can refer to a quantity or activity that is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 50%, 100%, or 200% less than the quantity or activity for which the decreased quantity or activity is being compared. The terms “decreased,” “lowered,” “reduced,” and the like may be used interchangeably herein.

[0085] 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 this disclosure. 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 this disclosure, 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 this disclosure.IFF10169-WO-PCT

[0086] Numerical values and ranges may be presented herein with the numerical value being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrccitcd number can be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. For example, in connection with a numerical value, the term “about” refers to a range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in context. All values and ranges implicitly include the term “about” unless the context clearly dictates otherwise.I. METHODS AND COMPOSITIONS FOR CO-PRODUCING RICE PROTEIN AND RICE STARCH

[0087] Provided herein are methods for co-producing rice protein and rice starch including the use of an enzyme composition. The enzyme compositions for use according the methods described herein are useful for separating rice protein from rice starch such that both rice protein and rice starch can be produced from a rice substrate in a single process. Thus, in an aspect is provided a method for co-producing rice starch and rice protein, including contacting a rice slurry with an enzyme composition to produce a treated slurry.A. Enzyme Compositions

[0088] As described in Section IV, it was surprisingly found that enzymes, e.g., proteases, and combinations of enzymes, e.g., proteases, cellulases, and / or hemicellulases, could facilitate the separation of rice starch and protein from a rice substrate, including at pHs lower than those used in a traditional alkaline method, e.g., less than pH 11. The ability to use lower pHs may improve the quality of extracted rice protein and / or rice starch. Thus, the enzyme compositions contemplated for use according to the methods provided herein include one or more of a protease, a cellulase, or a hemicellulase. For simplicity, enzyme composition is used herein to refer to combinations of enzymes as well as single enzymes. For example, enzyme composition may refer to one or more of the enzymes described herein.

[0089] In some embodiments, the enzyme composition includes a protease described herein. In some embodiments, the enzyme composition includes a cellulase described herein and a protease described herein. In some embodiments, the enzyme composition includes a protease described herein and a hemicellulase described herein. In some embodiments, the enzyme composition includes a protease described herein, a cellulase described herein, and a hemicellulase described herein. In some embodiments, the enzyme composition includes a protease described herein and a xylanase described herein. In some embodiments, the enzyme composition includes a cellulaseIFF10169-WO-PCT described herein and a xylanase described herein. In some embodiments, the enzyme composition includes a cellulase described herein, a protease described herein, and a xylanase described herein. In some embodiments, the enzyme composition includes a composition as shown in Table E2. See, Section IV, below. In some embodiments, the enzyme composition is an enzyme composition including a composition and a protease as shown in Table E3. Sec Section IV. i. Proteases

[0090] In some embodiments, the enzyme composition includes a protease. In some embodiments, the protease is an alkaline protease. In some embodiments, the protease is a non- alkaline protease. In some embodiments, the protease is a recombinant protease.

[0091] In some embodiments, the protease has the amino acid sequence set forth by SEQ ID NO: 1, or has at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth by SEQ ID NO: 1. In some embodiments, the protease has an amino acid sequence with at least 60% sequence identity to SEQ ID NO: 1. In some embodiments, the protease has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, the protease has an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the protease has an amino sequence with at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the protease has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the protease has an amino acid sequence with at least 96% sequence identity to SEQ ID NO: 1. In some embodiments, the protease has an amino acid sequence with at least 97% sequence identity to SEQ ID NO: 1. In some embodiments, the protease has an amino acid sequence with at least 98% sequence identity to SEQ ID NO: 1. In some embodiments, the protease has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the protease has the amino acid sequence set forth by SEQ ID NO: 1.

[0092] In some embodiments, the protease has the amino acid sequence set forth by SEQ ID NO: 2, or has at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth by SEQ ID NO: 2. In some embodiments, the protease has an amino acid sequence with at least 60% sequence identity to SEQ ID NO: 2. In some embodiments, the protease has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 2. In some embodiments, the protease has an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the protease has an amino sequence with at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the protease has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the protease has an amino acid sequence with at least 96% sequence identityIFF10169-WO-PCT to SEQ ID NO: 2. In some embodiments, the protease has an amino acid sequence with at least 97% sequence identity to SEQ ID NO: 2. In some embodiments, the protease has an amino acid sequence with at least 98% sequence identity to SEQ ID NO: 2. In some embodiments, the protease has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the protease has the amino acid sequence set forth by SEQ ID NO: 2.

[0093] In some embodiments, the protease has the amino acid sequence set forth by SEQ ID NO: 3, or has at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth by SEQ ID NO: 3. In some embodiments, the protease has an amino acid sequence with at least 60% sequence identity to SEQ ID NO: 3. In some embodiments, the protease has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 3. In some embodiments, the protease has an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, the protease has an amino sequence with at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, the protease has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 3. In some embodiments, the protease has an amino acid sequence with at least 96% sequence identity to SEQ ID NO: 3. In some embodiments, the protease has an amino acid sequence with at least 97% sequence identity to SEQ ID NO: 3. In some embodiments, the protease has an amino acid sequence with at least 98% sequence identity to SEQ ID NO: 3. In some embodiments, the protease has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 3. In some embodiments, the protease has the amino acid sequence set forth by SEQ ID NO: 3.

[0094] As described above, the proteases provided herein may be used alone in an enzyme composition or in combination with other enzymes, e.g., as described herein. ii. Cellulase

[0095] In some embodiments, the enzyme composition includes a cellulase. In some embodiments, the cellulase includes a cellobiohydrolase (E.C. 3.2.1.91, E.C. 3.2.1.74, and / or E.C. 3.2.1.176), an endoglucanase (E.C. 3.2.1.4 and / or E.C. 3.2.1.6), a beta-glucosidase (E.C. 3.2.1.21), or any combination thereof. In some embodiments, the cellulase includes a cellobiohydrolase I (CBHI), a cellobiohydrolase II (CBHII), an endoglucanase I (EGI), an endoglucanase II (EGII), a beta-glucosidase (BGL), or any combination thereof. In some embodiments, the cellulase is a recombinant cellulase.

[0096] In some embodiments, the enzyme composition includes an endoglucanase. In some embodiments, the endoglucanase has the amino acid sequence set forth by SEQ ID NO: 6, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 6. In some embodiments,IFF10169-WO-PCT the endoglucanase has an amino acid sequence with at least 60% sequence identity to SEQ ID NO: 6. In some embodiments, the endoglucanase has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 6. In some embodiments, the endoglucanase has an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, the endoglucanase has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, the endoglucanase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, the endoglucanase has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 6. In some embodiments, the endoglucanase has the amino acid sequence set forth by SEQ ID NO: 6.

[0097] In some embodiments, the endoglucanase has the amino acid sequence set forth by SEQ ID NO: 7, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7. In some embodiments, the endoglucanase has an amino acid sequence with at least 60% sequence identity to SEQ ID NO: 7. In some embodiments, the endoglucanase has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 7. In some embodiments, the endoglucanase has an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 7. In some embodiments, the endoglucanase has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 7. In some embodiments, the endoglucanase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 7. In some embodiments, the endoglucanase has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 7. In some embodiments, the endoglucanase has the amino acid sequence set forth by SEQ ID NO: 7. In some embodiments, the percent identity to SEQ ID NO: 7 refers to a percent identity to a mature form of the sequence set forth by SEQ ID NO: 7. In some embodiments, the mature form of SEQ ID NO: 7 is amino acids 29 to 242 of the amino acid sequence set forth by SEQ ID NO: 7.

[0098] In some embodiments, the endoglucanase has the amino acid sequence set forth by SEQ ID NO: 8, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8. In some embodiments, the endoglucanase has an amino acid sequence with at least 60% sequence identity to SEQ ID NO: 8. In some embodiments, the endoglucanase has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 8. In some embodiments, the endoglucanase has an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 8. In some embodiments, the endoglucanase has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, the endoglucanase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, the endoglucanase has an amino acidIFF10169-WO-PCT sequence with at least 99% sequence identity to SEQ ID NO: 8. In some embodiments, the endoglucanase has the amino acid sequence set forth by SEQ ID NO: 8.

[0099] In some embodiments, the cellulase is derived from a fungus. In some embodiments, the cellulase is derived from a strain of Aspergillus. In some embodiments, the strain of Aspergillus is a strain of Aspergillus aurantiacus, Aspergillus nigcr, or Aspergillus oryzac. In some embodiments, the cellulase is derived from a strain of Chrysosporium. In some embodiments, the strain of Chrysosporium is a strain of Chrysosporium iucknowense. In some embodiments, the cellulase is derived from a strain of Humicola. In some embodiments, the strain of Humicola is a strain of Humicola insolens. In some embodiments, the cellulase is derived from a strain of Penicillium. In some embodiments, the strain of Penicillium is a strain of Penicillium emersonii, Penicillium funiculosum, or Penicillium oxalicum. In some embodiments, the cellulase is derived from a strain of Talaromyces. In some embodiments, the strain of Talaromyces is a strain of Talaromyces aurantiacus or Talaromyces emersonii. In some embodiments, the cellulase is derived from a strain of Trichoderma. In some embodiments, the strain of Trichoderma is a strain of Trichoderma reesei. In some embodiments, the cellulase is derived from a strain of Trichoderma reesei.

[0100] In some embodiments, the fungus from which the cellulase is derived is a host cell. In some embodiments, the fungus is a recombinant fungus (e.g., host cell) that expresses, overexpresses, or does not express one or more cellulases. In some embodiments, the expressed or over-expressed cellulase is a recombinant cellulase. In some embodiments, the expressed or overexpressed cellulase is a heterologous cellulase. In some embodiments, the over-expressed cellulase is a native cellulase. In some embodiments, native, heterologous, and / or recombinant cellulase are derived from the fungus.

[0101] In some embodiments, the cellulase is contained in a cultured cell material, e.g., a cultured cell material from a fungus. In some embodiments, the cellulase is contained in a cultured cell material, e.g., a cultured cell material from a fungus where the fungus is a host cell (e.g., a recombinant fungus). In some embodiments, the cultured cell material is a whole broth. In some embodiments, the cultured cell material is clarified.

[0102] In some embodiments, the fungus from which the cellulase is derived also expresses a hemicellulase. In some embodiments, the hemicellulase is a xylanase, an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a pectate lyase, a xylosidase, or any combination thereof. In some embodiments, the hemicellulase includes a xylanase. In some embodiments, the hemicellulase includes an esterase.IFF10169-WO-PCTIn some embodiments, the esterase is a feruloyl esterase (E.C. 3.1.1.73). In some embodiments, the esterase is an acetylxylan esterase (E.C. 3.1.1.72). In some embodiments, the hemicellulase includes an arabinofuranosidase (E.C. 3.2.1.55). In some embodiments, the hemicellulase is a native hemicellulase, e.g., a native xylanase, esterase, arabinofuranosidase. In some embodiments, the hcmiccllulasc is a recombinant hcmiccllulasc, e.g., a recombinant xylanase, esterase, arabinofuranosidase. In some embodiments, the fungus from which the cellulase is derived is a host cell that expresses, over-expresses, or does not express a hemicellulase. In some embodiments, the expressed or over-expressed hemicellulase is a recombinant hemicellulase. In some embodiments, the over-expressed hemicellulase is a native hemicellulase.

[0103] In some cases, for example if the cellulase is a cultured cell material, the cellulase includes one or more hemicellulases. In some embodiments, the one or more hemicellulases include native and / or recombinant hemicellulases.

[0104] In some embodiments, the cellulases provided herein may be used alone in an enzyme composition or in combination with other enzymes, e.g., as described herein.Hi. Hemicellulases

[0105] In some embodiments, the enzyme composition includes a hemicellulase. For example, in some cases, the enzyme composition includes a hemicellulase that is not or is in addition to a hcmiccllulasc that may be part of a cultured cell material including cellulases. In some embodiments, the hemicellulase is a xylanase, an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a pectate lyase, a xylosidase, or any combination thereof. In some embodiments, the hemicellulase includes a xylanase. In some embodiments, the hemicellulase includes an arabinofuranosidase (E.C. 3.2.1.55). In some embodiments, the hemicellulase includes an esterase. In some embodiments, the esterase is a feruloyl esterase (E.C. 3.1.1.73). In some embodiments, the esterase is an acetylxylan esterase (E.C. 3.1.1.72). In some embodiments, the hemicellulase is a native hemicellulase. In some embodiments, the hemicellulase is a recombinant hemicellulase, e.g., a recombinant xylanase, esterase, arabinofuranosidase.

[0106] In some embodiments, the hemicellulase is not natively or recombinantly expressed by a fungus from which the cellulases are derived. Thus, in some cases, the enzyme composition includes a hemicellulase that is not contained in a cultured cell material. In some embodiments, the enzyme composition includes a hemicellulase, as described herein, that is native to a fungus from which the cellulase is derived and a hemicellulase that is heterologous to the fungus fromIFF10169-WO-PCT which the cellulases are derived. In some embodiments, the hemicellulase is a heterologous xylanase. In some embodiments, the hemicellulase is a recombinant xylanase.

[0107] In some embodiments, the enzyme composition includes a xylanase. In some embodiments, the enzyme composition includes a GH10 xylanase. In some embodiments, the xylanase has the amino acid sequence set forth by SEQ ID NO: 4, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth by SEQ ID NO: 4. In some embodiments, the xylanase has an amino acid sequence with at least 60% sequence identity to SEQ ID NO: 4. In some embodiments, the xylanase has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 4. In some embodiments, the xylanase has an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 4. In some embodiments, the xylanase has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, the xylanase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 4. In some embodiments, the xylanase has an amino acid sequence with at least 96% sequence identity to SEQ ID NO: 4. In some embodiments, the xylanase has an amino acid sequence with at least 97% sequence identity to SEQ ID NO: 4. In some embodiments, the xylanase has an amino acid sequence with at least 98% sequence identity to SEQ ID NO: 4. In some embodiments, the xylanase has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 4. In some embodiments, the xylanase has the amino acid sequence set forth by SEQ ID NO: 4. In some embodiments, the percent identity to SEQ ID NO: 4 refers to a percent identity to a mature form of the sequence set forth by SEQ ID NO: 4. In some embodiments, the mature form of SEQ ID NO: 4 is amino acids 24 to 328 of the amino acid sequence set forth by SEQ ID NO: 4.

[0108] In some embodiments, the xylanase has the amino acid sequence set forth by SEQ ID NO: 5, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth by SEQ ID NO: 5. In some embodiments, the xylanase has an amino acid sequence with at least 60% sequence identity to SEQ ID NO: 5. In some embodiments, the xylanase has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 5. In some embodiments, the xylanase has an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 5. In some embodiments, the xylanase has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 5. In some embodiments, the xylanase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, the xylanase has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 5. In some embodiments, the xylanase has the amino acid sequence set forth by SEQ ID NO: 5.IFF10169-WO-PCT

[0109] In some embodiments, the hemicellulases provided herein may be used alone in an enzyme composition or in combination with other enzymes, e.g., as described herein.B. Methods of Using Enzyme Compositions in the Co-Production Process

[0110] As described above, the enzyme compositions provided herein are useful for improving the separation of rice protein and rice starch from a rice substrate. In some embodiments, the methods provided herein allow for the production of a hydrolyzed rice protein or hydrolyzed rice protein isolate. Therefore, in some embodiments, the enzyme composition provided herein is contacted with a rice slurry from which rice protein and rice starch are to be extracted. In some embodiments, the rice slurry is produced by milling a rice substrate. Rice substrates suitable for the methods described herein include any form of polished or unpolished rice, such as whole grains, broken rice, rice grits and rice flour, and / or any plant part. In some embodiments, the rice substrate is a polished rice. In some embodiments, the rice substrate is a whole grain rice. In some embodiments, the rice substrate is a broken rice. In some embodiments, the enzyme composition provided herein is contacted with the rice substrate. i. Rice Substrate Milling and Treated Rice Substrates

[0111] Rice substrate milling may be accomplished using any suitable mill known in the art. In some embodiments, the rice substrate is milled to a desired particle size prior to dispersion in water or wet ground during processing. In some embodiments, the rice substrate is milled in the presence of water. In some embodiments, the rice substrate is milled in the presence of an alkali solution. In some embodiments, the rice substrate is milled in the presence of an alkali solution and water. In some embodiments, the milled rice substrate, such as rice flour, is slurried. In some embodiments, the milled rice substrate is slurried with water. Thus, in some embodiments, the milled rice substrate is slurried with water to create a rice slurry. In some embodiments, the milled rice substrate is slurried with an alkali solution to create a rice slurry. In some embodiments, the milled rice substrate is slurried with an alkali solution and water to create a rice slurry. Examples of alkali solutions include, but are not limited to, strong bases, Caustic, and ammonia. In some embodiments, the alkali solution is a strong base. In some embodiments, the alkali solution is Caustic. In some embodiments, the alkali solution is ammonia. In some embodiments, the rice slurry includes i) about 10 to about 55% ds, ii) about 20 to about 50% ds; iii) about 25 to about 45% ds; iv) about 20 to about 40% ds; v) about 20 to about 35% ds; or vi) about 30 to 35% ds.

[0112] In some embodiments, the rice substrate is contacted with an enzyme composition described herein during milling. In some embodiments, a rice substrate contacted with an enzyme composition described herein during, for example, a milling step, may be referred to herein as a treated rice substrate or a treated substrate.IFF10169-WO-PCT

[0113] In some cases, when the rice substrate is contacted with an enzyme composition including a cellulase and a protease, the rice substrate is milled in the presence of water. In some cases, when the rice substrate is contacted with an enzyme composition including a protease and a hemicellulase (e.g., a xylanase), the rice substrate is milled in the presence of water. In some cases, when the rice substrate is contacted with an enzyme composition including a cellulase, a protease, and a hemicellulase (e.g., a xylanase), the rice substrate is milled in the presence of water. In some cases, when the rice substrate is contacted with an enzyme composition including a cellulase and a protease, the rice substrate is milled in the presence of an alkali solution. In some cases, when the rice substrate is contacted with an enzyme composition including a protease and a hemicellulase (e.g., xylanase), the rice substrate is milled in the presence of an alkali solution. In some cases, when the rice substrate is contacted with an enzyme composition including a cellulase, a protease, and a hemicellulase (e.g., xylanase), the rice substrate is milled in the presence of an alkali solution.

[0114] In some cases, where the rice substrate is contacted with an enzyme composition including a protease, the rice substrate may be milled in the presence of an alkali solution. In some cases, where the rice substrate is contacted with a protease only, the rice substrate is milled in the presence of an alkali solution. In some embodiments, when the rice substrate is contacted with a cellulase and / or hcmiccllulasc only, the rice substrate is milled in the presence of water. ii. Rice Slurry and Treated Slurry

[0115] The slurried rice produced from the milling process, and optionally contacted with an enzyme composition during milling, may be contacted with an enzyme composition provided herein to improve the separation of rice protein and rice starch. Thus, in some embodiments, the rice slurry is contacted with an enzyme composition provided herein. In some embodiments, the rice slurry is contacted with an enzyme composition because the rice substrate was contacted with the enzyme composition during milling and the enzyme composition is therefore present in the rice slurry. A rice slurry that has been contacted with an enzyme composition described herein may be referred to interchangeably as a treated slurry or a treated rice slurry. In some embodiments, the contacting the rice slurry with the enzyme composition produces a treated slurry. A treated slurry, as used herein, includes an enzyme composition described herein and a rice slurry. In some embodiments, the contacting of the rice slurry occurs in a reaction tank unit. In some embodiments, the reaction tank unit is a reaction tank unit described in Section II.

[0116] In some embodiments, the rice substrate is milled and a rice slurry is produced, and the produced rice slurry is then contacted with the enzyme composition described herein. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a neutral pH.IFF10169-WO-PCTIn some embodiments, the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 4 to about 7.5. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 4.5 to about 7.5. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 5 to about 7.5. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 5.5 to about 7.5. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 6 to about 7.5. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 6.5 to about 7.5. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 7 to about 7.5. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 5 to about 7. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 6 to about 7.

[0117] In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 7.5 to about 10.5. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 7.5 to about 10. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 8 to about 10. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 9 to about 10.

[0118] In some embodiments, the pH of the contacted rice slurry (treated slurry) is adjusted. In some embodiments, the pH of the treated rice slurry is adjusted by addition of a base. In some embodiments, the pH is adjusted to be basic. In some embodiments, the pH of the treated slurry is adjusted to a pH in a range of about 7.5 to about 10.5. In some embodiments, the pH of the treated slurry is adjusted to a pH in a range of about 7.5 to about 10. In some embodiments, the pH of the treated slurry is adjusted to a pH in a range of about 8 to about 10.5. In some embodiments, the pH of the treated slurry is adjusted to a pH in a range of about 8 to about 10.

[0119] In some embodiments, the pH of the contacted rice slurry (treated slurry) is adjusted. In some embodiments, the pH of the treated rice slurry is adjusted by addition of an acid. In some embodiments, the pH is adjusted to be acidic. In some embodiments, the pH of the treated slurry is adjusted to a pH in a range of about 4 to about 7.5. In some embodiments, the pH of the treated slurry is adjusted to a pH in a range of about 4.5 to about 7.5. In some embodiments, the pH of the treated slurry is adjusted to a pH in a range of about 5 to about 7.5. In some embodiments, the pH of the treated slurry is adjusted to a pH in a range of about 5.5 to about 7.5. In some embodiments, the pH of the treated slurry is adjusted to a pH in a range of about 6 to about 7.5.IFF10169-WO-PCTIn some embodiments, the pH of the treated slurry is adjusted to a pH in a range of about 6.5 to about 7.5. In some embodiments, the pH of the treated slurry is adjusted to a pH in a range of about 7 to about 7.5. In some embodiments, the pH of the treated slurry is adjusted to a pH in a range of about 5 to about 7. In some embodiments, the pH of the treated slurry is adjusted to a pH in a range of about 6 to about 7.

[0120] In some embodiments, the pH adjustment of the treated slurry occurs at least 10, 20, 30, 40, or 50 minutes after the rice slurry is contacted with the enzyme composition. In some embodiments, the pH adjustment of the treated slurry occurs at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours after the rice slurry is contacted with the enzyme composition. In some embodiments, the pH adjustment of the treated slurry occurs at least 1, 2, 3, 4, 5, 6, or 7 hours after the rice slurry is contacted with the enzyme composition. In some embodiments, the pH adjustment of the treated slurry occurs at least 5 hours after the rice slurry is contacted with the enzyme composition. In some embodiments, the pH adjustment of the treated slurry occurs at least 3 hours after the rice slurry is contacted with the enzyme composition. In some embodiments, the pH adjustment of the treated slurry occurs at least 2 hours after the rice slurry is contacted with the enzyme composition. In some embodiments, the pH adjustment of the treated slurry occurs at least 1 hour after the rice slurry is contacted with the enzyme composition. In some embodiments, the pH adjustment of the treated slurry occurs at least 30 minutes after the rice slurry is contacted with the enzyme composition. In some embodiments, the pH is changed and / or maintained throughout the duration of the incubation. Thus, in some cases, the treated slurry may be pH adjusted more than once. In some embodiments, the pH is adjusted at regular intervals or continuously. In some embodiments, the pH is monitored manually or automatically and the pH is adjusted when and if it deviates from a target pH or pH range.

[0121] In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a temperature in the range of about 30 to 55°C. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a temperature in the range of about 40 to 55°C. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a temperature in the range of about 45 to 55°C. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a temperature in the range of about 50 to 55°C. In some embodiments, the contacting the rice slurry with the enzyme composition occurs at a temperature below 30°C.

[0122] In some embodiments, the temperature of the treated slurry is adjusted. For example, in some cases, the rice slurry is contacted with the enzyme composition at a given temperature and the temperature of the rice slurry contacted with the enzyme composition (treated slurry) isIFF10169-WO-PCT adjusted to a different temperature, e.g., a higher or lower temperature. In some embodiments, the temperature is adjusted to a higher temperature than the temperature at which the contacting of the enzyme composition with the rice slurry occurred. For example, the temperature is adjusted to a range described herein. In some embodiments, the temperature is adjusted to a lower temperature than the temperature at which the contacting of the enzyme composition with the rice slurry occurred.

[0123] In some embodiments, when the rice slurry is contacted with the enzyme composition at a temperature below or about 30°C, the treated slurry is heated to a temperature in a range of about 30 to 55°C, about 40 to 55°C, about 45 to 55°C, or about 50 to 55°C. In some embodiments, the heating of the treated slurry occurs at least 10, 20, 30, 40, or 50 minutes after the rice slurry is contacted with the enzyme composition. In some embodiments, the heating of the treated slurry occurs at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours after the rice slurry is contacted with the enzyme composition. In some embodiments, the heating of the treated slurry occurs at least 1, 2, 3, 4, 5, 6, or 7 hours after the rice slurry is contacted with the enzyme composition. In some embodiments, the heating of the treated slurry occurs at least 5 hours after the rice slurry is contacted with the enzyme composition. In some embodiments, the heating of the treated slurry occurs at least 3 hours after the rice slurry is contacted with the enzyme composition. In some embodiments, the heating of the treated slurry occurs at least 2 hours after the rice slurry is contacted with the enzyme composition. In some embodiments, the heating of the treated slurry occurs at least 1 hour after the rice slurry is contacted with the enzyme composition. In some embodiments, the heating of the treated slurry occurs at least 30 minutes after the rice slurry is contacted with the enzyme composition. In some embodiments, the temperature is changed and / or maintained throughout the duration of the incubation. Thus, in some cases, the treated slurry may be temperature adjusted more than once. In some embodiments, the temperature is adjusted at regular intervals or continuously. In some embodiments, the temperature is monitored manually or automatically and the temperature is adjusted when and if it deviates from a target temperature or temperature range.

[0124] In some embodiments, the pH and / or temperature adjustments are executed for purposes of modulating, e.g., enhancing, reducing, or maintaining, the function of enzymes in the enzyme composition. In some embodiments, adjusting the pH and / or temperature may allow different enzymes to act, e.g., increase or decrease activity, at different times during an incubation period.

[0125] To allow the enzyme composition described herein to act on the rice slurry after contact, the enzyme composition and contacted rice slurry may be incubated. Thus, in some embodiments, the treated slurry is incubated for at least 10, 20, 30, 40, or 50 minutes after the riceIFF10169-WO-PCT slurry is contacted with the enzyme composition. In some embodiments, the treated slurry is incubated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours after the rice slurry is contacted with the enzyme composition. In some embodiments, the treated slurry is incubated for at least 1, 2, 3, 4, 5, 6, or 7 hours after the rice slurry is contacted with the enzyme composition. In some embodiments, the treated slurry is incubated for at least 5 hours after the rice slurry is contacted with the enzyme composition. In some embodiments, the treated slurry is incubated for at least 3 hours after the rice slurry is contacted with the enzyme composition. In some embodiments, the treated slurry is incubated for at least 2 hours after the rice slurry is contacted with the enzyme composition. In some embodiments, the treated slurry is incubated for at least 1 hour after the rice slurry is contacted with the enzyme composition. In some embodiments, the treated slurry is incubated for at least 30 minutes after the rice slurry is contacted with the enzyme composition.

[0126] In some embodiments, adjustment to pH and / or temperature are performed during the incubation period to modify enzyme activity.

[0127] In some embodiments, the rice substrate is contacted during milling and the produced rice slurry is also contacted with the enzyme composition. In some embodiments, the method includes one or more or a plurality of contacts with an enzyme composition described herein. In some embodiments, the one or more or a plurality of contacts are with the same and / or different enzyme compositions. In some embodiments, the one or more or a plurality of contacts arc with the same enzyme composition. In some embodiments, the one or more or a plurality of contacts are with different enzyme compositions.

[0128] In some cases, the rice substrate is contacted with the enzyme composition during milling. In some embodiments, the rice slurry is contacted with the enzyme composition (e.g., after milling). In some embodiments, the rice substrate is contacted with the enzyme composition during milling and the resulting rice slurry is also contacted with the enzyme composition. In some embodiments, the enzyme composition contacted with the rice substrate and enzyme composition contacted with the rice slurry are the same enzyme compositions. In some embodiments, the enzyme composition contacted with the rice substrate and the enzyme composition contacted with the rice slurry are different enzyme compositions. In some embodiments, the rice slurry is contacted with two or more enzyme compositions, e.g., contacting at a first time point and then contacting at a second, later time point during incubation. In some embodiments, the enzyme compositions are the same. In some embodiments, the enzyme compositions are different. In some embodiments, the pH and / or temperature may be adjusted before and / or after the contacting with the enzyme compositions.Hi. Processing Rice Protein and Rice Starch StreamsIFF10169-WO-PCT

[0129] In some embodiments, the method includes separating the treated slurry to produce a protein fraction and a starch fraction. For example, in some embodiments, after the rice slurry is contacted with the enzyme composition, and, optionally, pH adjusted, temperature adjusted, and / or incubated as described herein, the treated slurry is capable of being separated into rice starch and rice protein streams that can be independently processed to produce a rice starch, c.g., a dry rice starch, and a rice protein, e.g., a dry rice protein. In some embodiments, the rice protein stream is processed to produce a hydrolyzed rice protein or hydrolyzed rice protein isolate, e.g., a dry hydrolyzed rice protein or dry hydrolyzed rice protein isolate. The separated protein and starch may be referred to as fractions or streams. For example, stream may be used to describe the output of a unit operation or process step of the method.

[0130] In some embodiments, the rice protein and rice starch streams may be processed independently to produce a rice protein and a rice starch. Thus, in some embodiments, the method includes separating the treated slurry to produce a protein stream and a starch stream. To facilitate separation, in some cases, the treated slurry may be diluted. For example, in some embodiments, the treated slurry is diluted before separating the treated slurry into a protein stream and a starch stream. In some embodiments, the treated slurry may be diluted during the separating into a protein stream and a starch stream. For example, in some cases, the method of separating the treated slurry into the two streams may include diluting the treated slurry in the process of separation. In some cases, the treated slurry may be diluted one or more times before the separating process. In some embodiments, the treated slurry may be diluted one or more times during the separating process. In some embodiments, the separating is accomplished by multiple separating steps. For example, in some cases, a multistage separation process gives rise to the protein and starch streams, e.g., multiple centrifuging steps give rise to the different streams. It is contemplated that any type of system capable of separating the treated slurry into a protein stream and a starch stream may be used. In some embodiments, the separating of the treated slurry is accomplished using a separation unit as described in Section II below.

[0131] As indicated supra, the separated protein and rice streams may be independently processed to produce a final product of rice starch, e.g., dry rice starch, and rice protein, e.g., dry rice protein. Such processing may include the additional method steps of concentrating and / or purifying the protein and starch streams, independently. Thus, in some embodiments, the method includes concentrating and / or purifying the protein stream. In some embodiments, the method includes concentrating the protein stream. In some embodiments, the protein content of the concentrated protein stream is greater than about 1 %, 5%, 10%, 20%, 30%, 40%, or 50%. In some embodiments, the protein content of the concentrated protein stream is greater than about 1 %, 5%,IFF10169-WO-PCT or 10%. In some embodiments, the protein content of the concentrated protein stream is greater than about 10%, 20%, 30%, 40%, or 50%. In some embodiments, the protein content of the concentrated protein stream is in the range of about 1% to 60%, in the range of about 1% to 50%, in the range of about 1% to 45%, and in the range of about 1% to 40%. In some embodiments, the protein content of the concentrated protein stream is in the range of about 5% to 60%, in the range of about 5% to 50%, in the range of about 5% to 45%, and in the range of about 5% to 40%. In some embodiments, the protein content of the concentrated protein stream is in the range of about 10% to 60%, in the range of about 10% to 50%, in the range of about 20% to 45%, and in the range of about 30% to 40%. In some embodiments, the method includes purifying the protein stream. In some embodiments, the protein content of the purified protein stream is greater than about 60%, 63%, 65%, 68%, 70%, or 75%. In some embodiments, the protein content of the purified protein stream is about 65% or greater. In some embodiments, the protein content of the purified protein stream is about 70% or greater. In some embodiments, the protein content of the purified protein stream is about 75% or greater. In some embodiments, the protein content of the purified protein stream is about 80% or greater. In some embodiments, the protein content of the purified protein stream is about 90% or greater. In some embodiments, the protein content of the purified protein stream is about 95% or greater.

[0132] In some embodiments, the method includes concentrating and purifying the protein stream. In some embodiments, for example when both concentrating and purifying the protein stream, the concentrating the protein stream occurs before the purifying. Thus, in some cases, the purifying of the protein stream is performed on the protein stream that has undergone concentration. In some embodiments, the protein content achieved by purification is made possible by first concentrating the protein stream. It is contemplated that any type of system capable of concentrating the protein stream may be used. In some embodiments, concentrating the protein stream is accomplished using a concentration unit as described in Section II. Similarly, any type of system capable of purifying the protein stream may be used. In some embodiments, purifying the protein stream is accomplished using a purifying unit as described in Section II.

[0133] In some embodiments, the method includes concentrating and / or purifying the starch stream. In some embodiments, the method includes concentrating the starch stream. In some embodiments, the method includes purifying the starch stream. In some embodiments, the method includes concentrating and purifying the starch stream. In some embodiments, for example when both concentrating and purifying the starch stream, the concentrating the starch stream occurs before the purifying. Thus, in some cases, the purifying of the starch stream is performed on the starch stream that has undergone concentration. It is contemplated that any type of system capableIFF10169-WO-PCT of concentrating the starch stream may be used. In some embodiments, concentrating the starch stream is accomplished using a concentration unit as described in Section II. Similarly, any type of system capable of purifying the starch stream may be used. In some embodiments, purifying the starch stream is accomplished using a purifying unit as described in Section II.

[0134] The rice protein and / or rice starch, c.g., processed independently according to one or more of the steps described herein, may be dried to produce a dry rice protein and / or dry rice starch. In some embodiments, the dried rice protein and / or dried rice starch is a final product that may be packaged for subsequent shipping and use. Thus, in some embodiments, the method includes drying the protein stream to produce a dry rice protein. In some embodiments, the protein stream that is dried has been processed, e.g., concentrated and / or purified, as described herein. It is contemplated that the protein stream may be dried using conventional and well-known techniques, see, e.g., Section II. In some embodiments, the drying of the protein stream is accomplished using a dryer unit as described in Section II. In some embodiments, the moisture content of the dry rice protein will be below about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the moisture content of the dry rice protein will be below about 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the dry rice protein will be dried to a moisture content below 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the moisture content will be between about 1% to 12%. In some embodiments, the moisture content will be between about 1% to 10%. In some embodiments, the moisture content will be between about 1% to 5%. In some embodiments, the moisture content will be between about 2% to 5%. In some embodiments, the dry rice protein is a powder. In some embodiments, the dry rice protein is dry hydrolyzed rice protein or dry hydrolyzed rice protein isolate.

[0135] Likewise, in some embodiments, the method includes drying the starch stream to produce a dry rice starch. In some embodiments, the starch stream that is dried has been processed, e.g., concentrated and / or purified, as described herein. It is contemplated that the starch stream may be dried using conventional and well-known techniques, see e.g., Section II. In some embodiments, the drying of the starch stream is accomplished using a dryer unit as described in Section II.

[0136] In some embodiments, the method further includes one or more dilution steps or washing steps, e.g., of the protein fraction or the starch fraction, before, after, or during any one of the concentrating, purifying, and / or drying steps.

[0137] In some embodiments, the method may further include contacting the concentrated and / or purified protein stream with a second enzyme or second enzyme composition. The furtherIFF10169-WO-PCT contacting may act as a second hydrolysis step and / or function to remove unwanted compounds, e.g., anti-nutrients. In some embodiments, the method further includes a second enzyme reaction step. In some cases, the method further includes a second protein hydrolyzing step. In some embodiments, the method further includes a step for removing unwanted compounds, e.g., antinutrients, for example phytic acid. In some embodiments, the concentrated and / or purified protein stream is contacted with an enzyme composition (e.g., a second enzyme composition) described in Section 1-A. In some embodiments, the concentrated and / or purified protein stream is contacted with a protease. In some embodiments, the protease is a glutamine-specific protease e.g., EC 3.5.1.44 (e.g., a glutaminase). In some embodiments, the protease is a proline-specific protease, e.g. EC 3.4.21.26. In some embodiments, the concentrated and / or purified protein stream is contacted with a phytase. In some embodiments, the concentrated and / or purified protein stream is contacted with a protease, e.g., a glutamine-specific protease and / or proline-specific protease, and a phytase. In some embodiments, the concentrated and / or purified protein stream contacted with a second enzyme or second enzyme composition produces a reacted protein, e.g., a reacted concentrated protein stream and / or a reacted purified protein stream. In some embodiments, the concentrated protein stream contacted with a second enzyme or second enzyme composition produces a hydrolyzed concentrated protein, e.g., a hydrolyzed concentrated protein stream. In some embodiments, the purified protein stream contacted with a second enzyme or second enzyme composition produces a hydrolyzed purified protein, e.g., a hydrolyzed purified protein stream.

[0138] In some embodiments, the method further includes heating the treated slurry, the protein stream, the starch stream, and / or the reacted protein stream to deactivate the enzyme composition. In some embodiments, the method further includes heating the treated slurry to deactivate the enzyme composition. In some embodiments, the method further includes heating the protein stream to deactivate the enzyme composition. In some embodiments, the method further includes heating the starch stream to deactivate the enzyme composition. In some embodiments, the method further includes heating the reacted protein stream to deactivate the enzyme composition. In some cases, the treated slurry, protein stream, and / or starch stream may be heated at any point during the process after the incubation has occurred. In some embodiments, the heating of the protein stream may occur after contacting with the second enzyme composition, e.g., heating the reacted protein stream. In some embodiments, the treated slurry, protein stream, and / or starch stream may be heated more than once to deactivate contacted enzymes. For example, the protein stream may be heated before and after contact with a second enzyme composition. In some cases, the treated slurry is heated, the protein stream is heated, the starch stream, and / or the reacted protein stream are heated. In some embodiments, the heating is to at least about 70, 75,IFF10169-WO-PCT80, 85, 90, 95, or 100°C. In some embodiments, the heating is to at least about 75°C. In some embodiments, the heating is to at least about 80°C. In some embodiments, the heating is to at least about 85°C. In some embodiments, the heating is to at least about 90°C. In some embodiments, the heating is to at least about 95°C. In some embodiments, the temperature is maintained for at least about 5, 10, 15, 20, 25, or 30 minutes. In some embodiments, the temperature is maintained for at least about 5 minutes. In some embodiments, the temperature is maintained for at least about 10 minutes. In some embodiments, the temperature is maintained for at least about 15 minutes. In some embodiments, the temperature is maintained for at least about 20 minutes.

[0139] The rice protein, e.g., dry rice protein, obtained according to the methods herein may have improved characteristics over a protein fraction obtained by starch processing methods using conventional alkaline methods. In some embodiments, the rice protein obtained according to the methods described herein has greater solubility at alkaline pH levels and particularly at pH levels of 10.0 as compared to the solubility of proteins in protein residues obtained from conventional rice starch processes techniques, e.g., alkaline processing. In some embodiments, the solubility at pH 10 will be at least 20%, at least 40% and at least 50%. In some embodiments, the rice protein obtained according to the methods provided herein include proteins having greater solubility at acidic pH levels, such as at pH levels of 2.0, compared to the solubility of proteins in protein residues obtained from conventional rice starch processes techniques, e.g., alkaline processing. In some embodiments, the solubility of the rice protein at pH 2.0 will be at least 15%, at least 20%, at least 25% and at least 30%.

[0140] In some embodiments, the rice protein obtained according to the methods provided herein will have an amino acid composition wherein the percent of certain amino acids will be increased as compared to the amino acid profile of the protein fraction of a residue obtained from conventional rice starch processes techniques, e.g., alkaline processing and isoelectric precipitation. For example, in some embodiments, the amount of glutamic acid, valine, leucine, proline, methionine, arginine aspartic acid, alanine, or all or any combination thereof will be greater in a rice protein obtained according to the methods provided herein.

[0141] In some embodiments, the methods provided herein maintain or increase starch yield, e.g., compared to an existing rice starch extraction process that does not include rice protein extraction described herein. In some embodiments, the methods provided herein maintain rice starch structure, e.g., compared to an existing rice starch extraction process that does not include rice protein extraction described herein. In some embodiments, the methods provided herein improve rice starch structure, e.g., compared to an existing rice starch extraction process that does not include rice protein extraction described herein.IFF10169-WO-PCT

[0142] The methods provided herein may be carried out using a system described herein. For example, the methods for processing the protein stream to produce a dry rice protein may be accomplished using the systems described in Section II.IL SYSTEM FOR PRODUCING RICE PROTEIN

[0143] In an aspect is provided a system for producing a rice protein. In some embodiments, the system for producing the rice protein produces a hydrolyzed rice protein or hydrolyzed rice protein isolate. The system described herein is suitable for use according to the methods provided herein. Sec, Section I. For example, the system for producing rice protein may be placed in operable contact with an existing rice starch process containing multiple unit operations for extracting rice starch from a rice substrate. For example, in some cases, the system for producing rice protein according to the methods provided herein connects to a unit, e.g., a separation unit, present in an existing rice starch extraction process of a rice starch extraction plant, such that the system for producing rice protein can be readily implemented in the existing rice starch extraction process. Implementation of the rice protein production system into the existing starch extraction process may transform the existing rice starch production process into a system for co-producing rice starch and rice protein in a single process by allowing starch and protein streams to be independently processed to produce a rice starch product and a rice protein product. Thus, in an aspect is provided a system for producing rice protein, including a concentration unit and / or a purification unit, and a dryer unit. In some embodiments, the system includes a concentration unit and a dryer unit. In some embodiments, the system includes a purification unit and a dryer unit. In some embodiments, the system includes a concentration unit, a purification unit, and a dryer unit. In some embodiments, the system includes one or more concentration units, one or more purification units, and a dryer unit. In some embodiments, the system further includes a reaction unit. See, e.g., FIG. 6.

[0144] In an aspect is provided a system for producing rice protein, including a concentration unit, a purification unit, and a dryer unit, where the concentration unit is in operable contact with the purification unit, and the purification unit is in operable contact with the dryer unit, where the concentration unit receives a protein stream and produces a concentrated protein stream, the purification unit receives the concentrated protein stream from the concentration unit to produce a purified protein stream, and the dryer unit receives the purified protein stream from the purification unit and produces a dry rice protein. In some embodiments, the system is a continuous system. FIG. 5 shows an exemplary system for producing a rice protein, and the interface of the system with an existing starch extraction process. In an aspect is provided a system for producing rice protein, including a concentration unit, a purification unit, a reaction unit, and a dryer unit,IFF10169-WO-PCT where the concentration unit is in operable contact with the purification unit, and the purification unit is in operable contact with the reaction unit, and the reaction unit is in operable contact with a dryer unit, where the concentration unit receives a protein stream and produces a concentrated protein stream, the purification unit receives the concentrated protein stream from the concentration unit to produce a purified protein stream, the reaction unit receives the purified protein stream to produce a reacted protein stream, and the dryer unit receives the reacted protein stream from the reaction unit and produces a dry rice protein. In some embodiments, the system is a continuous system. FIG. 6 also shows an exemplary system for producing a rice protein, and the interface of the system with an existing starch extraction process.

[0145] With reference to FIG. 5, the system for producing rice protein 110 includes a concentration unit 24. In some embodiments, the concentration unit is the unit of the system for producing rice protein that is operably connected with a unit in an existing starch extraction process. In some embodiments, the concentration unit 24 is in operable contact with a separation unit 14 of the existing rice starch extraction process. In some embodiments, the operable contact may be facilitated by hosing and a pump. In some embodiments, the operable contact may be facilitated by a pipeline. Any suitable means for operable connectivity according to the types of units used is contemplated herein. In some embodiments, the operable contact includes a tank into which the output of the separation unit is delivered, and the output present in the tank is delivered to the concentration unit by way of hosing and / or pumps. In some embodiments, the output present in the tank is delivered to the concentration unit by way of a pipeline. In some embodiments, the separation unit 14 of the starch extraction process receives a treated slurry described above, either directly or indirectly, e.g., as a result of other intervening unit operations, and separates the treated slurry into a starch stream and a protein stream. In some embodiments, the treated slurry is delivered to the separation unit from a reaction tank unit 12 in which the rice slurry is contacted with an enzyme composition described herein. In some embodiments, the reaction tank unit 12 includes temperature and / or pH sensors. In some embodiments, the reaction tank unit 12 is temperature controlled. In some embodiments, the reaction tank unit 12 includes a water jacket. In some embodiments, the reaction tank unit 12 is pH controlled. In some embodiments, the reaction tank unit 12 includes an agitator mechanism and / or is a continuous stirred tank reactor and / or a recirculation loop. In some embodiments, the reaction tank unit 12 includes an inlet for dosing one or more enzymes or an enzyme composition described herein. It will be appreciated that the treated slurry may undergo additional processing, for example, but not limited to, concentrating, filtering, straining, diluting, and / or washing, prior to reaching the separation unit 14 for separation into the starch stream 122 and protein stream 120. In some embodiments, theIFF10169-WO-PCT separation unit 14 includes one or more of a disk stack centrifuge, a decanter bowl centrifuge, a filtering centrifuge, a vortex separator, a gravity settler, a pressure filter (e.g. a nutsche filter), a vacuum filter (e.g. a belt filter), a mechanical pressure filter (e.g. a filter press, screw press), or a cross flow membrane filter. In some embodiments, the separation unit 14 includes separation units in scries. In some embodiments, the separation unit 14 includes two or more of the same type of separation unit. In some embodiments, the separation unit 14 includes two or more different types of separation units. In some embodiments, the separation unit 14 includes any combination, e.g., one or more identical and / or one or more different, separation units. In some embodiments, the concentration unit 24 receives the protein stream 120 from the separation unit 14.

[0146] In some embodiments, the concentration unit 24 concentrates, e.g., as described herein (see, Section I), the protein stream received from the separation unit 14. For example, in some cases, the concentration unit 24 up-concentrates the protein stream received from the separation unit 14. In some embodiments, the protein stream is concentrated to a level described in Section I. In some embodiments, the concentration unit includes one or more of a strainer, a cross flow membrane filter, a dynamic cross flow membrane filter, a disk stack centrifuge, a decanter bowl centrifuge, a vortex separator, a gravity setter, a pressure filter, a vacuum filter, a mechanical pressure filter, an evaporator, or any combination thereof. In some embodiments, the cross flow membrane filter is a microfiltration membrane, an ultrafiltration membrane, a nanofiltration membrane, or a reverse osmosis membrane. In some embodiments, the dynamic cross flow membrane filter is a microfiltration membrane, an ultrafiltration membrane, a nanofiltration membrane, or a reverse osmosis membrane. In some embodiments, the concentration unit 24 includes two or more different types of concentration units described herein. In some embodiments, the concentration unit 24 includes any combination, e.g., one or more identical and / or one or more different, concentration units described herein. In some embodiments, the concentration unit includes a strainer and a cross flow membrane filter. In some embodiments, the cross flow membrane filter includes a microfiltration membrane, an ultrafiltration membrane, a nanofiltration membrane, or a reverse osmosis membrane. In some embodiments, the concentration unit includes a strainer and a dynamic cross flow membrane filter. In some embodiments, the dynamic cross flow membrane filter includes a microfiltration membrane, an ultrafiltration membrane, a nanofiltration membrane, or a reverse osmosis membrane. In some embodiments, when two or more concentration units are included in the system, the concentration units may be in series.

[0147] Downstream of the system’s concentration unit 24, the concentrated protein 124 may be supplied to a purification unit 26. In some embodiments, the purification unit produces aIFF10169-WO-PCT purified protein 126. Thus, in some embodiments, the concentration unit 24 is in operable contact with a purification unit 26. In some embodiments, the operable contact may be facilitated by hosing and a pump. In some embodiments, the operable contact may be facilitated by a pipeline. Any suitable means for operable connectivity according to the types of units used is contemplated herein. In some embodiments, the operable contact includes a tank into which the output of the concentration unit is delivered, and the output present in the tank is delivered to the purification unit 26 by way of hosing and / or pumps. In some embodiments, the output is delivered by way of a pipeline. Any suitable means for operable connectivity according to the types of units used is contemplated herein. In some embodiments, the purification unit 26 purifies the concentrated protein to produce a purified protein 126. In some embodiments, the purification unit performs diafiltration. Diafiltration refers to a filtration process that separates and purifies a solution by removing smaller molecules and retaining larger molecules. Diafiltration makes use of membranes that are permeable to molecules, e.g., micro-molecules, such as nanofiltration membranes, ultrafiltration membranes, or microfiltration membranes. It will be appreciated that the membrane for use depends on the composition of the materials to be separated. In some embodiments, the process of diafiltration includes adding a buffer solution or water to the concentrate to replace the water lost during filtration. In such cases, the concentration of the molecules of interest is kept constant while diluting the unwanted smaller molecules. Thus, in some embodiments, the purification unit 26 includes an electrodialysis system, an ion exchange resin system, a cross flow membrane filtration system, or a dynamic cross flow membrane filtration system, e.g., to perform diafiltration. In some embodiments, the purification unit 26 includes one or more of an electrodialysis system, an ion exchange resin system, a cross flow membrane filtration system, or a dynamic cross flow membrane filtration system. In some embodiments, the cross flow membrane filtration system includes a microfiltration membrane, an ultrafiltration membrane, a nanofiltration membrane, or a reverse osmosis membrane. In some embodiments, the dynamic cross flow membrane filtration system comprises a microfiltration membrane, an ultrafiltration membrane, a nanofiltration membrane, or a reverse osmosis membrane. It will be appreciated that the difference in membrane filters reflects a difference in membrane pore size, with microfiltration being the largest and reverse osmosis being the smallest. In some embodiments, the purification unit 26 includes two or more different types of purification units. In some embodiments, the purification unit 26 includes any combination, e.g., one or more identical and / or one or more different, purification units. In some embodiments, when two or more purification units are included in the system, the purification units may be in series.IFF10169-WO-PCT

[0148] In some embodiments, the concentration unit 24 includes a strainer and the purification unit 26 includes a cross flow membrane filtration system or a dynamic cross flow membrane filtration system. In some embodiments, the concentration unit 24 includes a strainer and a cross flow membrane filtration system, and the purification unit 26 includes a cross flow membrane filtration system or a dynamic cross flow membrane filtration system, c.g., which performs diafiltration. In some embodiments, the system for producing rice protein includes two or more pairs of operably connected concentration units 24 and purification units 26. thus, in some embodiments, the system includes one, two, three, or more pairs of a concentration unit 24 and purification unit 26. In some embodiments, the concentration unit 24 and purification unit 26 of a pair are in operable contact, e.g., connected in series. In some embodiments, the concentration unit 24 and purification unit 26 of a pair are in operable contact with one another and in operable contact with a second pair of concentration and purification units. Thus, in some cases, the pairs of concentration and purification units are in operable contact with a downstream pair of concentration and purification units. In some embodiments, the purification unit of the upstream pair of concentration unit and purification unit is in operable contact with the concentration unit of the downstream pair of concentration and purification units such that the output from the upstream pair is received by the concentration unit of the downstream pair. This arrangement of pairs may be repeated as needed to achieve a desirable rice protein product.

[0149] In some embodiments, a pair of concentration unit 24 and purification unit 26 includes the same membrane filter, e.g., microfiltration, ultrafiltration, nanofiltration, or reverse osmosis. In some embodiments, a pair of concentration unit 24 and purification unit 26 includes a different membrane filter. In some embodiments, when two or more pairs of a concentration unit 24 and purification unit 26 are used in the system, a pair of concentration unit and purification unit in the system may use the same membrane filter which is different from the membrane filters of one or more other pairs. For example, in cases where more than one pair of concentration unit 24 and purification unit 26 is included in the system, the first pair may have the same membrane that includes a larger pore size than the second pair of a concentration unit and a purification unit. In some embodiments, the concentration unit 24 includes a strainer. In some embodiments, for example when two or more pairs of a concentration unit 24 and a purification unit 26 are present in the system, only the concentration unit 24 of the first pair of a concentration unit 24 and a purification unit includes a strainer.

[0150] The system for producing rice protein provided herein may include a dryer unit 28. In some embodiments, the dryer unit 28 receives a concentrated protein 124 from the concentration unit 24. Thus, in some embodiments, the dryer unit 28 is in operable contact with the concentrationIFF10169-WO-PCT unit 26. In some embodiments, the dryer unit 28 receives the purified protein 126 from the purification unit 26. Thus, in some embodiments, the purification unit 26 is in operable contact with a dryer unit 28. In some embodiments, the dryer unit 28 is in operable contact with the concentration unit 24 via the purification unit 26. In some embodiments, the operable contact may be facilitated by hosing and a pump. In some embodiments, the operable contact is facilitated by way of a pipeline. Any suitable means for operable connectivity according to the types of units used is contemplated herein. In some embodiments, the operable contact includes a tank into which the output of the concentration unit or purification unit is delivered, and the output present in the tank is delivered to the dryer unit 28 by way of hosing and / or pumps. In some embodiments the output is delivered by way of a pipeline. Any suitable means for operable connectivity according to the types of units used is contemplated herein. In some embodiments, the dryer unit produces a dry rice protein 30 by drying the purified protein 126 (or concentrated protein 124). In some embodiments, the dryer unit 28 includes a convection dryer. Non-limiting examples of convection dryers include flash dryers, spray dryers, and the like. In some embodiments, the dryer is a conduction dryer. Non-limiting examples of conduction dryers include vacuum dryers, thin film drum dryers, and the like. In some embodiments, the dryer is a radiant dryer. Non-limiting examples of radiant dryers include IR dryers, high vacuum freeze dryers, and the like. In some embodiments the dryer is a convection dryer, a conduction dryer, a radiant dryer, or any combination thereof.

[0151] In some embodiments, the system for producing rice protein further includes a reaction unit 32, for example, as shown in FIG. 6. In some embodiments, the reaction unit 32 is the same type of unit as described for the reaction tank unit 12. For example, enzymes may be received by the reaction unit 32 in the same way as described for the reaction tank unit 12. However, in reaction unit 32 the contacting occurs with the concentrated and / or purified protein and produces a reacted protein stream 128. In some embodiments, the reaction unit 32 receives the concentrated protein stream 124 from concentration unit 24, and the contacting of the concentrated protein stream with the second enzyme or second enzyme composition as described herein produces a reacted protein, e.g., a reacted concentrated protein stream. In some embodiments, the reaction unit 32 receives the purified protein stream 126 from purification unit 26, and the contacting of the purified protein stream with the second enzyme or second enzyme composition as described herein produces a reacted purified protein 128. In some embodiments, the purified protein stream is produced from a concentrated protein stream by the purification unit. In some embodiments, the purified protein stream is produced from a protein stream received from the separation unit 14. It will beIFF10169-WO-PCT appreciated that the reaction tank unit 12 and reaction unit 32 may be the same or different according the embodiments depending on the needs of the second reaction step.III. PRODUCTS

[0152] The rice protein produced according to the methods described herein may be used in various products, including, for example, food and beverage products, functional nutrition and health products, cosmetics and personal care products, pharmaceutical products, pet and animal nutrition products, and industrial products. Thus, in an aspect is provided a product including a rice protein produced according to the described methods. The role of rice protein in finished products varies with each type of product, such as consumer products or feed products.

[0153] Rice protein can have several functions in finished food products, including emulsification, foam stability, sedimentation stability (i.e., precipitation, suspension), and body (e.g., viscosity, thickness). In some embodiments, the rice protein effects finished food product taste, aroma, and color. In some embodiments, the rice protein may be used in any food product, including but not limited to snacks, bakery products, confectionery products, meat or meatreplacement products, dairy or dairy -like products, cheese or cheese-like products, beverages, and sauces. In some embodiments, the rice protein may be used to enrich or increase protein content in foods.

[0154] In some embodiments, the rice protein may be used in cosmetic and personal care products. In some embodiments, the cosmetic and personal care product is a skin care product. In some embodiments, the cosmetic and personal care product is a hair care product.

[0155] In some embodiments, the rice protein may be used in or as a functional nutrition and health product. In some embodiments, the rice protein may be used in clinical nutrition, nutrition supplement, a functional beverage, a therapeutic nutrition product, a personal care formulation, or an infant formula (e.g., hypoallergenic infant formula).

[0156] In some embodiments, the rice protein may be used for pharmaceutical applications, for example in a pharmaceutical product. In some embodiments, the rice protein is used as a carrier or a film (e.g., an edible film), for example, for drug delivery. In some embodiments, the rice protein may be used for encapsulation. In some embodiments, the rice protein may be used in or as a nutraceutical.

[0157] In some embodiments, the rice protein may be used in pet and animal nutrition applications. For example, the rice protein may be used to increase the protein content of pet food or livestock feed. Thus, in some embodiments, the rice product is used in pet food. In some embodiments, the rice protein is used in animal feed.IFF10169-WO-PCT

[0158] In some embodiments, the rice protein may be used in industrial applications. For example, the rice protein may be used as a fermentation substrate, for example, to increase nitrogen sources for microbial growth

[0159] It will be appreciated that the molecular weight of the rice protein may be used to determine which product the rice protein may be used in. In some embodiments, the methods and systems provided herein allow the molecular weight of the rice protein to be tailored to a specific use. In some embodiments, rice protein produced according to the methods provided herein and having a molecular weight of less than 5 kDa (kilodaltons) may be used in a product described herein. In some embodiments, rice protein produced according to the methods provided herein and having a molecular weight of greater than 5 kDa may be used in products described herein. In some embodiments, the rice protein used in products is a hydroylzed rice protein or hydrolyzed rice protein isolate.

[0160] In some embodiments, the rice protein for use in products requires a level of purity. In some embodiments, the rice protein for use in products is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more pure. In some embodiments, the rice protein for use in products is at least about 20% pure. In some embodiments, the rice protein for use in products is at least about 30% pure. In some embodiments, the rice protein for use in products is at least about 40% pure. In some embodiments, the rice protein for use in products is at least about 50% pure. In some embodiments, the rice protein for use in products is at least about 60% pure. In some embodiments, the rice protein for use in products is at least about 70% pure. In some embodiments, the rice protein for use in products is at least about 75% pure. In some embodiments, the rice protein for use in products is at least about 80% pure. In some embodiments, the rice protein for use in products is at least about 85% pure. In some embodiments, the rice protein for use in products is at least about 90% pure. In some embodiments, the rice protein for use in products is at least about 95% pure. In some embodiments, the rice protein for use in products is at least about 96% pure. In some embodiments, the rice protein for use in products is at least about 97% pure. In some embodiments, the rice protein for use in products is at least about 98% pure. In some embodiments, the rice protein for use in products is at least about 99% pure.EXEMPLARY EMBODIMENTS

[0161] Among the provided embodiments are:1. A method for co-producing rice starch and rice protein, comprising contacting a rice slurry with an enzyme composition to produce a treated slurry, wherein the enzyme composition comprises a protease.IFF10169-WO-PCT2. The method of embodiment 1, wherein the enzyme composition further comprises a cellulase.3. The method of embodiment 2, wherein the cellulase comprises a cellobiohydrolase, an endoglucanase, a beta-glucosidase, or any combination thereof.4. The method of embodiment 2 or embodiment 3, wherein the cellulase is derived from a fungus.5. The method of any one of embodiments 2-4, wherein the cellulase is derived from a strain of Trichoderma or a strain of Penicillium.6. The method of any one of embodiments 1-5, wherein the protease is an alkaline protease.7. The method of any one of embodiments 1-6, wherein the protease comprises an amino acid sequence set forth by SEQ ID NOs: 1, 2, or 3, or an amino acid sequence having at least 70, 80, 90, 95, 98, or 100% sequence identity to the sequence set forth by SEQ ID NOs: 1, 2, or 3.8. The method of any one of clams 1-7, wherein the enzyme composition comprises a hemicellulase, optionally a xylanase, an esterase, an arabinofuranosidase, or any combination thereof.9. The method of any one of clams 1-8, wherein the enzyme composition further comprises a xylanase.10. The method of embodiment 8 or embodiment 9, wherein the xylanase comprises an amino acid sequence set forth by SEQ ID NOs: 4 or 5, or an amino acid sequence having at least 70, 80, 90, 95, 98, or 100% sequence identity to the sequence set forth by SEQ ID NOs: 4 or 5.11. The method of any one of embodiments 1-10, wherein the rice slurry is produced by milling a rice substrate.12. The method of embodiment 11, wherein the rice substrate is polished rice.13. The method of embodiment 11 or embodiment 12, wherein the rice substrate is broken rice.14. The method of any one of embodiments 11-13, wherein the rice substrate is contacted with the enzyme composition during milling.15. The method of any one of embodiments 11-14, wherein the rice slurry is contacted with the enzyme composition after milling.16. The method of any one of embodiments 1-15, wherein the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 8 to about 10.17. The method of any one of embodiments 1-15, wherein the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 5 to about 7.IFF10169-WO-PCT18. The method of any one of embodiments 1-17, comprising adjusting a pH of the treated slurry to a pH in a range of about 8 to about 10.19. The method of embodiment 18, wherein the adjusting the pH of the treated slurry occurs at least 30 minutes after the rice slurry is contacted with the enzyme composition.20. The method of embodiment 18 or embodiment 19, wherein the adjusting the pH of the treated slurry occurs at least 1 hour after the rice slurry is contacted with the enzyme composition.21. The method of any one of embodiments 1-20, wherein the contacting the rice slurry with the enzyme composition occurs at a temperature in a range of about 30 to 55°C.22. The method of any one of embodiments 1-20, wherein the contacting the rice slurry with the enzyme composition occurs at a temperature below 30°C.23. The method of any one of embodiments 1-20 and 22, wherein the contacting the rice slurry with the enzyme composition occurs at a temperature below 30°C to produce the treated slurry, and the treated slurry is heated to a temperature in a range of about 30 to 55 °C.24. The method of embodiment 23, wherein the treated slurry is heated at least 30 minutes after the rice slurry is contacted with the enzyme composition.25. The method of embodiment 23 or embodiment 24, wherein the treated slurry is heated at least 1 hour after the rice slurry is contacted with the enzyme composition.26. The method of any one of embodiments 1-25, wherein the treated slurry is incubated for about 2 to about 8 hours.27. The method of any one of embodiments 1-26, comprising separating the treated slurry to produce a protein stream and a starch stream.28. The method of embodiment 27, wherein the treated slurry is diluted before and / or during the separating.29. The method of embodiment 27 or embodiment 28, comprising concentrating and / or purifying the protein stream.30. The method of any one of embodiments 27-29, comprising drying the protein stream to produce a dry rice protein.31. The method of any one of embodiments 27-30, comprising concentrating and / or purifying the starch stream.32. The method of any one of embodiments 27-31, comprising drying the starch stream to produce a dry rice starch.33. A system for producing rice protein, comprising a concentration unit and a dryer unit, wherein the concentration unit is in operable contact with the dryer unit; and wherein theIFF10169-WO-PCT concentration unit receives a protein stream and produces a concentrated protein, and the dryer unit receives the concentrated protein and produces a dry rice protein.34. The system of embodiment 33, wherein the concentration unit comprises one or more of a strainer, a cross flow membrane filtration system, a dynamic cross flow membrane filtration system, a disk stack centrifuge, a decanter bowl centrifuge, a vortex separator, a gravity setter, a pressure filter, a vacuum filter, a mechanical pressure filter, an evaporator, or any combinations thereof.35. The system of embodiment 33 or embodiment 34, wherein the system comprises a purification unit in operable contact with the concentration unit and the dryer unit, and wherein the purification unit receives the concentrated protein from the concentration unit, purifies the concentrated protein stream to produce a purified protein, and the dryer unit receives the purified protein from the purification unit and produces a dry rice protein.36. The system of embodiment 35, wherein the purification unit comprises one or more of a cross flow membrane filtration system, a dynamic cross flow membrane filtration system, an ion exchange resin system, or an electrodialysis system.37. The system of any one of embodiments 34-36, wherein the cross flow membrane filtration system and / or the dynamic cross flow membrane filtration system comprises, independently, a microfiltration membrane, an ultrafiltration membrane, a nanofiltration membrane, or reverse osmosis membrane.38. The system of any one of embodiments 35-37, wherein the system comprises one or more pairs of a concentration unit and a purification unit.39. The system of any one of embodiments 33-38, wherein the concentration unit is in further operable contact with a separation unit, wherein the separation unit is comprised in a rice starch extraction system, and wherein the separation unit receives a rice slurry contacted with an enzyme composition and produces the protein stream and a starch stream.40. The system of embodiment 39, wherein the separation unit comprises one or more of a disk stack centrifuge, a decanter bowl centrifuge, a filtering centrifuge, a vortex separator, a gravity settler, a pressure filter, a vacuum filter, a mechanical pressure filter, a dynamic cross flow membrane, or a cross flow membrane filter.IV. EXAMPLES

[0162] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.Example 1: Proteases for Rice Protein ExtractionIFF10169-WO-PCT

[0163] This Example describes the use of proteases for extracting rice protein.A. Methods

[0164] Rice slurry was prepared in a 1:2 ratio (8.5 gram rice and 16.5 gram water) to create a 25 gram slurry. Slurry pH was adjusted according to protease requirements, and 1% weight / weight (w / w) of protease was added to designated tubes. The slurry was incubated at 55 °C for 4 hours, and finally centrifuged at 4000 rpm for 15 minutes. The supernatant was collected and the protein concentration in supernatant was estimated using the Kjeldahl method. The relative yield was calculated based on the protein recovery in Control (assuming control = 100%). The Control condition did not include a protease enzyme and was performed at pH 11.5.B. Results

[0165] Protein Content'. The data shown in Table El demonstrates that proteases may be used to increase recoverable rice protein at lower pH.

[0166] Table El: Shows the relative yield of protein in supernatant for each protease and no enzyme at a pH of 10 compared to the Control condition (no enzyme; pH 11.5). The relative yield is the average of two experiments, except for No enzyme at pH 10, which represents a single experiment.

[0167] Conclusion: These data support the ability of proteases to improve rice protein extraction at lower pH, which will reduce the potential damage to rice proteins.Example 2: Enzyme Combinations for Rice Protein Extraction

[0168] This Example describes enzyme compositions for extracting rice protein.A. Methods

[0169] Rice slurry was prepared in a 1 :2 ratio (8.5 gram rice and 16.5 gram water) to create a 25 gram slurry. Slurry pH was adjusted to 5 and 1% w / w of an enzyme composition shown in Table E2 and 1% w / w of a protease were added to designated tubes (See, Table E3). The slurry was incubated at 55°C for 2 hours before the slurry pH was raised from 5 to 10 and further incubated for 4 hours at 55°C. The slurry was then centrifuged at 4000 rpm for 15 minutes. The supernatant was collected and the protein concentration in supernatant was estimated using the Kjeldahl method. The relative yield was calculated based on the protein recovery in ControlIFF10169-WO-PCT(assuming control = 100%). The Control condition did not include any enzymes and was performed at pH 11.5. Experiments were performed in triplicate.

[0170] Table E2: Exemplary enzyme compositions.B. Results

[0171] Protein Content'. The data shown in Table E3 demonstrate proteases may be used to increase recoverable protein at lower pH.

[0172] Table E3: Shows the relative yield (%) of protein compared to the Control condition(no enzyme) at pH 1 1.5.Composition Protease § Relative protein :IFF10169-WO-PCT

[0173] Conclusion'. These data support the ability of combinations of proteases, cellulases, and hemicellulases, such as xylanases, to improve rice protein extraction at lower pH, which will reduce the potential damage to rice proteins, at relatively high yield compared to control.Example 3: Method for Rice Protein and Starch Extraction

[0174] This Example describes an enzyme composition including cellulases, hemicellulases, and proteases for extracting rice protein and rice starch.A. Methods

[0175] Alkaline Method: 8.5 grams of dewatered rice flour was resuspended in 16.5 grams of 0.18N NaOH, resulting in a 25 gram slurry. The initial slurry pH was recorded at 11.5. The slurry was mixed thoroughly using a vortex mixer and then incubated at 45 °C for 3 hours (h). Following incubation, 56.54 milliliters (mL) of water were added to replicate the washing step, and the slurry was mixed again. The mixture was then centrifuged at 3000g for 30 seconds (sec). The resulting supernatant was collected in a separate tube, while the pellet (starch cake) was subjected to moisture analysis using a moisture analyzer. The remaining starch cake was placed in an oven dryer at 45 °C overnight to remove any remaining moisture. The pH of the supernatant was measured using a pH meter.

[0176] Enzymatic method: 8.5 grams of dewatered rice flour was resuspended in 16.5 grams of RO water, resulting in a 25 gram slurry. The initial slurry pH was recorded to be approximately pH 5.5. Next, 1% weight / weight (w / w) of Comp9 (see, Table E2 above) and 1% w / w alkaline protease (SEQ ID NO: 1) were added to the slurry, which was then mixed thoroughly using a vortex mixer. The mixture was incubated at 45 °C for 1 hour. After completing the first hour, the pH of the slurry was raised to pH 10 by adding 83 microliters (pL) of 10N NaOH, followed by further incubation at 45°C for 2 hours. Subsequently, 56.54 mL of water was added to the slurry to replicate the washing step, and the mixture was mixed well. Finally, the slurry was centrifugedIFF10169-WO-PCT at 3000g for 30sec, supernatant collected in a separate tube. The remaining pellet (starch cake) was subjected to moisture analysis using a moisture analyzer. The remaining starch cake was placed in an oven dryer at 45°C to remove remaining moisture. Protein in supernatant and starch cake were analyzed through Kjeldahl method. The pH of supernatant in enzyme-treated slurry was measured using a pH meter.B. Analysis

[0177] A series of analyses were performed to evaluate protein recovery and starch cake properties for both methods. Protein content in supernatant and starch cake was determined using the Kjeldahl method. Moisture of starch cake was analysed through a Moisture Analyzer. The viscosity profile for the slurry during the 3 h reaction was determined using a Rapid Visco Analyzer (PerkinElmer Inc.. Waltham, MA, USA). Starch damage was assessed using a Starch Damage Assay Kit (Product-Code: K-DAM; Megazyme, Bray, Ireland). Following the assay kit protocol, 100 milligrams (mg) dried starch was resuspended in 1 mb of pre-equilibrated solution- 1 (a- Amylase, 50 U / mL) and incubated at 40°C for 10 minutes. Subsequently, 8 mL of dilute sulphuric acid solution (0.2% volume / volume (v / v)) was added to the mixture and mixed well. The reaction mixture was centrifuged at 3000 rpm (1000 g) for 5 minutes (min) and supernatant was transferred in 0.1 mL aliquots into fresh tubes. 0.1 mL of solution-2 (Amyloglucosidase 20 U / mL) was added to the aliquotcd supernatant and 4 mL of GOPOD reagent was further added. The absorbance at 510 nm was then measured.C. Results

[0178] pH-. The pH of supernatant at the end of the reactions was 11.2 for the alkaline method and 7.9 for the enzymatic method.

[0179] Protein Content'. The enzymatically treated slurry exhibited approximately 25% more protein in the supernatant and 13% less protein in the starch cake when compared to the alkaline- treated slurry (FIG. 1C). FIG. IB illustrates the absolute protein content in grams, while FIG. 1A presents the Kjeldahl result in grams per 100 grams. The data shown in FIGs. 1A-1C demonstrate that the enzymatic method results in a higher protein concentration in the solution and a lower concentration in the starch cake.

[0180] Moisture Content: The starch cake produced using the alkaline method had a higher moisture content compared to the starch cake produced according to the enzymatic method (FIG. 2). Without being bound by theory, at pH 11.5, starch granules may swell and retain more water leading to a higher moisture content. However, under the enzymatic method, the slurry has a pH of 10, which is relatively less harsh than pH 11.5. Without being bound by theory, the starch granules may experience less swelling and hold less water. A lower moisture content in theIFF10169-WO-PCT enzymatically treated starch cake can be advantageous in the drying process, by drying more quickly, resulting in reduced electric consumption during drying.

[0181] Viscosity: Using a Rapid Visco Analyzer (RVA), the viscosity of the enzymatically treated slurry was found to be lower than that of the alkaline -treated slurry during and at the end of the reaction (FIGs. 3A-3B).

[0182] Starch Damage: The starch damage assay revealed that the starch cake obtained using the enzyme method had 50% less damage compared to the starch cake obtained through the alkaline method (FIG. 4).

[0183] Conclusion: These data support the ability of the enzymatic method to extract rice protein and rice starch with beneficial features, such as lower pH, lower moisture, lower viscosity, and less starch damage, compared to alkaline extraction methods.Example 4: Protein Solubility of Enzyme Method vs Conventional Method

[0184] This example describes the protein solubility for the Enzyme Method versus the conventional method (Alkaline Method).A. Methods

[0185] Alkaline Method: A slurry was prepared by mixing 5.5 g of milled rice flour with 19.5 g of 0.16 N NaOII solution to achieve a dry solids (DS) content of 22 % w / w and a pll of approximately 12.5-12.6. The mixture was incubated at 37 °C for 3 hours with continuous shaking at 450 rpm. Post incubation, the slurry was diluted with 56.5 ml of Milli-Q water and subjected to centrifugation at 3000 x g for 30 seconds. The resulting supernatant, rich in protein, and the starch-rich pellet were separated and stored at -20 °C for further analysis.

[0186] Enzymatic method: A slurry was prepared by mixing 5.5 g of milled rice flour with19.5 g of diluted 0.16 N NaOH solution to achieve a dry solids (DS) content of 22 % w / w and a pH range of 10.0-10.3. Enzyme (protease, SEQ ID NO:1) was added at a concentration of 1 % relative to the rice flour weight. The slurry was incubated at 45 °C for 3 hours with continuous shaking at 450 rpm. To maintain the target pH and support enzymatic activity while minimizing microbial contamination, 1 N NaOH was added at 30-60 minute intervals throughout the incubation. After 3 hours, the slurry was diluted with 56.5 ml of Milli-Q water and centrifuged at 3000 x g for 30 seconds. The resulting supernatant (protein-rich fraction) and pellet (starch-rich fraction) were separated. The supernatant was subjected to heat treatment at 92 °C for 5 minutes to deactivate the enzyme. All samples were stored at -20 °C until further analysis.

[0187] Negative Control: The negative control process was conducted replicating the enzymatic extraction method described above, with the exception of enzyme usage. Specifically,5.5 g of milled rice flour was mixed with 19.5 g of 0.16 N diluted NaOH solution to adjust the pHIFF10169-WO-PCT to 10.0-10.3. The resulting slurry was incubated at 45 °C for 3 hours under agitation at 450 rpm. Following incubation, the slurry was diluted with 56.5 ml of Milli-Q water and centrifuged at 3000 x g for 30 seconds. The supernatant (protein-rich fraction) and pellet (starch-rich fraction) were separated and stored at -20 °C for subsequent analysisB. Analysis

[0188] Total Nitrogen / Protein Estimation: Protein content was measured using the Kjeldahl method (Nx6.25) with an automated Kjeldahl system from Gerhardt (C. Gerhardt GmbH & Co. KG, Konigswinter, Germany). Briefly, 1 g of sample was digested with 0.5 g cupric sulfate, 5 g potassium sulfate, and 20 ml concentrated sulfuric acid at 370-380 °C for 2 hours and 30 minutes using the Kjeldtherm digestion unit. After cooling, the digest was transferred to the Vapodest unit for automated distillation and titration using 40% sodium hydroxide, 4% boric acid, and 0.1 N HC1 respectively. Nitrogen and protein content were calculated based on formula of total nitrogen content fed on the Kjeldahl system. Total Nitrogen=1.4VNW, where, W stands for the sample’s weight (in grams), V is the titration’s acid used (in ml), N = Standard acid normality.C. Results

[0189] Protein Solubility: As shown in Table E4 below, compared to the alkaline extraction method, which was set at 100%, the enzymatic approach solubilized a similar amount of rice protein. In contrast, the negative control — identical conditions to enzymatic reaction without enzyme — yielded minimal protein recovery. While the alkaline method requires a harsh pH of 12.5 or higher, the enzymatic method achieved comparable protein extraction at a milder pH of 10-10.3 and 45-55 °C. Under the same pH and temperature conditions, the absence of enzyme resulted in significantly lower protein recovery.

[0190] Table E4: Protein solubility (%) by method of extraction.

[0191] Conclusion: The Enzymatic Method achieved similar protein solubility to the Alkaline Method, and significantly higher solubility than negative control at similar pH.IFF10169-WO-PCTExample 5: Molecular Weight Difference of Rice Protein for Enzyme Method vs Conventional Method

[0192] This example describes the difference in the molecular weight profile for the protein solubilized by the Enzyme Method vs the convention method (Alkaline Method).A. Methods

[0193] The Alkaline Method and the Enzyme Method are described in Example 4.B. Analysis

[0194] Molecular Weight Distribution: The molecular weight of the samples was determined using the GPC method of HPLC with an 1260 Infinity II Bio-SEC System by Agilent USA using the TSKgel G2000 SWXL 300mmx7.8mm (Tosoh Bioscience LLC, Tokyo, Japan). Deionized water was used to dilute the sample in the ratio of Ig / L, followed by filtering with 0.45 m syringe filter. The elution buffer was the acetonitrile buffer as 40% Acetonitrile +60% water +0.05% TFA. The flow rate of 0.5 ml was used during the elution process. The analysis used a lOpl injection volume and ran for the 60 min at column temperature of 30 °C. The detection wavelength was 220 nm. Bovine serum albumin (MW 67 kDa), ovalbumin (MW 43 kDa), phosphatase (MW 32 kDa), Cytochrome C (12.4 kDa), aprotinin (MW 6.5 kDa), bacitracin (1.4 kDa), Gly-Gly-Tyr-Arg (451 Da; SEQ ID NO: 9), Gly-Gly-Gly (189 D) were molecular weight (MW) standards. The calibration curve was shown R2 of 0.99 and below: y = - 0.11311gMW+7.071 (where y is the retention time of standard).C. Results

[0195] Molecular Weight Profile: As shown in FIG. 7, compared to the Alkaline Method, the Enzyme Method produced a distinct and consistent molecular weight profile, predominantly yielding peptides below 5 kDa. Major fractions included 180-500 Da (-50%), 500-1000 Da (-30%), and 1-5 kDa (-15%). In contrast, the alkaline method generated larger protein fractions, with -75% above 12 kDa, followed by smaller proportions in the 1-5 kDa and 5-10 kDa ranges.

[0196] Conclusion: The Enzyme Method can achieve protein with a molecular weight of all fractions of less than 5kDa compared to the Alkaline Method which had a majority of fractions greater than 5kDa.Example 6: Flexibility in Use of Proteases

[0197] This example describes the protein solubility and molecular weight profile with the use of a second protease.A. MethodsIFF10169-WO-PCT

[0198] The Enzyme Method used is as described in Example 4. The enzymes used were SEQ ID NOs: 1 and 3.B. Analysis

[0199] Total Nitrogen / Protein Estimation: Methods are described in Example 4.

[0200] Molecular Weight Distribution: Methods arc described in Example 5.C. Results

[0201] The Enzyme Method of protein extraction used and described in Examples 4 and 5 offers flexibility to use various proteases with consistent results. FIG. 8 demonstrates comparable protein solubility across different proteases, matching the recovery observed in Example 4. This indicates that various proteases may be used according to the Enzyme Method to achieve yield.

[0202] The Enzyme Method consistently produced a molecular weight distribution with different proteases that was similar to that shown in Example 5, with no significant variation. See FIG. 9. This demonstrates the reliability of the Enzyme Method in generating protein fractions predominantly below 5 kDa even with different proteases, with major components in the ranges of 180-500 Da (-50%), 500-1000 Da (-30%). and 1-5 kDa (-15%).

[0203] Conclusion: Alternative proteases can be used to deliver similar protein solubility and protein molecular weight profile of less than 5kDa

[0204] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure. Although the invention may be described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art or related fields are intended to be within the scope of the following claims.IFF10169-WO-PCTSEQUENCE LISTINGIFF10169-WO-PCT

Claims

IFF10169-WO-PCTCLAIMSWhat is claimed is:1 . A method for co-producing rice starch and rice protein, comprising contacting a rice slurry with an enzyme composition to produce a treated slurry, wherein the enzyme composition comprises a protease.

2. The method of claim 1, wherein the enzyme composition consists essentially of a protease.

3. The method of claim 1, wherein the enzyme composition further comprises a cellulase.

4. The method of claim 3, wherein the cellulase comprises a cellobiohydrolase, an endoglucanase, a beta-glucosidase, or any combination thereof.

5. fhe method of claim 3 or claim 4, wherein the cellulase is derived from a fungus.

6. The method of any one of claims 3-5, wherein the cellulase is derived from a strain of Trichoderma or a strain of Penicillium.

7. The method of any one of claims 1-6, wherein the protease is an alkaline protease and / or a non-alkaline protease.

8. The method of any one of claims 1-7, wherein the protease comprises an amino acid sequence set forth by SEQ ID NO: 1 , 2, or 3, or an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO: 1, 2, or 3.

9. The method of any one of claims 1 and 3-8, wherein the enzyme composition further comprises a hemicellulase, optionally a xylanase, an esterase, an arabinofuranosidase, or any combination thereof.

10. The method of any one of claims 1 and 3-9, wherein the enzyme composition further comprises a xylanase.IFF10169-WO-PCT11. The method of any one of claims 1-10, wherein the rice slurry is produced by milling a rice substrate.

12. The method of claim 11, wherein the rice substrate is polished rice.

13. The method of claim 1 1 or claim 12, wherein the rice substrate is broken rice.

14. The method of any one of claims 11-13, wherein the rice substrate is contacted with the enzyme composition during milling.

15. The method of any one of claims 11-14, wherein the rice slurry is contacted with the enzyme composition after milling.

16. The method of any one of claims 1-15, wherein the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 8 to about 10.

17. The method of any one of claims 1-15, wherein the contacting the rice slurry with the enzyme composition occurs at a pH in a range of about 5 to about 7.

18. The method of any one of claims 1-17, comprising adjusting a pH of the treated slurry to a pH in a range of about 8 to about 10.

19. The method of claim 18, wherein the adjusting the pH of the treated slurry occurs at least 30 minutes after the rice slurry is contacted with the enzyme composition.

20. The method of claim 18 or claim 19, wherein the adjusting the pH of the treated slurry occurs at least 1 hour after the rice slurry is contacted with the enzyme composition.

21. The method of any one of claims 1-20, wherein the contacting the rice slurry with the enzyme composition occurs at a temperature in a range of about 30 to about 55°C.

22. The method of any one of claims 1-20, wherein the contacting the rice slurry with the enzyme composition occurs at a temperature below 30°C.

23. The method of any one of claims 1-20 and 22, wherein the contacting the rice slurry with the enzyme composition occurs at a temperature below 30°C to produce the treated slurry,IFF10169-WO-PCT and the treated slurry is heated to a temperature in a range of about 30 to about 55°C.

24. The method of claim 23, wherein the treated slurry is heated at least 30 minutes after the rice slurry is contacted with the enzyme composition.

25. The method of claim 23 or claim 24, wherein the treated slurry is heated at least 1 hour after the rice slurry is contacted with the enzyme composition.

26. The method of any one of claims 1-25, wherein the treated slurry is incubated for about 2 to about 8 hours.

27. The method of any one of claims 1-26, comprising separating the treated slurry to produce a protein stream and a starch stream.

28. The method of claim 27, wherein the treated slurry is heated to at least about 80°C, optionally for at least about 10 min, before and / or during the separating.

29. The method of claim 27 or claim 28, wherein the treated slurry is diluted before and / or during the separating.

30. The method of any one of claims 27-29, comprising concentrating and / or purifying the protein stream.

31. The method of any one of claims 27-30, comprising contacting the protein stream with a second enzyme or second enzyme composition.

32. The method of claim 31, wherein the second enzyme or second enzyme composition comprises a protease and / or a phytase, optionally wherein the protease is a glutaminespecific protease and / or a proline-specific protease.

33. The method of any one of claims 27-32, wherein the treated slurry, the protein stream, and / or the starch stream is heated to at least about 80°C, optionally for at least about 10 min.IFF10169-WO-PCT34. The method of any one of claims 27-33, comprising drying the protein stream to produce a dry rice protein.

35. The method of claim 34, wherein the dry rice protein is a dry hydrolyzed rice protein isolate.

36. The method of any one of claims 37-35, comprising concentrating and / or purifying the starch stream.

37. The method of any one of claims 27-36, comprising drying the starch stream to produce a dry rice starch.

38. A rice protein produced according to the method of any one of claims 1-37.

39. The rice protein of claim 38, wherein the rice protein has a molecular weight of less than 5 kilodaltons.

40. The rice protein of claim 38 or claim 39, wherein the rice protein is at least 70% pure.

41. A product comprising a rice protein of any one of claims 38-40 or produced according to the method of any one of claims 1-37, wherein the product is a food product, a beverage product, a functional nutrition product, a health ingredient product, a cosmetic product, a personal care product, a pharmaceutical product, a pet food product, an animal nutrition product, or a product for industrial application.

42. Use of a rice protein of any one of claims 38-40 or produced according to the method of any one of claims 1-37 in a food product, a beverage product, a functional nutrition product, a health ingredient product, a cosmetic product, a personal care product, a pharmaceutical product, a pet food product, an animal nutrition product, or a product for industrial application.

43. A system for producing rice protein, comprising a concentration unit and a dryer unit, wherein the concentration unit is in operable contact with the dryer unit; and wherein the concentration unit receives a protein stream and produces a concentrated protein, and the dryer unit receives the concentrated protein and produces a dry rice protein.IFF10169-WO-PCT44. The system of claim 43, wherein the concentration unit comprises one or more of a strainer, a cross flow membrane filtration system, a dynamic cross flow membrane filtration system, a disk stack centrifuge, a decanter bowl centrifuge, a vortex separator, a gravity setter, a pressure filter, a vacuum filter, a mechanical pressure filter, an evaporator, or any combination thereof.

45. The system of claim 43 or claim 44, further comprising a purification unit in operable contact with the concentration unit and the dryer unit, wherein the purification unit receives the concentrated protein from the concentration unit, purifies the concentrated protein stream to produce a purified protein, and the dryer unit receives the purified protein from the purification unit and produces a dry rice protein.

46. The system of claim 45, wherein the purification unit comprises one or more of a cross flow membrane filtration system, a dynamic cross flow membrane filtration system, an ion exchange resin system, or an electrodialysis system.

47. The system of any one of claims 44-46, wherein the cross flow membrane filtration system and / or the dynamic cross flow membrane filtration system comprises, independently, a microfiltration membrane, an ultrafiltration membrane, a nanofiltration membrane, or reverse osmosis membrane.

48. The system of any one of claims 45-47, wherein the system comprises one or more pairs of a concentration unit and a purification unit.

49. The system of any one of claims 43-48, wherein the concentration unit is in further operable contact with a separation unit, wherein the separation unit is comprised in a rice starch extraction system, and wherein the separation unit receives a rice slurry contacted with an enzyme composition and produces the protein stream and a starch stream.

50. The system of claim 49, wherein the separation unit comprises one or more of a disk stack centrifuge, a decanter bowl centrifuge, a filtering centrifuge, a vortex separator, a gravity settler, a pressure filter, a vacuum filter, a mechanical pressure filter, a dynamic cross flow membrane, or a cross flow membrane filter.IFF10169-WO-PCT51. The system of any one of claims 45-50, further comprising a reaction unit in operable contact with the purification unit and the dryer unit; wherein the reaction unit receives a purified protein from the purification unit and produces a reacted protein, and the dryer unit receives the reacted protein from the reaction unit and produces a dry rice protein.

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