WO2026008449A2

WO2026008449A2 - A process for producing a fermentation product and a concentrated protein co-product

Info

Publication number: WO2026008449A2
Application number: PCT/EP2025/068082
Authority: WO
Inventors: Mohan Bilekalahalli HUCHEGOWDA; Pandurang Shinde
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2024-07-04
Filing date: 2025-06-26
Publication date: 2026-01-08
Anticipated expiration: 2027-01-04

Abstract

A process for producing a fermentation product and a concentrated protein co-product from a starch-containing material.

Description

A PROCESS FOR PRODUCING A FERMENTATION PRODUCT AND A CONCENTRATED PROTEIN CO-PRODUCT

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a process for producing a fermentation product and a concentrated protein co-product from a starch/fiber-containing material.

BACKGROUND OF THE INVENTION

The production of ethanol for fuel, beverages and industrial use is a major industry. Ethanol has widespread application, including for use as a gasoline additive or as a straight liquid fuel.

Processes for producing fermentation products, such as ethanol, from a starch or lignocellulose containing material are well known in the art. The preparation of the starch containing material such as corn or rice for utilization in such fermentation processes typically begins with grinding the corn in a dry-grind or wet-milling process. Wet-milling processes involve fractionating the grain/kernels into different components where only the starch fraction enters the fermentation process. Dry-grind processes involve grinding the grain/kernels into meal and mixing the meal with water and enzymes. Generally, two different kinds of dry-grind processes are used. The most commonly used process, often referred to as a "conventional process," includes grinding the starch-containing grain and then liquefying gelatinized starch at a high temperature using typically a bacterial alpha-amylase, followed by simultaneous saccharification and fermentation (SSF) carried out in the presence of a glucoamylase and a fermentation organism. Another well-known process, often referred to as a "raw starch hydrolysis" process (RSH process), includes grinding the starch-containing grain and then simultaneously saccharifying and fermenting granular starch below the initial gelatinization temperature typically in the presence of an acid fungal alphaamylase and a glucoamylase.

In a process for producing ethanol from grains/kernel, following SSF orthe RSH process, the liquid fermentation products are recovered from the fermented mash (often referred to as “beer mash”), e.g., by distillation, which separates the desired fermentation product, e.g. ethanol, from other liquids and/or solids. The remaining fraction is referred to as “whole stillage”. Whole stillage typically contains about 10 to 20% solids. The whole stillage is separated into a solid and a liquid fraction, e.g., by centrifugation. The separated solid fraction is referred to as “wet cake” (or “wet grains”) and the separated liquid fraction is referred to as “thin stillage”. Wet cake and thin stillage contain about 30%-35% and 6-10% solids, respectively. Wet cake, with optional additional dewatering, is used as a component in animal feed or is dried to provide “Distillers Dried Grains” (DDG) used as a component in animal feed. Thin stillage is typically evaporated to provide evaporator condensate and syrup or may alternatively be recycled to the slurry tank as “backset”. Evaporator condensate may either be forwarded to a methanator before being discharged and/or may be recycled to the slurry tank as “cook water”. The syrup may be blended into DDG or added to the wet cake before or during the drying process, which can comprise one or more dryers in sequence, to produce DDGS (Distillers Dried Grain with Solubles). Syrup typically contains about 25% to 35% solids. Oil can also be extracted from the thin stillage and/or syrup as a by-product for use in biodiesel production, as a feed or food additive or product, or other bio-renewable products.

WO 2010/138110 A1 is directed to a method for producing a high protein corn meal from a whole stillage byproduct produced in a corn dry-milling process for making ethanol and a system therefore. The method therein includes separating the whole stillage byproduct into an insoluble solids portion and a thin stillage portion. The thin stillage portion is separated into a protein portion and a water soluble solids portion.

However, there is still a need for improved processes for producing ethanol and to increase alcohol and/or by-product yields, such as protein.

SUMMARY OF THE INVENTION

In a first aspect the present invention relates to a process for producing a fermentation product and a concentrated protein co-product from a starch-containing material, comprising the steps of:

(a) liquefying a starch-containing material at a temperature above the initial gelatinization temperature of the starch with an alpha-amylase to produce a liquefied mash;

(b) subjecting the liquefied mash to a protein concentration process to produce a concentrated protein co-product, a soluble starch stream and a sugar stream;

(c) saccharifying the soluble starch stream and the sugar stream with a glucoamylase to produce a fermentable sugar;

(d) fermenting the sugar with a fermenting organism to produce the fermentation product, wherein at least one separation step is performed after the liquefaction in step (a) resulting in a liquid portion comprising soluble starch/ oligosacchrides and an insoluble portion comprising solids from the liquefied mash, said insoluble portion undergoing said protein concentration process.

In a second aspect the present invention relates to a process for producing a fermentation product and a concentrated protein co-product from a starch-containing material, comprising the steps of:

(a) liquefying a starch-containing material at a temperature above the initial gelatinization temperature of the starch with an alpha-amylase to produce a first liquefied mash;

(b) separating the slurry into a first insoluble solids portion and a first liquid portion comprising a soluble starch/ oligosaccharides stream;

(c) liquefying the insoluble solids portion at a temperature above the initial gelatinization temperature of the starch with an alpha-amylase to produce a second liquefied mash;

(d) saccharifying the second liquefied mash to produce a second saccharified mash in presence of a glucoamylase, alpha-amylase, pullulanase, lysophospholipase, cellulase, xylanse, arabinofuranisidase, and/or hemicellualse;

(e) separating the second saccharified mash into a second insoluble solids portion and a second liquid portion comprising a first sugar stream;

(f) optionally washing the second insoluble solids portion to produce a third insoluble solids portion and third liquid portion comprising a second sugar stream;

(g) drying the third insoluble solids portion to produce a concentrated protein coproduct;

(h) saccharifying the soluble starch/oligosacchrides stream and optionally the first and second sugar streams to produce a fermentable sugar in presence of a glucoamylase and optionally alpha-amylase;

(i) fermenting the sugar with a fermenting organism to produce the fermentation product.

Brief Description of the Figures

Figure 1 shows the process of concentrated protein co-product.

DEFINITIONS

In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Reference to "about" a value or parameter herein includes aspects that are directed to that value or parameter perse. For example, description referring to "about X" includes the aspect "X".

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

Alpha-amylase: Alpha-amylases (EC 3.2.1.1) are a group of enzymes which catalyze the hydrolysis of starch and other linear and branched 1 ,4 glucosidic oligo- and polysaccharides. The skilled person will know how to determine alpha-amylase activity. It may be determined, e.g., by measuring residual activity after stressing the sample at pH 4.0 using a commercial alpha-amylase activity assay kit, such as kits containing G7-pNP substrate and alpha-Glucosidase, e.g., manufactured by Roche/Hitachi (cat. No.11876473) or Sigma-Aldrich (Catalog number MAK009).

Alpha-Amylase having raw starch activity, means an alpha-amylase (EC 3.2.1.1) having activity against raw starch (non-gelatinized starch), and preferably comprising of Carbohydrate-Binding Module Family 20 or 26 (CBM20 or CBM26) (URL: http://www.cazy.org/Carbohydrate-Binding-Modules.html). Many relevant raw starch amylases to be applied in the process of the invention are acid stable fungal alpha-amylases.

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

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

Cellulolytic enzyme activity can be determined by measuring the increase in production/release of sugars during hydrolysis of a cellulosic material by cellulolytic enzyme(s) under the following conditions: 1-50 mg of cellulolytic enzyme protein/g of cellulose in pretreated corn stover (PCS) (or other pretreated cellulosic material) for 3- 7 days at a suitable temperature such as 40°C-80°C, e.g., 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C, and a suitable pH, such as 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0, compared to a control hydrolysis without addition of cellulolytic enzyme protein. Typical conditions are 1 ml reactions, washed or unwashed PCS, 5% insoluble solids (dry weight), 50 mM sodium acetate pH 5, 1 mM MnSO4, 50°C, 55°C, or60°C, 72 hours, sugar analysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

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

Expression vector: The term “expression vector” means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.

Endoglucanase: The term “endoglucanase” means an endo-1 ,4-(1 ,3;1 ,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. Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481). For purposes of the present invention, endoglucanase activity is determined using carboxymethyl cellulose (CMC) as substrate according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5, 40°C. Fragment: The term “fragment” means a polypeptide having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide, wherein the fragment has pectin lyase activity.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase” or “Family GH61” or “GH61” means a polypeptide falling into the glycoside hydrolase Family 61 according to Henrissat, 1991 , A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat and Bairoch, 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695-696. The enzymes in this family were originally classified as a glycoside hydrolase family based on measurement of very weak endo-1 ,4-beta-D- glucanase activity in one family member. The structure and mode of action of these enzymes are non-canonical and they cannot be considered as bona fide glycosidases. However, they are kept in the CAZy classification on the basis of their capacity to enhance the breakdown of lignocellulose when used in conjunction with a cellulase or a mixture of cellulases. The GH61 polypeptides have recently been classified as lytic polysaccharide monooxygenases (Quinlan et al., 2011 , Proc. Natl. Acad. Sci. USA 208: 15079-15084; Phillips et al., 2011 , ACS Chem. Biol. Q: 1399-1406; Lin et al., 2012, Structure 20: 1051-1061) and are designated “Auxiliary Activity 9” or “AA9” polypeptides.

Fermentation product: “Fermentation product” means a product produced by a process including fermenting using a fermenting organism. Fermentation products include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B12, beta-carotene); and hormones. In a preferred embodiment the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry. Preferred beer types comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer. In an embodiment the fermentation product is ethanol.

Fermenting organism: “Fermenting organism” refers to any organism, including bacterial and fungal organisms, especially yeast, suitable for use in a fermentation process and capable of producing the desired fermentation product. Glucoamylase: The term glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is defined as an enzyme, which catalyzes the release of D-glucose from the non-reducing ends of starch or related oligo- and polysaccharide molecules.

GH5 polypeptide: refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 5 in the database of Carbohydrate-Active EnZymes (CAZymes) (http://www.cazy.org/).

GH5_21 xylanase: “GH5_21 xylanase” is an abbreviation for Glycoside Hydrolase Family 5 subfamily 21 endo-beta-1, 4-xylanases that possess a three- dimensional structure characterized by a (P / a) 8 barrel and use a glutamine residue as a catalytic nucleophile/base.

GH5_35 xylanase: “GH5_35 xylanase” is an abbreviation for Glycoside Hydrolase Family 5 subfamily 35 endo-beta-1, 4-xylanases that possess a three- dimensional structure characterized by a ( / a) 8 barrel and use a glutamine residue as a catalytic nucleophile/base.

GH8 polypeptide: refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 5 in the database of Carbohydrate-Active EnZymes (CAZymes) (http://www.cazy.org/).

GH30 polypeptide: refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 30 in the database of Carbohydrate-Active EnZymes (CAZymes) (http://www.cazy.org/).

GH10 polypeptide: refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 10 in the database of Carbohydrate-Active EnZymes (CAZymes) available at http://www.cazy.org/. (Lombard, V.; Golaconda Ramulu, H.; Drula, E.; Coutinho, P. M.; Henrissat, B. (21 November 2013). "The carbohydrate-active enzymes database (CAZy) in 2013". Nucleic Acids Research. 42 (D1): D490-D495 Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (January 2009). "The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics". Nucleic Acids Res. 37 (Database issue): D233-8).

GH11 polypeptide refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 11 in the database of Carbohydrate-Active EnZymes (CAZymes).

GH62 polypeptide: refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 62 in the database of Carbohydrate-Active EnZymes (CAZymes). GH43 polypeptide: refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 43 in the database of Carbohydrate-Active EnZymes (CAZymes).

GH51 polypeptide: refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 51 in the database of Carbohydrate-Active EnZymes (CAZymes).

Hydrolytic enzymes or hydrolase/polypeptide with hydrolase activity: “Hydrolytic enzymes” refers to any catalytic protein that use water to break down substrates. Hydrolytic enzymes include alpha-amylase (EC 3.2.1.1), glucoamylase (EC 3.2.1.3), cellulases (EC 3.2.1.4), xylanases (EC 3.2.1.8) arabinofuranosidases (EC 3.2.1.55 (Non-reducing end alpha-L-arabinofuranosidases); EC 3.2.1.185 (Nonreducing end beta-L-arabinofuranosidases) cellobiohydrolase I (EC 3.2.1.150), cellobiohydrolase II (EC 3.2.1.91), cellobiosidase (EC 3.2.1.176), beta-glucosidase (EC 3.2.1.21), beta-xylosidases (EC 3.2.1.37).

Initial gelatinization temperature: "Initial gelatinization temperature" means the lowest temperature at which gelatinization of the starch commences. Starch heated in water begins to gelatinize between 50 degrees Celsius and 75 degrees Celsius; the exact temperature of gelatinization depends on the specific starch, and can readily be determined by the skilled artisan. Thus, the initial gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions. In the context of this disclosure the initial gelatinization temperature of a given starch-containing material is the temperature at which birefringence is lost in 5 percent of the starch granules using the method described by Gorinstein. S. and Lii. C, Starch/Starke, Vol. 44 (12) pp. 461-466 (1992).

Raw Starch Material: The term “raw starch material” means primary starch- based grains, which has not been subjected to temperatures above the initial gelatination temperature for starch, e.g., non-gelatinized starch.

Raw Starch Hydrolysis (RSH): The term “raw starch hydrolysis” means the degradation of starch to polysaccharides from primary starch-based grains which has not been subjected to temperatures above the initial gelatination temperature. Raw starch hydrolysis (RSH) processes are well-known in the art.

Starch: The term “starch” means any material comprised of complex polysaccharides of plants, composed of glucose units that occurs widely in plant tissues in the form of storage granules, consisting of amylose and amylopectin, and represented as (CeH Osjn, where n is any number.

S8A Protease: The term “S8A protease” means an S8 protease belonging to subfamily A. Subtilisins, EC 3.4.21.62, are a subgroup in subfamily S8A. The S8A protease hydrolyses the substrate Suc-Ala-Ala-Pro-Phe-pNA. The release of p- nitroaniline (pNA) results in an increase of absorbance at 405 nm and is proportional to the enzyme activity.

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

For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment).

Xylanases/polypeptide with xylanase activity: The term “xylanase” means a 1 ,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1 ,4-beta-D-xylosidic linkages in xylans. Xylanase activity can be determined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C. One unit of xylanase activity is defined as 1.0 pmole of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6. Xylanases can be found in, e.g., the GH5, GH30, GH10, and GH11 families.

Thin Stillage: “Thin stillage” refers to centrate separated from whole stillage that is pumped toward the evaporators to be concentrated into syrup. Whole Stillage: "Whole stillage" includes the material that remains at the end of the distillation process after recovery of the fermentation product, e.g., ethanol.

DETAILED DESCRIPTION

It is an object of the present invention to provide a process for producing fermentation product from a starch-containing material.

Particularly, it is an object of the present invention to provide a process for producing a fermentation product and a concentrated protein co-product from a starch- containing material.

The process of the invention is particularly suitable for the conventional starch to ethanol industry, which is well-known in the art and relies on producing fermentation products, such as ethanol from gelatinized starch-containing material. The starch containing material is typically dry-milled before the liquefaction step.

This type of process includes a liquefaction step and sequentially or simultaneously performed saccharification and fermentation steps. The conventional process for producing fermentation products from starch-containing material therefore comprises the steps of:

(a) liquefying starch-containing material in the presence of an alpha-amylase;

(b) saccharifying the liquefied material obtained in step (a) using a glucoamylase;

(c) fermenting using a fermenting organism.

The present invention combines the above process in the conventional space with a protein purification process, extraction protein from an insoluble fraction isolated after the liquefaction step and at the same time increases the overall ethanol yield.

The process of the present invention comprises at least one separation step performed after the liquefaction step, resulting in a liquid portion comprising soluble starch/oligosacchrides and an insoluble portion comprising solids from the liquefied mash, said insoluble portion undergoing a protein concentration process, and the liquid portion comprising soluble starch/oligosacchrides undergoing saccharification and fermentation to produce ethanol.

Dry milling processes are well-known in the art, and generally involve the step of grinding/milling whole cereal grains in a dry or substantially dry state. The production of ethanol in accordance with a dry milling process generally includes the main process steps of grinding/milling whole cereal grains to produce a meal, and subjecting the meal to liquefaction, saccharification, fermentation, and optionally distillation to produce ethanol. Whole corn grains are the preferred starting raw material for ethanol production; however, other cereal grains may also be used, including, for example, milo, wheat and barley. Liquefaction is a process in which the long-chained starch is degraded into oligosaccharides. Liquefaction processes are well-known in the art and are usually performed by enzymatic or acid hydrolysis. Preferably, liquefaction is performed by treating the meal with an effective amount of an alpha-amylase. Liquefaction is often carried out at a temperature of about 105 to 120°C. for about 5 to 15 minutes followed by a lower temperature holding period of about 1 to 3 hours at >85°C.

Saccharification is a process in which the oligosaccharides resulting from liquefaction are converted by hydrolysis to fermentable sugars. The hydrolysis is preferably preformed enzymatically by addition of a glucoamylase, alone or in combination with other enzymes, such as alpha-glucosidase, acid alpha-amylase and/or pullulanase. Saccharification processes are also well-known in the art. A full saccharification process may be about 40-92 hours, and is often carried out at temperatures from about 30 to 60°C. However, it is often more preferred to do a presaccharification step, lasting for about 40 to 720 minutes, and then to do a complete saccharification process during fermentation in simultaneous saccharification and fermentation (SSF) or simultaneous liquefaction, saccharification, and fermentation (LSF).

In a first aspect, the invention relates to a process for producing fermentation products and a concentrated protein co-product from a starch-containing material, comprising the steps of:

(d) fermenting the sugar with a fermenting organism to produce the fermentation product, wherein at least one separation step is performed after the liquefaction in step (a) resulting in a liquid portion comprising soluble starch/ oligosaccharides and an insoluble portion comprising solids from the liquefied mash, said insoluble portion undergoing said protein concentration process.

The protein extraction and concentration process preferably comprise several additional process steps, such as at least a liquefaction step, a saccharification step, and one or more separation steps as shown in Figure 1 . Therefore, in a second aspect the present invention relates to a process for producing a fermentation product and a concentrated protein co-product from a starch- containing material, comprising the steps of:

(b) separating the slurry into a first insoluble solids portion and a first liquid portion comprising a soluble starch/oligosacchrides stream;

(h) saccharifying the soluble starch/oligosacchrides stream and optionally the first and second sugar streams to produce a fermentable sugar in presence of a glucoamylase, and optionally an alpha-amylase,

In an embodiment, the process of the invention further comprises, prior to the step a), a steps of: reducing the particle size of the starch-containing material, and forming a slurry comprising the starch-containing material and water.

Any suitable starch-containing material may be used. The material is selected based on the desired fermentation product. Examples of starch-containing materials, include without limitation, barley, cassava, millet, sorghum, oats, potatoes, rice, wheat, and maize, or any mixture thereof.

In a preferred embodiment, starch-containing material is rice.

In an embodiment, the separation is done using centrifugation, filtration, decantation and/or cloth filtration.

Following the grinding step, which is preferably dry-grinding, the particle size is reduced to between 0.05 to 3.0 mm, preferably 0.1 -0.5 mm, or so that at least 30 percent, preferably at least 50 percent, more preferably at least 70 percent, even more preferably at least 90 percent of the starch-containing material fit through a sieve with a 0.05 to 3.0 mm screen, preferably 0.1 -0.5 mm screen.

In another embodiment at least 50 percent, preferably at least 70 percent, more preferably at least 80 percent, especially at least 90 percent of the starch-containing material fit through a sieve.

The aqueous slurry may contain from 10-55 w/w- percent dry solids (DS), preferably 25- 45 w/w- percent dry solids (DS), more preferably 30-40 w/w- percent dry solids (DS) of starch-containing material.

The slurry is heated to above the gelatinization temperature and an alphaamylase may be added to initiate liquefaction (thinning). The slurry may be heated to above the initial gelatinization temperature. The slurry may optionally be jet-cooked to further gelatinize the starch in the slurry before adding alpha-amylase during liquefying step (a). Jet cooking can be performed at temperatures ranging from 100 °C to 120 °C for up to at least 15 minutes.

Liquefaction may in an embodiment be carried out as a three-step hot slurry process. The slurry may be heated to between 60-105 degrees centigrade, at a pH of 4-6, and alpha-amylase, optionally together with a protease, a carbohydrate-source generating enzyme, such as a glucoamylase, a phospholipase, a phytase, and/or pullulanase, are added to initiate liquefaction (thinning).

The pH used during liquefying step (a) may range from 4 to 6, from 4.5 to 5.5, or from 4.8 to 5.2. Preferably, the pH is at least 4.5, at least 4.6, at least 4.7, at least 4.8, at least 4.9, at least 5.0, or at least 5.1.

The liquefaction for performing liquefying step (a) may range from 30 minutes to 5 hours, from 1 hour to 3 hours, or 90 minutes to 150 minutes. Preferably, the time is at least 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 2 hours.

The temperature used during liquefying step (a) may range from 70°C to 110°C, such as from 75°C to 105°C, from 80°C to 100°C, from 85°C to 95°C, or from 88°C to 92°C. Preferably, the temperature is at least 70°C, at least 80°C, at least 85°C, at least 88°C, or at least 90°C.

Saccharification step (b) may be carried out using conditions well known in the art. For instance, a full saccharification process may last up to from about 40 to about 92 hours, however, it is common only to do a pre-saccharification of typically 40-720 minutes at a temperature between 30-65 degrees centigrade, typically about 60 degrees centigrade, followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation process (SSF process). Saccharification is typically carried out at a temperature from 20-75 degrees centigrade, in particular 40-70 degrees centigrade, typically around 60 degrees centigrade, and at a pH between 4 and 5, normally at about pH 4.5. The most widely used process to produce a fermentation product, especially ethanol, is a simultaneous saccharification and fermentation (SSF) process, in which there is no holding stage for the saccharification, meaning that a fermenting organism, such as yeast, and enzyme(s), may be added together. SSF may typically be carried out at a temperature from 25 degrees centigrade to 40 degrees centigrade, such as from 28 degrees centigrade to 35 degrees centigrade, such as from 30 degrees centigrade to 34 degrees centigrade, preferably around about 32 degrees centigrade In an embodiment fermentation is ongoing for 6 to 120 hours, in particular 24 to 96 hours.

Distillation is a process of separating ethanol from the fermented mash, preferably, by evaporation. The vapours are preferably driven off by applying direct heat to the fermented mash. The vapours are collected, condensed and recovered as a liquid and may be redistilled to increase the ethanol concentration. Because ethanol has a higher vapor pressure than water, the vaporization of water and ethanol results in a liquid higher in ethanol. Through condensation, a highly concentrate distillate is obtained. Normal distillation results in a liquid with a purity of about 95 volume-% ethanol (190 proof). For fuel ethanol, the final proof must approach 200. To accomplish this result, the ethanol may be subjected to further dehydration steps.

Stillage is a product which remains after mash has been converted to sugar, fermented and distilled into ethanol. Stillage can be separated into two fractions, such as, by centrifugation or screening: (1) wet grain (solid phase) and (2) the thin stillage (supernatant). The solid fraction or distillers' wet grains (DWG) can be pressed to remove excess moisture and then dried to produce distillers' dried grains (DDG). After ethanol has been removed from the liquid fraction, the remaining liquid can be evaporated to concentrate the soluble material into condensed distillers' solubles (DS) or dried and ground to create distillers' dried solubles (DDS). DDS is often mixed with DDG to form distillers' dried grains with solubles (DDGS). DDG, DDGS, and DWG are collectively referred to as distillers' grains.

In an embodiment the concentrated protein coproduct recovery is above 50% w/w, such as 60% w/w, such as 70% w/w, such as 80% w/w.

In an embodiment, the concentrate protein co-product purity is at least 65% w/w, at least 70% w/w, at least 75% w/w, at least 80% w/w, at least 85% w/w, such as at least 90% w/w.

The present invention contemplates the use of thermostable enzymes during liquefying step (a). It is well known in the art to use various thermostable enzymes during liquefying step (a), including, for example, thermostable alpha-amylases, thermostable glucoamylases, thermostable endoglucanases, thermostable lipases, thermostable phytase, thermostable proteases, thermostable pullulanases, and/or thermostable xylanases. The present invention contemplates the use of any thermostable enzyme in liquefying step (a). The published patent applications listed below describe activity assays for determining whether a candidate thermostable enzyme contemplated for use in liquefying step (a) will be deactivated at a temperature contemplated for liquefying step (a).

Examples of suitable thermostable alpha-amylases and guidance for using them in liquefying step (a) include, without limitation, the alpha-amylases described in WO94/18314, WO94/02597, WO 96/23873, WO 96/23874, WO 96/39528, WO 97/41213, WO 97/43424, WO 99/19467, WO 00/60059, WO 2002/010355, WO 2002/092797, WO 2009/149130, WO 2009/61378, WO 2009/061379, WO

2009/061380, WO 2009/061381 , WO 2009/098229, WO 2009/100102, WO

2010/115021 , WO2010/115028, WO 2010/036515, WO 2011/082425, WO 2013/096305, WO 2013/184577, WO 2014/007921 , WO 2014/164777, WO

2014/164800, WO 2014/164834, WO 2019/113413, WO 2019/113415, WO

2019/197318 (each of which is incorporated herein by reference).

Suitable commercially available alpha-amylases includes Liquozyme® LpH, Liquozyme® SC, Liquozyme® Supra, Fortiva® Edge Alpha, Spezyme® HT, Spezyme® Ethyl, Spezyme® Fred.

In an embodiment the alpha-amylase added during liquefaction is selected from the group consisting of:

(a) an alpha-amylase comprising or consisting of the polypeptide of SEQ I D NO: 1 ;

(b) an alpha-amylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 1 ;

(c) a fragment of the polypeptide of (a) or (b); wherein the polypeptide has alphaamylase activity.

In an embodiment, the alpha-amylase added during liquefaction is selected from the group consisting of:

(a) an alpha-amylase comprising or consisting of the polypeptide of SEQ ID NO: 2;

(b) an alpha-amylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 2; c) a fragment of the polypeptide of (a) or (b); wherein the polypeptide has alphaamylase activity.

(a) an alpha-amylase comprising or consisting of the polypeptide of SEQ ID NO: 1 and SEQ ID NO: 2;

(b) an alpha-amylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 1 and SEQ ID NO: 2; c) a fragment of the polypeptide of (a) or (b); wherein the polypeptide has alphaamylase activity.

Examples of suitable thermostable glucoamylases include, without limitation, the glucoamylases described in WO 2011/127802, WO 2013/036526, WO 2013/053801 , WO 2018/164737, WO 2020/010101 , and WO 2022/090564 (each of which is incorporated herein by reference).

Examples of suitable thermostable endoglucanases include, without limitation, the endoglucanases described in WO 2015/035914 (which is incorporated herein by reference)

Examples of suitable thermostable lipases include, without limitation, the lipases described in WO 2017/112542 and WO 2020/014407 (which are both incorporated herein by reference).

Examples of suitable thermostable phytases include, without limitation, the phytases described in WO 1996/28567, WO 1997/33976, WO 1997/38096, WO 1997/48812, WO 1998/05785, WO 1998/06856, WO 1998/13480, WO 1998/20139, WO 1998/028408, WO 1999/48330, WO 1999/49022, WO 2003/066847, WO 2004/085638, WO 2006/037327, WO 2006/037328, WO 2006/038062, WO

2006/063588, WO 2007/112739, WO 2008/092901 , WO 2008/116878, WO

2009/129489, and WO 2010/034835 (each of which is incorporated herein by reference). Commercially available phytase containing products include BIO-FEED PHYTASE™, PHYTASE NOVO™ CT or L, LIQMAX or RONOZYME™ NP, RONOZYME® HIPHOS, RONOZYME® P5000 (CT), NATUPHOS™ NG 5000.

Examples of suitable thermostable proteases include, without limitation, the proteases described in WO 1992/02614, WO 98/56926, WO 2001/151620, WO 2003/048353, WO 2006/086792, WO 2010/008841 , WO 2011/076123, WO 2011/087836, WO 2012/088303, WO 2013/082486, WO 2014/209789, WO 2014/209800, WO 2018/098124, WO2018/118815 A1 , and WO2018/169780A1 (each of which is incorporated herein by reference).

Suitable commercially available protease containing products include AVANTEC AMP®, FORTIVA REVO®, FORTIVA HEM I®.

Examples of suitable thermostable pullulanases include, without limitation, the pullulanases described in WO 2015/007639, WO 2015/110473, WO 2016/087327, WO 2017/014974, and WO 2020/187883 (each of which is incorporated herein by reference in its entirety). Suitable commercially available pullulanase products include PROMOZYME 400L, PROMOZYME™ D2 (Novozymes A/S, Denmark), OPTIMAX L- 300 (Genencor Int. , USA), and AMANO 8 (Amano, Japan).

Examples of suitable thermostable xylanases include, without limitation, the xylanases described in WO 2017/112540 and WO 2021/126966 (each of which is incorporated herein by reference). Suitable commercially available thermostable xylanase containing products include FORTIVA HEMI®.

The enzyme(s) described above are to be used in effective amounts in the processes of the present invention. Guidance for determining effective amounts of enzymes to be used in liquefying step (a) can be found in the published patent applications cited for each of the different thermostable liquefaction enzymes, along with guidance for performing activity assays for determining the activity of those enzymes.

In a preferred embodiment, saccharification may be performed at temperatures ranging from 20 °C to 75 °C, from 30 °C to 70 °C, or from 40 °C to 65 °C. Preferably, the saccharification temperature is at least about 50 °C, at least about 55 °C, or at least about 60 °C.

In a preferred embodiment, saccharification may occur at a pH ranging from 4 to 5. Preferably, the pH is about 4.5.

In a preferred embodiment, saccharification may last from about 24 hours to about 72 hours.

In a preferred embodiment, fermentation may last from 6 to 120 hours, from 24 hours to 96 hours, or from 35 hours to 60 hours.

In a preferred embodiment, Simultaneous Saccharification and Fermentation (SSF) may be performed at a temperature from 25 °C to 40 °C, from 28 °C to 35 °C, or from 30 °C to °C, at a pH from 3.5 to 5 or from 3.8 to 4.3., for 24 to 96 hours, 36 to 72 hours, or from 48 to 60 hours. Preferably, SSF is performed at about 32 °C, at a pH from 3.8 to 4.5 for from 48 to 60 hours. The present invention contemplates the use of enzymes during saccharifying step and/or fermenting step. It is well known in the art to use various enzymes during saccharifying step and/or fermenting step, including, for example, alpha-amylases, alpha-glucosidases, beta-amylases, beta-glucanases, beta-glucosidases, cellobiohydrolases, endoglucanases, glucoamylases, lipases, lytic polysaccharide monooxygenases (LPMOs), maltogenic alpha-amylases, pectinases, peroxidases, phytases, proteases, and trehalases.

The enzymes used in saccharifying step and/or fermenting step may be added exogenously as mono-components or formulated as compositions comprising the enzymes. The enzymes used in saccharifying step and/or fermenting step may also be added via in situ expression from the fermenting organism (e.g., yeast).

Examples of suitable alpha-amylases include, without limitation, the alphaamylases described in WO 2004/055178, WO 2006/069290, WO 2013/006756, WO 2013/034106, WO 2013/044867, WO 2021/163011 , and WO 2021/163030 (each of which is incorporated herein by reference).

Examples of suitable glucoamylases include, without limitation, the glucoamylases described in WO 1984/02921 , WO 1992/00381 , WO 1999/28448, WO 2000/04136, WO 2001/04273, WO 2006/069289, WO 2011/066560, WO 2011/066576, WO 2011/068803, WO 2011/127802, WO 2012/064351 , WO

2013/036526, WO 2013/053801 , WO 2014/039773, WO 2014/177541 , WO

2014/177546, WO 2016/062875, WO 2017/066255, and WO 2018/191215 (each of which is incorporated herein by reference).

Examples of suitable compositions comprising alpha-amylases and glucoamylases include, without limitation, the compositions described in WO 2006/069290, WO 2009/052101 , WO 2011/068803, and WO 2013/006756 (each of which is incorporated by reference herein). Commercially available compositions comprising glucoamylase include AMG 200L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™ FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA, SPIRIZYME™ EXCEL, SPIRIZYME ACHIEVE and AMG™ E (from Novozymes A/S); OPTIDEX™ 300, GC480, GC417 (from DuPont-Genencor); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900, G-ZYME™ and G990 ZR (from DuPont-Genencor).

Examples of suitable beta-glucanases include, without limitation, the beta- glucanases described in WO 2021/055395 (which is incorporated herein by reference).

Examples of suitable beta-glucosidases include, without limitation, the betaglucosidases described in WO 2005/047499, WO 2013/148993, WO 2014/085439 and WO 2012/044915 (each of which is incorporated herein by reference). Examples of suitable cellobiohydrolases include, without limitation, the cellobiohydrolases described in WO 2013/148993, WO 2014/085439, WO 2014/138672, and WO 2016/040265 (each of which is incorporated herein by reference).

Examples of suitable endoglucanases include, without limitation, the endoglucanases described in WO 2013/148993 and WO 2014/085439 (both of which are incorporated herein by reference).

Examples of suitable maltogenic alpha-amylases are described in US Patent nos. 4,598,048, 4,604,355 and 6,162,628, which are hereby incorporated by reference.

Examples of suitable lipases include, without limitation, the lipases described in WO 2017/112533, WO 2017/112539, and WO 2020/076697 (each of which is incorporated herein by reference).

Examples of suitable LPMOs include, without limitation, the LPMOs described in WO 2013/148993, WO 2014/085439, and WO 2019/083831 (each of which is incorporated herein by reference).

Examples of suitable phytases include, without limitation, the phytases described in WO 2001/62947 (which is incorporated herein by reference).

Examples of suitable pectinases include, without limitation, the pectinases described in WO 2022/173694 (which is incorporated herein by reference).

Examples of suitable peroxidases include, without limitation, the peroxidases described in WO 2019/231944 (which is incorporated herein by reference).

Examples of suitable proteases include, without limitation, the proteases described in WO 2017/050291 , WO 2017/148389, WO 2018/015303, and WO 2018/015304 (each of which is incorporated herein by reference).

Examples of suitable trehalases include, without limitation, the trehalases described in WO 2016/205127, WO 2019/005755, WO 2019/030165, and WO 2020/023411 (each of which is incorporated herein by reference).

In a preferred embodiment, the alpha-amylase is of fungal or bacterial origin.

In a preferred embodiment, the alpha-amylase is a fungal acid stable alphaamylase.

A fungal acid stable alpha-amylase is an alpha-amylase that has activity in the pH range of 3.0 to 7.0 and preferably in the pH range from 3.5 to 6.5, including activity at a pH of about 4.0, 4.5, 5.0, 5.5, and 6.0.

In a preferred embodiment the alpha-amylase is present and/or added in saccharification and/or fermentation is derived from a strain of the genus Rhizomucor, preferably a strain the Rhizomucor pusillus, such as one shown in SEQ ID NO: 3 in WO 2013/006756, such as a Rhizomucor pusillus alpha-amylase hybrid having an Aspergillus niger linker and starch-binding domain, such as the one shown in SEQ ID NO: 3 herein, or a variant thereof.

In an embodiment, the alpha-amylase is an acid stable amylase of fungal origin, preferably from a stain of Aspergillus, preferably A. niger, A. awamori, or A. oryzae or a strain of Rhizomucor, preferably Rhizomucor pusilus.

In an embodiment, the alpha-amylase is of bacterial origin. E.g., derived from a strain of Bacillus sp.

Most preferably the alpha-amylase comprises a CBM20 or CBM26.

In an embodiment the alpha-amylase catalytic domain is derived from Rhizomucor, such as a strain of Rhizomucor pusillus, such as the one comprised in in SEQ ID NO: 3 herein, and optionally comprising the substitutions G128D + D143N using SEQ ID NO: 3 for numbering.

In an embodiment, the glucoamylase used in saccharification or simultaneous saccharification and fermentation (SSF) is of fungal origin, preferably from a stain of Aspergillus, preferably A. niger, A. awamori, or A. oryzae or a strain of Trichoderma, preferably T. reeser, or a strain of Talaromyces, preferably T. emersonii or a strain of Trametes, preferably T. cingulata, or a strain of Pycnoporus, preferable P. sanguineus, or a strain of Gloeophyllum, such as G. sepiarium or G. trabeum, or a strain of the Nigrofomes.

In an embodiment the glucoamylase is derived from Gloeophyllum, such as a strain of Gloeophyllum sepiarium, such as the one shown in SEQ ID NO: 4.

In an embodiment the glucoamylase is selected from the group consisting of:

(a) a glucoamylase comprising or consisting of the polypeptide of SEQ ID NO: 4;

(b) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 4;

(c) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acids 1-556 of SEQ ID NO: 4; d) a fragment of the polypeptide of (a), (b), or (c); wherein the polypeptide has glucoamylase activity.

Glucoamylases may in an embodiment be added to the saccharification and/or fermentation in an amount of 0.0001-20 AGU/g DS, preferably 0.001-10 AGU/g DS, especially between 0.01-5 AGU/g DS, such as 0.1-2 AGU/g DS. Commercially available compositions comprising glucoamylase include AMG 200L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™ FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA, SPIRIZYME™ EXCEL and AMG™ E (from Novozymes A/S); OPTIDEX™ 300, GC480, GC417 (from DuPont.); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900, G- ZYME™ and G990 ZR (from DuPont).

Protease Present and/or Added During Liquefaction

According to the invention a thermostable protease may be optionally present and/or added during liquefaction together with an alpha-amylase, such as a thermostable alpha-amylase, and optionally a carbohydrate-source generating enzyme, in particular a thermostable glucoamylase, and/or optionally a pullulanase.

Proteases are classified on the basis of their catalytic mechanism into the following groups: Serine proteases (S), Cysteine proteases (C), Aspartic proteases (A), Metallo proteases (M), and Unknown, or as yet unclassified, proteases (U), see Handbook of Proteolytic Enzymes, A. J. Barrett, N.D. Rawlings, J. F. Woessner (eds), Academic Press (1998), in particular the general introduction part.

In a preferred embodiment the thermostable protease used according to the invention is a “metallo protease” defined as a protease belonging to EC 3.4.24 (metalloendopeptidases); preferably EC 3.4.24.39 (acid metallo proteinases).

To determine whether a given protease is a metallo protease or not, reference is made to the above “Handbook of Proteolytic Enzymes” and the principles indicated therein. Such determination can be carried out for all types of proteases, be it naturally occurring or wild-type proteases; or genetically engineered or synthetic proteases.

Protease activity can be measured using any suitable assay, in which a substrate is employed, that includes peptide bonds relevant for the specificity of the protease in question. Assay-pH and assay-temperature are likewise to be adapted to the protease in question. Examples of assay-pH-values are pH 6, 7, 8, 9, 10, or 11. Examples of assay-temperatures are 30, 35, 37, 40, 45, 50, 55, 60, 65, 70 or 80°C.

In an embodiment the thermostable protease has at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 100% of the protease activity of the Protease 196 variant or Protease Pfu determined by the AZCL-casein assay.

There are no limitations on the origin of the protease used in a process of the invention as long as it fulfills the thermostability properties defined below.

In one embodiment the protease is of fungal origin. The protease may be a variant of, e.g., a wild-type protease as long as the protease has the thermostability properties defined herein. In a preferred embodiment the thermostable protease is a variant of a metallo protease as defined above. In an embodiment the thermostable protease used in a process of the invention is of fungal origin, such as a fungal metallo protease, such as a fungal metallo protease derived from a strain of the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670 (classified as EC 3.4.24.39).

The thermostable protease may also be derived from any bacterium as long as the protease has the thermostability properties defined according to the invention.

In an embodiment the thermostable protease is derived from a strain of the bacterium Pyrococcus, such as a strain of Pyrococcus furiosus (pfu protease). Fermenting Organisms

Suitable fermenting organisms able to ferment, i.e., convert, sugars, such as arabinose, glucose, maltose, and/or xylose, directly or indirectly into the desired fermentation product, such as ethanol. Examples of fermenting organisms include fungal organisms, such as yeast. Preferred yeast includes strains of Saccharomyces spp., in particular, Saccharomyces cerevisiae.

Examples of commercially available yeast includes, e.g., RED STAR™ and ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI (available from Fleischmann’s Yeast, USA), SUPERSTART and THERMOSACC™ fresh yeast (available from Ethanol Technology, Wl, USA), BIOFERM AFT and XR (available from NABC - North American Bioproducts Corporation, GA, USA), GERT STRAND (available from Gert Strand AB, Sweden), FERMIOL (available from DSM Specialties) and Innova® Achieve® D; Innova® Apex; Innova® Excel T; Innova® PT; Innova® Force; Innova® Ultra-L (available from Novozymes, Denmark). Other useful yeast strains are available from biological depositories such as the American Type Culture Collection (ATCC) or the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), such as, e.g., BY4741 (e.g., ATCC 201388); Y108-1 (ATCC PTA.10567) and NRRL YB-1952 (ARS Culture Collection). Still other S. cerevisiae strains suitable as host cells DBY746, [Alpha][Eta]22, S150-2B, GPY55-15Ba, CEN.PK, USM21 , TMB3500, TMB3400, VTT-A-63015, VTT-A-85068, VTT-c-79093 and their derivatives as well as Saccharomyces sp. 1400, 424A (LNH-ST), 259A (LNH- ST) and derivatives thereof.

As used herein, a “derivative” of strain is derived from a referenced strain, such as through mutagenesis, recombinant DNA technology, mating, cell fusion, or cytoduction between yeast strains. Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, may be described with reference to a suitable host organism and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art can apply the teachings and guidance provided herein to other organisms. For example, the metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species.

The fermenting organism may be Saccharomyces strain, e.g., Saccharomyces cerevisiae strain produced using the method described and concerned in US patent no. 8,257,959-BB. In one embodiment, the recombinant cell is a derivative of a strain Saccharomyces cerevisiae CIBTS1260 (deposited under Accession No. NRRL Y- 50973 at the Agricultural Research Service Culture Collection (NRRL), Illinois 61604 U.S.A.).

The fermenting organism may also be a derivative of Saccharomyces cerevisiae strain NMI V14/004037 (See, WO2015/143324 and WO2015/143317 each incorporated herein by reference), strain nos. V15/004035, V15/004036, and V15/004037 (See, WO 2016/153924 incorporated herein by reference), strain nos. V15/001459, V15/001460, V15/001461 (See, WO2016/138437 incorporated herein by reference), strain no. NRRL Y67342 (See, WO2018/098381 incorporated herein by reference), strain nos. NRRL Y67549 and NRRL Y67700 (See, WO 2019/161227 incorporated herein by reference), or any strain described in WO2017/087330 (incorporated herein by reference).

The fermenting organisms may comprise one or more heterologous polynucleotides encoding an alpha-amylase, glucoamylase, protease and/or cellulase. Examples of alpha-amylase, glucoamylase, protease and cellulases suitable for expression in the fermenting organism are known in the art (See, WO2021/231623 incorporated herein by reference).

The fermenting organism may be in the form of a composition comprising a fermenting organism and a naturally occurring and/or a non-naturally occurring component.

The fermenting organism may be in any viable form, including crumbled, dry, including active dry and instant, compressed, cream (liquid) form etc. In one embodiment, the fermenting organism (e.g., a Saccharomyces cerevisiae yeast strain) is dry yeast, such as active dry yeast or instant yeast. In one embodiment, the fermenting organism is crumbled yeast. In one embodiment, the fermenting organism is a compressed yeast. In one embodiment, the fermenting organism is cream yeast.

In one embodiment is a process comprising a fermenting organism described herein (e.g., a Saccharomyces cerevisiae yeast strain), and one or more of the components selected from the group consisting of: surfactants, emulsifiers, gums, swelling agent, and antioxidants and other processing aids.

The process described herein may comprise a fermenting organism described herein (e.g., a Saccharomyces cerevisiae yeast strain) and any suitable surfactants. In one embodiment, the surfactant(s) is/are an anionic surfactant, cationic surfactant, and/or nonionic surfactant.

The process described herein may comprise a fermenting organism described herein (e.g., a Saccharomyces cerevisiae yeast strain) and any suitable emulsifier. In one embodiment, the emulsifier is a fatty-acid ester of sorbitan. In one embodiment, the emulsifier is selected from the group of sorbitan monostearate (SMS), citric acid esters of monodiglycerides, polyglycerolester, fatty acid esters of propylene glycol.

In one embodiment, the process comprises a fermenting organism described herein (e.g., a Saccharomyces cerevisiae yeast strain), and Olindronal SMS, Olindronal SK, or Olindronal SPL including composition concerned in European Patent No. 1 ,724,336 (hereby incorporated by reference). These products are commercially available from Bussetti, Austria, for active dry yeast.

The process described herein may comprise a fermenting organism described herein (e.g., a Saccharomyces cerevisiae yeast strain) and any suitable gum. In one embodiment, the gum is selected from the group of carob, guar, tragacanth, arabic, xanthan and acacia gum, in particular for cream, compressed and dry yeast.

The process described herein may comprise a fermenting organism described herein (e.g., a Saccharomyces cerevisiae yeast strain) and any suitable swelling agent. In one embodiment, the swelling agent is methyl cellulose or carboxymethyl cellulose.

The process described herein may comprise a fermenting organism described herein (e.g., a Saccharomyces cerevisiae yeast strain) and any suitable anti-oxidant. In one embodiment, the antioxidant is butylated hydroxyanisol (BHA) and/or butylated hydroxytoluene (BHT), or ascorbic acid (vitamin C), particular for active dry yeast.

Suitable concentrations of the viable fermenting organism during fermentation, such as SSF, are well known in the art or can easily be determined by the skilled person in the art. In one embodiment the fermenting organism, such as ethanol fermenting yeast, (e.g., Saccharomyces cerevisiae) is added to the fermentation medium so that the viable fermenting organism, such as yeast, count per mL of fermentation medium is in the range from 105 to 1012, preferably from 107 to 1010, especially about 5x107. Fermentation Products

The term “fermentation product” means a product produced by a process including a fermentation step using a fermenting organism. Fermentation products contemplated according to the invention include alcohols (e.g., ethanol, methanol, butanol; polyols such as glycerol, sorbitol and inositol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B12, beta-carotene); and hormones. In a preferred embodiment the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry. Preferred beer types comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer. Preferably processes of the invention are used for producing an alcohol, such as ethanol. The fermentation product, such as ethanol, obtained according to the invention, may be used as fuel, which is typically blended with gasoline. However, in the case of ethanol it may also be used as potable ethanol.

Recovery

Subsequent to fermentation, or SSF, the fermentation product may be separated from the fermentation medium. The slurry may be distilled to extract the desired fermentation product (e.g., ethanol). Alternatively, the desired fermentation product may be extracted from the fermentation medium by micro or membrane filtration techniques. The fermentation product may also be recovered by stripping or other method well known in the art.

Drying of Wet Cake and Producing Concentrated Protein

After washing the second insoluble solids portion to produce a third insoluble solids portion; the wet cake, containing about 25-45 wt-%, preferably 30-38 wt-% dry solids, can be dried using methods such as a drum dryer, spray dryer, ring drier, fluid bed drier. The drying process produces “Protein Concentrate” (PC) a valuable feed ingredient for human consumption, and animals, such as livestock, poultry and fish. To enhance shelf-life and reduce transportation cost, it is preferred to maintain PC with a moisture about to 10-12 wt.-%. Additionally, care should be taken during drying to avoid denaturing proteins in the wet cake.

The invention is further disclosed in the following numbered paragraphs. Paragraph 1 . A process for producing a fermentation product and a concentrated protein co-product from a starch-containing material, comprising the steps of:

(d) fermenting the sugar with a fermenting organism to produce the fermentation product, wherein at least one separation step is performed after the liquefaction in step

(a) resulting in a liquid portion comprising soluble starch/oligosacchrides and an insoluble portion comprising solids from the liquefied mash, said insoluble portion undergoing said protein concentration process.

Paragraph 2. A process for producing a fermentation product and a concentrated protein co-product from a starch-containing material, comprising the steps of:

(b) separating the slurry into a first insoluble solids portion and a first liquid portion comprising a soluble starch/ oligosacchrides stream;

(g) drying the third insoluble solids portion to produce a concentrated protein coproduct; (h) saccharifying the soluble starch/oligosacchrides stream and optionally the first and second sugar streams to produce a fermentable sugar in presence of a glucoamylase, and optionally an alpha-amylase;

Paragraph 3. The process according to any one of the preceding paragraph, wherein the starch containing material is selected from group consisting of rice, wheat, maize, millet, sorghum, oats, barely, potatoes and casava.

Paragraph 4. The process according to paragraph 3, wherein the starch- containing material comprises rice.

Paragraph 5. The process according to any of the preceding paragraphs, wherein the starch containing material is obtained from a dry-milling process.

Paragraph 6. The process according to any of the preceding paragraphs, wherein the concentrated protein coproduct recovery is above 50% w/w, such as 60% w/w, such as 70% w/w, such as 80% w/w.

Paragraph 7. The process according to any of the preceding paragraphs, wherein the concentrate protein co-product purity is at least 65% w/w, at least 70% w/w, at least 75% w/w, at least 80% w/w, at least 85% w/w, such as at least 90% w/w.

Paragraph 8. The process according to paragraph 1 , wherein saccharification in step (c) and fermentation in step (d) is carried out sequentially or simultaneously.

Paragraph 9. The process according to paragraph 2, wherein saccharification in step (h) and fermentation in step (i) is carried out sequentially or simultaneously.

Paragraph 10. The process according to any of the preceding paragraphs, wherein the pH during liquefaction is between above 5.0-6.5, such as above 5.0-6.0, such as above 5.0-5.5, such as between 5.2-6.2, such as around 5.2, such as around 5.4, such as around 5.6, such as around 5.8.

Paragraph 11. The process according to any of the preceding paragraphs, wherein the temperature during liquefaction is in the range from 70-100°C, such as between 75-95°C, such as between 75-90°C, preferably between 80-90°C, such as around 85°C.

Paragraph 12. The process according to any of the preceding paragraphs, wherein the saccharification step is carried out at pH values between 4.0 and 6.0.

Paragraph 13. The process according to any of the preceding paragraphs, wherein saccharification is carried out at a temperature from 20-75°C, preferably from 40-70°C, such as around 60°C. Paragraph 14. The process according to any of the preceding paragraphs, wherein the fermentation product is recovered after fermentation, such as by distillation.

Paragraph 15. The process according to any of the preceding paragraphs, wherein the fermentation product is an alcohol, preferably ethanol, especially fuel ethanol, potable ethanol and/or industrial ethanol.

Paragraph 16. The process according to any of the preceding paragraphs, wherein the fermenting organism is yeast, preferably a strain of Saccharomyces, especially a strain of Saccharomyces cerevisae.

Paragraph 17. The process according to any of the preceding paragraphs, further wherein a pullulanase is present or added during saccharification.

Paragraph 18. The process according to any of the preceding paragraphs, further wherein a glucoamylase is present or added during saccharification.

Paragraph 19. The process according to any of the preceding paragraphs, further wherein a protease is present or added during liquefaction.

Paragraph 20. The process according to any of the preceding paragraphs, wherein the alpha amylase is of bacterial or fungal origin.

Paragraph 21 . The process according to any of preceding paragraphs, wherein the alpha amylase in step (a) is derived from Bacillus amyloliquefaciens or Bacillus stearothermophilus.

Paragraph 22. The process according any of preceding paragraphs, wherein the alpha amylase added during saccharification is derived from a group consisting of Aspergillus niger, Aspergillus terreus, Meripilus giganteus, Rhizomucor pusillus and combination thereof.

Paragraph 23. The process according to any of preceding paragraphs, wherein the alpha amylase applied in saccharification is derived from a group consisting of Aspergillus niger, Aspergillus terreus, Meripilus giganteus, Rhizomucor pusillus and combination thereof.

Paragraph 24. The process according to any of preceding paragraphs, wherein the glucoamylase is of fungal origin, preferably from a strain of Aspergillus, preferably A. niger, Aspergillus fumigatus, A. awamori, or A. oryzae or a strain of Trichoderma, preferably T. reeser, or a strain of Talaromyces, preferably T. emersonii or a strain of Trametes, preferably T. cingulata, or a strain of Pycnoporus, or a strain of Gloeophyllum, such as G. serpiarium or G. trabeum, or a strain of the Nigrofomes, Penicillium oxalicum or Humicloa insolens.

Paragraph 25. The process according to paragraph 17, wherein the pullulanase is derived from Bacillus deramificans or Bacillus acidopullulyticus. Paragraph 26. The process according to paragraph 19, wherein the protease is a variant of a metallo protease derived from a strain of the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670.

Paragraph 27. The process according to claim 19, wherein the protease is derived from a strain of Pyrococcus, preferably a strain of Pyrococcus furiosus.

Paragraph 28. The process of any one of preceding paragraphs, wherein the fermentation product is ethanol.

Paragraph 29. The process of any one of preceding paragraphs, wherein the fermenting organism is yeast.

Paragraph 30. The process of any one of preceding paragraphs, wherein separation is done using centrifugation, filtration, decantation and/or cloth filtration.

Paragraph 31. The process of any one of preceding paragraphs, wherein the alpha-amylase added during liquefaction is selected from the group consisting of:

(b) an alpha-amylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 1 ; c) a fragment of the polypeptide of (a) or (b); wherein the polypeptide has alphaamylase activity.

Paragraph 32. The process of any one of preceding paragraphs, wherein the alpha-amylase added during liquefaction is selected from the group consisting of:

(b) an alpha-amylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 2;

Paragraph 33. The process of any one of preceding paragraphs, wherein the alpha-amylase added during liquefaction is selected from the group consisting of:

Paragraph 34. The process of any one of preceding paragraphs, wherein the alpha-amylase added during saccharification is selected from the group consisting of:

(a) an alpha-amylase comprising or consisting of the polypeptide of SEQ ID NO: 3;

(b) an alpha-amylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 3;

(c) an alpha-amylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acids 1-556 of SEQ ID NO: 3; d) a fragment of the polypeptide of (a), (b), or (c); wherein the polypeptide has alpha-amylase activity.

Paragraph 35. The process of any one of preceding paragraphs, wherein the glucoamylase added during saccharification is selected from the group consisting of:

(a) a glucoamylase comprising or consisting of the polypeptide of SEQ ID NO: 4;

Paragraph 36. The process of any one of preceding paragraphs, wherein the pullulanase added in saccharification step (d) is selected from the group consisting of:

(a) a pullulanase comprising or consisting of the polypeptide of SEQ ID NO: 5;

(b) a pullulanase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 5; (c) a pullulanase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acids 1-556 of SEQ ID NO: 5; d) a fragment of the polypeptide of (a), (b), or (c); wherein the polypeptide has pullulanase activity.

Paragraph 37. The process of any one of preceding paragraphs, wherein the lysophospholipase added in saccharification step (d) is selected from the group consisting of:

(a) a xylanase comprising or consisting of the polypeptide of SEQ ID NO: 7;

(b) a xylanase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 7;

(c) a xylanase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acids 1-556 of SEQ ID NO: 7; d) a fragment of the polypeptide of (a), (b), or (c); wherein the polypeptide has xylanase activity.

Paragraph 38. The process of any one of preceding paragraphs, wherein the lysophospholipase added in saccharification step (d) is selected from the group consisting of:

(a) a lysophospholipase comprising or consisting of the polypeptide of SEQ ID NO: 6;

(b) a lysophospholipase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 6;

(c) a lysophospholipase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acids 1-556 of SEQ ID NO: 6; d) a fragment of the polypeptide of (a), (b), or (c); wherein the polypeptide has lysophospholipase activity.

Paragraph 39. The process of any one of preceding paragraphs, wherein the cellulase composition produced from Trichoderma reesei is added during saccharification. Paragraph 40. The process according to paragraph 2, further comprises of addition of protease before step (g).

Paragraph 41. A fermentation product and a concentrated protein co-product produced by a process as claimed in paragraphs 1-40.

Paragraph 42. The process according to paragraph 41 , wherein the fermentation product is an alcohol, an organic acid, a ketone, an amino acid, or a gas.

Paragraph 43. The process according to paragraph 42, wherein the alcohol is ethanol.

Paragraph 44: The process according to paragraph 43, wherein the protein coproduct us used in feed, food and/or beverage industry.

The invention is further illustrated by the following examples.

EXAMPLES

Materials & Methods

Materials:

Alpha-Amylase composition added during liquefaction comprising of an alphaamylase shown in as SEQ ID NO: 1 and an alpha-amylase shown in as SEQ ID NO: 2 herein.

Experimental saccharification composition added during saccharification step (d) comprising of alpha-amylase as shown in SEQ ID NO: 3 herein, glucoamylase as shown in as SEQ ID NO: 4 herein, pullulanase as shown in as SEQ ID NO: 5 herein, lysophospholipase as shown in as SEQ ID NO: 6 herein and xylanase as shown in as SEQ ID NO: 7 herein.

A whole cellulase composition produced from Trichoderma reesei Tv10 and comprising all cellulases normally produce from T. reesei. Also available as Celluclast®.

Example 1 : Rice protein concentrate

Rice samples from different region having brand name like Calrose Rice- from North America Region; Grod Ris- from Europe Region and Parboiled Ris from Europe Region.

Below table Represents the Proximate analysis of Rice samples received from different regions and the analysis was conducted based on AOAC methods.

AOAC method details are:

1 . Moisture Analysis: AOAC 972.20

2. Starch Analysis: AOAC 969.39

3. Protein Analysis: AOAC 992.23

4. Fat Analysis: AOAC 945.16

5. Ash Determination: AOAC 942.05

With three different variety of rice the initial Starch was in the range of 78 to 80% and Protein was in the range of 4 to 6%.

Rice Cleaning and Size Reduction (Particle Size): Rice is received from various sources and was pre-cleaned to remove stones, husks, straws, and iron metals. The cleaned rice was then milled using a Buhler Mill to achieve a particle size fine to coarse <300 to 1200 microns. The flour was sieved to the desired particle size and 5 to 100% of the flour was diverted to the protein extraction process, while the remaining portion was used for the conventional ethanol process.

Slurry Preparation: The desired particle size rice flour (<300 microns) was transferred to the slurry preparation tank with 30% dry solids (DS), and the slurry pH was adjusted to 5.8 while maintaining a conductivity of >400 microsiemens.

First Liquefaction: First Liquefaction has two steps: first is Jet cooking A and then Liquefaction A. Once the rice slurry was prepared, the initial 30% of alpha amylase (0.3 Kg/ TDS) was added before jet cooking A at 110 °C for 10 minutes. Then, the remaining 70% of alpha-amylase (alpha-amylase composition was added at 0.7 Kg/ TDS) was added before liquefaction A, which was incubated at 95 °C for 3.5 hours with constant mixing.

Filtration: After first Liquefaction, the liquefied slurry was transferred for solidliquid separation, where solids and liquids are separated by a cloth (cloth filtration) with a 50-micron pore size at a slurry temperature of 75 - 80 °C to achieve better separation. The wet cake portion is taken for further rice protein concentrate (RPC) process, and the liquid portion containing solubilized starch stream was sent to conventional fermenters for fermentation.

Wet Cake Slurry Preparation: Wet cake was transferred to the slurry preparation tank, and a 16% dry solids (DS) slurry was prepared by adjusting the slurry pH to 5.8 and conductivity to be greater than 400 microsiemens. Second Liquefaction: Second Liquefaction has two steps: first was Jet cooking B and then Liquefaction B. Once the 16% DS slurry was prepared, alpha-amylase (alpha-amylase composition was added at 1.5 Kg/ T DS) was added before jet cooking B at 110 °C for 10 minutes. Then, another dosage of alpha-amylase (alpha-amylase composition was added at 1 .5 Kg/ T DS) was added before liquefaction B, which was incubated at 95 °C for 2 hours with constant mixing.

Saccharification: After second Liquefaction, the liquefied slurry is cooled to 55 °C. Then, glucoamylase, alpha-amylase, pullulanase, lysophospholipase, cellulase, xylanase, arabinofuranisidase, and/or hemicellualse (Experimental saccharification composition added during saccharification comprising of alpha-amylase as shown in SEQ ID NO: 3 herein, glucoamylase as shown in as SEQ ID NO: 4 herein, pullulanase as shown in as SEQ ID NO: 5 herein, lysophospholipase as shown in as SEQ ID NO: 6 herein and xylanase as shown in as SEQ ID NO: 7 herein and supplemented with cellulases from T. reeser, each at a dosage of 1 .5 Kg/ TDS), are added and incubated for 18 hours at 55 °C with constant mixing to convert complex starch/ sugars into simpler fermentable sugars.

Filtration: After Saccharification, the saccharified slurry was transferred for solid-liquid separation, where solids and liquids are separated by a cloth with a 50- micron pore size at a slurry temperature of 75 - 80 °C to achieve better separation. The wet cake portion was taken for further washing process, and the liquid portion containing solubilized sugar stream was sent to conventional fermenters for fermentation.

Washing: After cloth filtration, the wet cake was further washed with hot water at 70 °C using a ratio of 1 :1.4, meaning that for every portion of wet cake, 1.4 portion of water are added. This process involves constant mixing for 30 minutes to remove sugars bound to protein molecules and further concentrate the protein content.

Filtration: After Washing the wet cake, the slurry was transferred for solid-liquid separation, where solids and liquids are separated by a cloth with a 50-micron pore size at a slurry temperature of 75 - 80 °C to achieve better separation. The protein rich wet cake portion was taken for drying process, and the liquid portion containing solubilized sugar stream was sent to conventional fermenters for fermentation.

Drying: Protein-rich rice wet cake was then transferred to a dryer and dried at 50°C, achieving a protein purity of 85%.

Fermentation: After each cloth filtration, the liquid portions rich in starch and sugars are combined and sent for fermentation leading to the production of ethanol. Rice Protein Concentrate (RPC) Steps and its intermediate product analysis Analysis of different components after each filtration process for soluble and insoluble byproducts was summarized as below. After First Liquefaction, the insoluble Protein purity was 54% in Calrose Rice as compared to 51 % in Grod Ris and 37% in Parboiled Ris. After second Liquefaction, the insoluble Protein purity was 83% in Calrose Rice as compared to 82% in Grod Ris and73% in Parboiled Ris. To remove the sugars formed after saccharification process in protein solids, washing step was introduced to get more protein purity, and after washing the protein purity was increased to 90% in Calrose Rice, 88% in Grod Ris and 83% in Parboiled Ris.

1. PROTEIN MASS BALANCE Protein extraction efficiency was calculated based on the initial protein quantity in the starting raw material rice and compared with the final protein quantity. Protein extraction efficiency was found to be in the range of 86 to 87% in all three different variety of Rice. 2. FERMENTATION Across three different varieties of Rice (Calrose Rice, Grod Ris and Parboiled Ris) more than 83% protein purity has been achieved using our enzyme application, with >86% recovery. Calrose Rice showed highest protein purity of 90.47% on dry basis as compared with Grod Ris (88.29%) and Parboiled Ris (83.22%). Overall ethanol yield >15.14 %v/v with 72 hours of fermentation. Different variety of Rice showed the efficiency of fermentation varied from 90 to 92% which are identical to conventional distilling process. Residual Sugar (RS) in the final fermented wash was in the range of 0.2 to 0.5%. Resistant Starch (RST) in the final fermented wash was in the range of 0.62 to 1.63%. By using different variety of rice across region, with our enzyme application we can increase the Rice Protein Concentration to >83% with15%v/v ethanol yield.

Claims

1 . A process for producing a fermentation product and a concentrated protein coproduct from a starch-containing material, comprising the steps of:

2. A process for producing a fermentation product and a concentrated protein coproduct from a starch-containing material, comprising the steps of:

3. The process according to any one of the preceding claims, wherein the starch containing material is selected from group consisting of rice, wheat, maize, millet, sorghum, oats, barely, potatoes and casava.

4. The process according to claim 3, wherein the starch-containing material comprises rice.

5. The process according to any of the preceding claims, wherein the starch containing material is obtained from a dry-milling process.

6. The process according to any of the preceding claims, wherein the concentrated protein coproduct recovery is above 50% w/w, such as 60% w/w, such as 70% w/w, such as 80% w/w.

7. The process according to any of the preceding claims, wherein the concentrate protein co-product purity is at least 65% w/w, at least 70% w/w, at least 75% w/w, at least 80% w/w, at least 85% w/w, such as at least 90% w/w.

8. The process according to any of the preceding claims, further wherein a pullulanase is present or added during saccharification.

9. The process according to any of the preceding claims, further wherein a glucoamylase is present or added during saccharification.

10. The process according to any of the preceding claims, further wherein a protease is present or added during liquefaction.

11. The process according to any of preceding claims, wherein the alpha amylase in step (a) is derived from Bacillus amyloliquefaciens or Bacillus stearothermophilus.

12. The process according any of preceding claims, wherein the alpha amylase applied in saccharification is derived from a group consisting of Aspergillus niger, Aspergillus terreus, Meripilus giganteus, Rhizomucor pusillus and combination thereof.

13. The process according to any of preceding claims, wherein the alpha amylase applied in saccharification is derived from a group consisting of Aspergillus niger, Aspergillus terreus, Meripilus giganteus, Rhizomucor pusillus and combination thereof.

14. The process according to any of preceding claims, wherein the glucoamylase is of fungal origin, preferably from a strain of Aspergillus, preferably A. niger, Aspergillus fumigatus, A. awamori, or A. oryzae; or a strain of Trichoderma, preferably T. reesei; or a strain of Talaromyces, preferably T. emersonii or a strain of Trametes, preferably T. cingulata, or a strain of Pycnoporus, or a strain of Gloeophyllum, such as G. serpiarium or G. trabeum, or a strain of the Nigrofomes, Penicillium oxalicum or Humicloa insolens.

15. A fermentation product and a concentrated protein co-product produced by a process as claimed in claims 1-14.