Back-end processing of co-products from ethanol plants

Abstract

Provided herein is a method of obtaining processed corn fermented protein (CFP), comprising: providing a composition that comprises mechanically separated CFP; removing water-soluble material from the composition; contacting the composition with an effective amount of an enzyme, a single cell organism that produces an enzyme, or a combination thereof, thereby producing enzymatically-treated CFP comprising enzymatic degradation products, wherein the enzyme is at least one of a proteolytic enzyme, a fiber-degrading enzyme, or a toxin-degrading enzyme; and removing enzymatic degradation products from the enzymatically-treated CFP, thereby forming processed CFP. Also provided herein is an animal feed comprising processed CFP (e.g., processed CFP made by a method described herein).

Description

6109.1001001 BACK-END PROCESSING OF CO-PRODUCTS FROM ETHANOL PLANTS RELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 636,913, filed on April 22, 2024. The entire teachings of the above application are incorporated herein by reference. BACKGROUND

[0002] The US ethanol industry developed during the interval of 1970-2016 in response to a series of forces in the political, energy market and financial arenas, as well as the natural resource sector. By 2014, it had achieved a production of greater than 12 billion gallons of ethanol (1). Non-ethanol fractions of the manufacturing stream were regarded typically as by-products possessing marginal value by ethanol producers. SUMMARY

[0003] Provided herein are methods of obtaining processed corn fermented protein (CFP), comprising: providing a composition that comprises mechanically separated CFP, wherein the mechanically separated CFP comprises water-soluble material and one or more of a fiber and an animal toxin; removing the water-soluble material; contacting the composition with an effective amount of an enzyme, a single cell organism that produces an enzyme, or a combination thereof, wherein the enzyme is at least one of a proteolytic enzyme, a fiber-degrading enzyme, or a toxin-degrading enzyme, thereby producing enzymatically-treated CFP comprising enzymatic degradation products; and removing the enzymatic degradation products from the enzymatically-treated CFP, thereby producing processed CFP. In some embodiments, the water-soluble material that is removed from the composition comprises a pH-sensitive macromolecular complex. In some embodiments, the method further comprises adjusting the pH of the composition (e.g., to assist in removing the pH-sensitive macromolecular complex). In some embodiments, the processed CFP is dried using spray drying.

[0004] Also provided herein are animal feeds comprising processed CFP (e.g., processed CFP made by a method described herein). - 1 - 4151998.v16109.1001001 BRIEF DESCRIPTION OF THE DRAWINGS

[0005] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0006] FIG.1: Schematic diagram of the evolution of dry grind ethanol production and subsequent modifications including those described in the present disclosure. (Panel A) – Initial components of corn-based ethanol production. (Panel B) – Development of mechanical separation processes #1 and #2 to fractionate corn slurry before fermentation (Mechanical Fractionation Process #1) or after fermentation and distillation (Mechanical Fractionation Process #2). In addition to mechanical fractionation processes #1 or #2, new enzymes and yeasts have been added to the corn mash before fermentation (referred to herein as a “front end processes”) which are indicated by the checkered arrow and dialog box located immediately to the right of the Panel B title. (Panel C) – The present disclosure is designed to provide cost effective improvements to the finished dried feed ingredient, which can be prepared, for example, by treating a wet slurry of unprocessed, or mechanically separated corn fermented protein (CFP) by tank-based processing using one or more enzymes that reduce the fiber, toxin and / or other antinutritional contents while increasing the overall protein content and digestibility (referred to herein as a “back-end process”). In addition, components of pH sensitive macromolecular complexes can be selectively removed from the CFP slurry to facilitate the activity of the enzyme(s) being added, thereby further improving the quality of the finished product, and facilitating its drying. As a result, the treated CFP slurry can either be dried in a more efficient manner (e.g., by spray drying) or incorporated directly into other downstream processes, such as the manufacture of extruded feeds.

[0007] FIG.2: General appearance of mechanically separated corn fermented protein (CFP) produced by a mechanical separation process (Fluid Quip Technologies, Cedar Rapids, IA) and its dried product. (Panel A) – mechanically separated CFP slurry containing a 70% moisture content; (Panel B) – Corresponding ring dried mechanically separated product containing a 10% moisture content; (Panel C) – “Wet” CFP product produced by adding sufficient water to increase the moisture content of dried mechanically separated CFP Protein to 70%.

[0008] FIG.3: General appearance of “wet” and dried processed CFP subjected to the present disclosure. (Panel A) – Wet undigested mechanically separated CFP corresponding to - 2 - 4151998.v16109.1001001 Panel A of FIG.2. Note that it’s quite crumbly and possesses the appearance of cookie dough. (Panel B) – Wet digested processed CFP subjected to a method of the present disclosure. Note its appearance is similar to peanut butter and is distinct from that of undigested mechanically separated CFP slurry. (Panel C) – Freeze dried undigested mechanically separated CFP; (Panel D) – Freeze dried digested processed CFP subjected to a method of the present disclosure. Samples A-D have each been subjected to a compression event using a thumb to illustrate differences in their texture and characteristics.

[0009] FIG.4: Graphical summary of digestion data shown in Table V. As compared to control (no digest values), the protein content (open circles – right vertical axis) increased whereas both the acid detergent content (filled columns – left axis) and neutral detergent content (open columns – left axis) decreased in processed CFP.

[0010] FIG.5: Changes in the protein content expressed on a dry matter basis of mechanically separated CFP slurry incubated for 16 hours with no (Control) or additions of cellulase / hemicellulase as described in Table VII.

[0011] FIG.6: Graphical summary of concentrations of various size carbohydrate fractions (DP1-10 and fructose) obtained from mechanically separated CFP slurries after 6 hours of digestion under present disclosure conditions. All values are normalized to quantities of carbohydrates present at Time 0 (without digestion). Table VII provides concentrations of enzymes used for Digests A, B and C.

[0012] FIG.7: SDS PAGE analysis of mechanically separated CFP protein slurry incubated with either no (Control), low (15 µg / 100gm of CFP protein) or high (90 µg / 100gm of CFP protein) for an interval of 30 minutes. All samples were processed for SDS-PAGE and compared to a mixture of standard proteins of known molecular mass (ladder). The bracket indicates protein bands that have been reduced in intensity while the leftward pointing arrow shows a smaller band that has increased in staining intensity.

[0013] FIG.8: Experimental format to treat mechanically separated CFP slurry with fumonisin esterase enzyme to detoxify the processed CFP protein product before drying.

[0014] FIG.9: Comparison of the increases in average weights of trout fed on of 4 different diets over an 8-week interval of time. Each time point represents changes in the 2- week interval as compared to the previous measurement. The error bars denote standard error of the mean.

[0015] FIG.10: Scatter plot of fish length (x axis) vs. body weight (y axis) for individual fish from each of the four test groups determined at the end of the trial. - 3 - 4151998.v16109.1001001

[0016] FIG.11: Processing and comparison of mechanically separated CFP slurry samples that were dried either by freeze drying with no heat exposure or ring drying with heat exposure. Note that both the coloration and darkness of the mechanically separated CFP product subjected to ring drying is more reddish and darker vs. its freeze-dried counterpart.

[0017] FIG.12: Comparison of protein, acid detergent fiber (ADF), neutral detergent fiber (NDF) and lysine content of mechanically separated CFP slurry samples dried either with no heat (freeze drying) or heat (ring drying) to obtain the dried CFP product. Shown are average values from 4 independent samples from plant #1 and a single sample from plant #2.

[0018] FIG.13: Comparison of Hunter Color analyses performed on samples described in FIGs.11 and 12. The spectrum of light-dark (L), green-red (A) and blue-yellow (B) measurements are shown at the top of the table. Each value represents the average of 4 independent measurements. Differences between ring dried and freeze dried CFP products are shown with reference to freeze dried values.

[0019] FIG.14: The decrease in Hunter Color Value L and increase in Color Value A displayed by dried mechanically separated CFP slurry with and without heat exposure correlates with a reduction in its lysine content.

[0020] FIG.15: Photograph of ring dried mechanically separated CFP products from two ethanol plants that were fractionated using mesh sieves with various size screens (#20-#100) followed by measuring the weight fraction of each size category within the sample (shown in box adjacent to each sample).

[0021] FIG.16: Comparison of chromatograms from size exclusion chromatography of water extracted fractions from larger #20 size particles (left panel) vs. smaller #80-#100 size particles (right panel) of Plant #2 mechanically separated CFP. Large downward pointing arrows denote the position of two macromolecular components of different sizes present in the left panel and minimally present on the right panel.

[0022] FIG.17: Experimental procedural diagram and resulting color changes to wet decanter cake when pH of mechanically separated CFP slurry is pH adjusted using either NaOH or HCl.

[0023] FIG.18: Overlaying of size exclusion chromatograms from supernatants derived from mechanically separated CFP slurry isolated after pH adjustments to either pH 7.6 or pH 5.7 as described in FIG.17. Identical equipment and protocols were used to generate FIG.16 data. - 4 - 4151998.v16109.1001001

[0024] FIG.19: Experimental protocol and results showing isolation of a pH sensitive macromolecular complex from mechanically separated CFP wet decanter cake supernatant.

[0025] FIG.20: Photograph comparing the appearance of freeze dried mechanically separated CFP vs. isolated pH sensitive complex obtained from mechanically separated CFP.

[0026] FIG.21: Simplified schematic diagram comparing the specifications and tank dimensions that achieve the same enzymatic digestion to produce a protein-rich corn co- product using either a single fermentation tank in an existing ethanol plant (Left Panel) or a single tank located at the back-end of an existing ethanol plant to enable the practice of the present disclosure on a commercial scale (Right Panel).

[0027] FIG.22: Schematic diagram of an example process of the disclosure, showing how the back-end process is distinct and separate from mechanical separation equipment such as the MSCTMprocess. Back-end process tanks receive mechanically separated CFP slurry from MSCTMprotein decanters to process CFP slurry creating processed CFP before its final centrifugation and drying in a spray dryer to yield improved dried processed CFP. Alternatively, the processed CFP can be sent to an existing ring dryer if desired. The rightward facing blue arrow shows removal of the macromolecular complex before drying of the processed CFP slurry and the return of more than 95% of the liquid in the form of dilute processed stillage to the “Backset Tank” for use in slurrying the incoming corn. DETAILED DESCRIPTION

[0028] A description of example embodiments follows.

[0029] The majority of ethanol production is derived from corn and occurs via a dry- grind process, the basic steps of which are shown in diagrammatic form illustrated in Panel A of FIG.1. After its testing and receipt, dried corn is ground and its starch component is subjected to liquefaction (conversion to soluble form), saccharification (conversion of starch to glucose) and fermentation under anaerobic conditions to yield an ethanol-containing mash. The ethanol is distilled from other components that are referred to as whole stillage. In this early ethanol industry production format (Panel A), whole stillage was fractionated via centrifuge to produce a liquid fraction called thin stillage that was either recycled to slurry incoming corn flour (small yellow leftward pointing arrow Panel A) or evaporated to a syrup and added to the heavy solids called distillers grains (DDGs) used commonly as cattle feed in wet or dry form (2). - 5 - 4151998.v16109.1001001

[0030] The basic format illustrated in Panel A of FIG.1 has been the subject of multiple improvements via both biotechnology and engineering additions during the interval of 2016 to the present. As illustrated in Panel B, application of such mechanical separation technology has provided increased revenue to corn ethanol producers via the manufacture of multiple new co-products derived from the fractionation of either corn slurry or whole stillage as well as increased ethanol yields (3). One such mechanical separation technology (Panel B - #1) is referred to as Fiber Separation Technology or FST (US Patent Nos. 11103811; 10800994; 11603507; 11427784; 0243143) and separates fiber components from corn slurry before fermentation and then feeds fiber-depleted slurry to the fermentation tank. The resulting fiber can be used for multiple fiber-related products including cellulosic ethanol production or fiber-based feeds for cattle produced as a stand-alone product or mixed with traditional DDGs.

[0031] Furthermore, the addition of specialized enzymes has increased the efficiency of the saccharification process and the use of new genetically engineered yeasts has further improved fermentation and ethanol yields. As an alternative to Process#1 that is shown in FIG.1, mechanical separation can be performed post fermentation and distillation using whole stillage as a substrate. In this processing mode (Process #2 – Panel B of FIG.1), whole stillage can be further separated into various forms via mechanical separation technology. These mechanically separated whole stillage co-products include: 1) distiller grains with solubles produced by fiber removal with some associated protein and its sequestration into DDGs with added thin stillage solubles, 2) distillers corn oil produced by removal of fat / oil from whole stillage, and 3) protein-enriched co-product known as corn fermented protein, abbreviated as CFP (e.g., produced by removal of fiber, oil and water from whole stillage) that contains a collection of various insoluble components including corn protein, yeast, residual fiber as well as a variety of compounds arising from either their synthesis by corn plants or possibly derived from other sources such as toxins from fungi or other organisms (see Process #2-Panel B of FIG.1).

[0032] CFP that is obtained (e.g., from an ethanol production plant) by fractionating the plant’s process stream either prior to fermentation (shown as Mechanical Fractionation Process #1 in FIG.1) or after fermentation and distillation (shown as Mechanical Fractionation Process #2 in FIG.1) yielding whole stillage that is separated using one or more separation methods utilized in the industry is referred to herein as “mechanically separated CFP”. This class of products are described under current Association of American - 6 - 4151998.v16109.1001001 Feed Control Officials (AAFCO) definitions 27.4, 27.527.6 and 27.8 as provided in https_distillersgrains_org / distillers-grains / , the contents of which are incorporated herein by reference in their entirety. Accordingly, “mechanically separated CFP” refers to CFP obtained directly by any suitable separation process applied to the ethanol plant’s process stream including whole stillage separated by methods such as, but not limited to, mechanical separation, flocculation, and electrostatic separation methods, or any combination of such methods. Mechanically separated CFP can occur in the form of a wet slurry, partially dried composition, or wholly dried composition (e.g., a dry powder). In some embodiments, mechanically separated CFP is the starting material, or feedstock, used to obtain “processed CFP” using the methods of the disclosure. In some embodiments, mechanically separated CFP comprises about 27 wt% to about 50 wt% crude protein (e.g., in the absence of treatment with auxiliary enzymes), such as about 27 wt% to about 30 wt% or about 46 wt% to about 50 wt%. In some embodiments, mechanically separated CFP comprises about 50 wt% to about 60 wt% crude protein (e.g., after treatment with auxiliary enzymes). In addition to crude protein, mechanically separated CFP can further comprise, for example, fiber, toxins, anti- nutritional components, oil, carbohydrates, water, and / or water-soluble material.

[0033] In contrast to the mechanically separated CFP starting material / feedstock for the methods of the present disclosure, the treated and altered CFP product that is produced by the methods of the present disclosure is referred to herein as “processed CFP”. Processed CFP provides benefits relative to mechanically separated CFP that include, but are not limited to, reduced amounts of fiber, toxin and / or antinutritional components (e.g., pH-sensitive complexes), reduced viscosity, and improved blending with other feed ingredients, etc. Processed CFP is produced initially in a wet form (e.g., a wet slurry, a wet cake) and can be dried (e.g., dehydrated) subsequently to produce partially dried CFP or wholly dried CFP (e.g., a dry processed CFP powder). The protein content of processed CFP will depend on the crude protein content of the mechanically separated CFP starting material / feedstock. In some embodiments, processed CFP comprises about 46 wt% to about 90 wt% protein or greater, such as, for example, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 80 wt% or about 90 wt% protein.

[0034] A person of ordinary skill in the art will appreciate that the principles, techniques and methods of the present disclosure can be readily applied to any protein-enriched corn co- product, regardless of whether the product is dried, partially dried or present in a wet slurry similar to that of CFP that is produced by ethanol plants using commonly used mechanical - 7 - 4151998.v16109.1001001 separation technologies (identified as Mechanical Fractionation Processes #1 and #2 in Panel B of FIG.1).

[0035] There are multiple mechanical separation technologies that have been developed for use in ethanol production. Non-limiting examples of such technologies are described in U.S. Patent No.9376504 – Hybrid Separation; U.S. Patent No.10233404; 10190076; 10160932; 87784333; – Methods for producing a high protein corn meal from a whole stillage byproduct and system therefore; U.S. Patent No.10226774 – Multi-zoned paddle screen apparatus; U.S. Patent No.9932246 – Pulse cavitation processor and method of using same; U.S. Patent No.88113973 – Apparatus and method for filtering a material from a liquid medium; U.S. Patent No.11,553,726 – Fractionated stillage separation. The entire contents of each of the aforementioned U.S. patents are incorporated herein by reference.

[0036] In order to further optimize the fractionation of fiber, oil and protein from whole stillage, a variety of specific fiber degrading enzymes are added to fermentation tanks as processing aids and are employed to degrade proteins (proteases), cellulose (cellulases), beta- glucans (ß-glucanases) and non-starch polysaccharides (xylanases) all of which are constituents of fiber matrices present in the corn kernel (4). These fiber degrading enzymes have been the subject of significant protein engineering efforts (5) to optimize them for enhancing ethanol co-product production singly or in combination (U.S. Patent App. Pub. Nos.: 2023 / 0265108 A1, 2023 / 0183667 A1, 2023 / 0148423 A1, 2023 / 0012672 A1, 2022 / 0279818 A1, the contents of which are incorporated herein by reference in their entireties). However, these specialized often unique “designer protein versions” of these enzymes used in corn ethanol plants are variants of enzymes derived from natural sources that are generally known by those skilled in the art. In this regard, such enzymes and enzymatic treatments have been utilized for a wide variety of applications and industries.

[0037] For their use as processing aids in ethanol fermentation, there is a need for the development and large-scale manufacture of specialized fiber degrading enzymes to facilitate such mechanical separation methods as presented above.

[0038] Another application that the present disclosure provides, which yields improvements and cost reduction or even elimination of such enzymes, involves the use of fiber degrading enzymes together with one or more additional enzymes, such as, for example, phytase, fumonisin esterase or fiber degrading enzymes that are presently added as a dry mixture to complete animal feeds (U.S. Patent App. Pub. No.2023 / 0357740 A1; 6-8, the contents of which are incorporated herein by reference). The use of enzymes in animal feeds - 8 - 4151998.v16109.1001001 are presently designed such that the respective enzymatic digestions of unwanted fiber, toxins and nutritional inhibitors are designed to occur only after the animal has ingested the feed where it is hydrated as part of the digestion process in the animal’s gastrointestinal tract. Such applications are used to address feed problems that are generally known by those skilled in the art where a high content of undigestible fiber and / or plant anti-nutritional compounds will reduce the feed’s nutrient digestibility properties and / or overall performance of many monogastric animals that ingest such feeds (6-11). Thus, the overall goal of these present-day enzyme additions to complete feeds is to enhance utilization of their dietary components particularly in feeds containing high dietary fiber and / or anti-nutritional components (6). Examples of patents describing the inclusion of fiber degrading enzymes into complete animal feeds for the purpose of increasing the nutrient utilization of feed components (examples include – U.S. Patent App. Pub. No.2023 / 0357740 A1; U.S. Patent No. 11241025; U.S. Patent No.10980249; U.S. Patent No.8815316; U.S. Patent No.8815315; U.S. Patent No.8409641; U.S. Patent No.7906310; U.S. Patent No.7662415; U.S. Patent No.7291493, the contents of each of the aforementioned patents are incorporated herein by reference in their entirety).

[0039] However, this general strategy of adding enzymes to complete feeds to degrade fiber and / or anti-nutritional components as well as toxins has some significant limitations. For example, addition of such enzymes to aquafeeds for fish that are extruded under high temperatures inactivates them. Application of these enzymes to the surfaces of pellets is not an efficient process (11). Moreover, enzyme digestion of ingredients present in complete feeds results in their digestive products being liberated directly into the animal’s gastrointestinal tract where they may produce a combination of deleterious and beneficial effects (12-14). As described below, the present disclosure presents the removal or reduction of the fiber, anti-nutritional and toxin content of feed ingredients and complete feeds by eliminating these compounds during the feed ingredient or complete feed manufacturing process.

[0040] Another significant challenge in the present-day production of corn-based feed ingredients such as CFP is the exposure of the mechanically separated CFP slurry to high temperature drying conditions that produces multiple unwanted changes that degrade the quality and appearance of the dried product. In general, after its mechanical separation from other whole stillage components, CFP is collected as a wet slurry that is then dried via commercial ring or drum dryer equipment. In order to achieve the desired moisture content of - 9 - 4151998.v16109.1001001 the final dried CFP product, the high moisture CFP slurry is exposed to elevated temperatures where water is removed but in order to facilitate this drying process, the partially dried CFP is recycled multiple times (encountering multiple exposures to high heat conditions). As is generally known by one skilled in the art, exposure of proteins to elevated temperatures in the presence of carbohydrate moieties (fiber as well as mono and polysaccharides) produce temporary and permanent chemical changes via Maillard reactions (15-18).

[0041] For the process of drying mechanically separated CFP slurry to a dried CFP product, unwanted Maillard reactions produce multiple challenges that include: changes in the coloration of dried CFP product making it darker and browner in color; loss of important amino acids possessing reactive amino groups such as lysine; and creation of crosslinks between protein and fiber moieties to create protein-carbohydrate fiber complexes.

[0042] All of these Maillard reaction processes are unwanted since they reduce the quality and digestibility of the final dried CFP product. As described below, the present disclosure has discovered a previously unknown reversible pH sensitive macromolecular complex that complicates the drying process for CFP and reduces its quality via Maillard reaction changes to CFP components.

[0043] The present disclosure is distinct from previous disclosures designed to either improve the overall process and efficiency of ethanol plant operations or used as feed additives that are designed to improve the performance of animals after their ingestion of specialized feeds.

[0044] It is important to note that an example embodiment of the present disclosure as shown in FIG.1, Panel C is distinct from all of the described disclosures as summarized above. These differences between the present disclosure and prior art include the following.

[0045] Embodiments of the present disclosure are not based on any mechanical separation and engineering improvements but rather on a fundamentally new concept of using a variety of biological processes that can occur individually or in concert to improve and / or alter existing co-products produced by ethanol plants prior to their present day finished product state.

[0046] Embodiments of the present disclosure are not designed as processing aids for existing ethanol plant production using specialized enzymes or compounds conforming to ethanol production requirements. Instead, the embodiments comprise a distinct “back-end” step that uses wet, partially dried or dried mechanically separated CFP as feedstock for the production of processed CFP. The “back-end” process can produce processed CFP - 10 - 4151998.v16109.1001001 independently of the constraints imposed by previous ethanol plant designs or process flow pathways.

[0047] Embodiments of the present disclosure do not require the use of specialized “designer” enzymes engineered to conform to the requirements of present-day ethanol production but rather can utilize naturally occurring enzymes or single cell organisms (e.g. bacteria or yeasts) where each is operated based on its individual biological or substrate requirements to optimize final improved co-product outcome.

[0048] Embodiments of the present disclosure add commercial value to existing ethanol plant co-products by removing unwanted compounds or adding valuable co-ingredients to an initial ethanol plant co-product to create processed CFP that can either be dried for sale as a feed ingredient or incorporated as a processed CFP slurry into the manufacture of complete formulated feeds.

[0049] Embodiments of the present disclosure allow for the simultaneous production of multiple value-added co-products from present day ethanol plants derived from a mechanically separated wet slurry of an existing ethanol co-product. This is possible by having multiple “back-end” processing units that are operated in parallel using the mechanically separated CFP product as feedstock. By contrast, present day applications to create such value-added ethanol co-products require these process operations to be performed in series in the form of “campaigns” by a single ethanol plant.

[0050] In the present disclosure, the discovery of a previously unknown pH-sensitive reversible macromolecular complex allows the adjustment of CFP slurry conditions to prevent the formation of this complex and facilitate the drying of processed CFP slurry and reduce its heat exposure. Alternatively, embodiments of the present disclosure allow for the efficient isolation of this complex and its separation from other processed CFP slurry components in order to more efficiently process CFP components with enzymes and increase its protein content. In addition, separation of this pH sensitive complex from other co- product(s) prevents the recycling of this macromolecular complex to liquid streams used to slurry ground corn flour at the start of the dry grind ethanol production process.

[0051] The present disclosure generally relates to methods of obtaining processed corn fermented protein (CFP). In some embodiments, the methods comprise one or more of: a) providing a composition (e.g., slurry, wet cake, powder) that comprises mechanically separated CFP; b) removing water-soluble material from the composition; c) contacting the composition with an effective amount of enzyme (e.g., naturally-occurring enzyme, artificial - 11 - 4151998.v16109.1001001 enzyme), a single cell organism that produces the enzyme, or a combination thereof, thereby producing enzymatically-treated CFP comprising enzymatic degradation products; and d) removing the enzymatic degradation products from the enzymatically-treated CFP, thereby forming processed CFP. In some embodiments, the composition further comprises one or more of fiber and animal toxin. In some embodiments, the enzyme is at least one of proteolytic enzyme, fiber-degrading enzyme, or toxin-degrading enzyme. The composition may be obtained as a co-product of ethanol production.

[0052] As used herein, enzymatically-treated CFP refers to CFP that is produced by contacting mechanically separated CFP with one or more enzymes (e.g., a toxin-degrading enzyme, a fiber-degrading enzyme, and / or a proteolytic enzyme) and / or enzyme-producing microorganisms. As a non-limiting example, contacting a composition with a fiber-degrading enzyme produces enzymatic degradation products comprising of sugars, oligosaccharides, and polysaccharides. Enzymatic degradation products may be removed before drying or processing into a feed. As a non-limiting example, the removal of degradation products can be readily accomplished by centrifugation of slurry material after its enzymatic digestion to obtain 1) a denser protein-enriched CFP material that is subsequently dried and 2) a liquid supernatant containing the digestion products that can be sent for downstream processing.

[0053] The toxin-degrading enzyme can be any enzyme that reduces the level of one or more toxins (e.g., mycotoxin, aflatoxin, ochratoxin A, patulin, fumonisin, zearalenone, nivalenol / deoxynivalenol, etc.) in mechanically separated CFP compositions of the present disclosure. Non-limiting examples of toxin-degrading enzymes include esterases, lipases, proteases, oxidases, amino acid oxidases, lactonohydrolases, peroxidases, lactoperoxidases, manganese peroxidases, epoxidases, polysaccharases and dehydrogenases, and combinations thereof. In some embodiments, the toxin-degrading enzyme is phytase, oxalate decarboxylase, or fumonisin esterase.

[0054] The fiber-degrading enzyme can be any enzyme that reduces one or more of cellulose content or non-starch polysaccharide (hemicellulose or xylan) content of compositions of the present disclosure. Non-limiting examples of fiber-degrading enzymes include cellulase, hemicellulase, pectinase, and laccase.

[0055] In some embodiments, the fiber-degrading enzyme is cellulase or hemicellulase, or a combination thereof. Compositions of the present disclosure may be contacted with cellulase and hemicellulase in a ratio of from about 1:1 to about 1:20 (e.g., about 1:2 to about 1: 20, about 1:2 to about 1: 15, about 1:4 to about 1:10, about 1:5 to about 1:9, about 1:6 to - 12 - 4151998.v16109.1001001 about 1:9, about 1:7 to about 1:8, etc.). In some embodiments, the ratio of cellulase to hemicellulase is about 1:7.6.

[0056] Proteolytic enzymes can be any enzyme that break downs (e.g., digests, degrades) protein. Non-limiting examples of proteolytic enzymes include serine proteases (e.g., proteinase K, trypsin, chymotrypsin, elastase), cysteine proteases (e.g., papain, calpain, lysosomal cathepsins), aspartic proteases (e.g., pepsin and rennin), and metallo-proteases (e.g., thermolysin and carboxypeptidase A). In some embodiments, the proteolytic enzyme is proteinase K.

[0057] A person of ordinary skill in the art can readily determine, using routine protocols and reagents, an effective amount of an enzyme, or microorganism producing an enzyme, to add to a composition of the invention to achieve degradation (e.g., partial degradation, complete degradation) of a target product (e.g., a toxin, a fiber, a protein) in the composition. In some embodiments, the effective amount of an enzyme, or microorganism producing an enzyme, is sufficient to degrade all or substantially all of the target product (e.g., a toxin, a fiber, a protein) in the composition. In some embodiments the effective amount of an enzyme, or microorganism producing an enzyme, is sufficient to degrade at least 50% of the target product (e.g., a toxin, a fiber, a protein) in the composition, for example, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% of the target product (e.g., a toxin, a fiber, a protein) in the composition.

[0058] For example, purified fumonisin esterase enzyme possessing an activity of 30,000 U / kg may be added directly to CFP during the production of processed CFP on a wt. / wt. ratio basis of 6 x 10-8gm enzyme / gm CFP in order to significantly reduce the content of fumonisin toxin present in mechanically separated CFP. Similarly, oxalate decarboxylase, a manganese- dependent enzyme capable of degrading oxalate may be added to CFP in either purified form or as Bacillus subtilis growth media containing bacterial biomass together with oxalate decarboxylase enzyme activity. Alternatively, bacteria, such as but not limited to Cupriavidus baslilensis, that possess the capability of degrading ochratoxin may be added directly to the mechanically separated CFP by addition of culture media containing the bacteria. The bacteria may be grown previously from a single colony inoculum using standard LB (Luria- Bertani) liquid media in a glass flask to attain an optical density (OD600) of 0.6.

[0059] In some embodiments, contacting the composition with an effective amount of an enzyme, a single cell organism that produces the enzyme, or a combination thereof, further comprises adjusting the pH of the composition. For example, the pH of the composition may - 13 - 4151998.v16109.1001001 be adjusted with a mineral acid such as hydrochloric acid or mineral base such as sodium hydroxide. In some embodiments, the pH of the composition is adjusted to a pH of from about 5.0 to about 7.5 (e.g., about 5.0 to about 7.0, about 5.5 to about 7.0, about 5.5 to about 6.5, etc.). In some embodiments, the pH of the composition is adjusted to about pH 6.0.

[0060] In some embodiments, when the composition is contacted with an effective amount of an enzyme, a single cell organism that produces the enzyme, or a combination thereof, the enzyme or single cell organism, or the combination thereof is in contact with the composition for about 0.5 hour to about 20 hours (e.g., about 0.5 hour to about 15 hours, about 0.5 hour to about 10 hours, about 1 hour to about 10 hours, about 1 hour to about 9 hours, about 1 hour to about 8 hours, about 1 hour to about 7 hours, about 2 hours to about 7 hours, about 2 hours to about 6 hours, about 3 hours to about 6 hours, or about 3 to about 5 hours). In some embodiments, the enzyme or single cell organism, or the combination thereof is in contact with the composition for about 4 hours. A person of ordinary skill in the art can readily determine, using routine protocols and reagents, a suitable amount of time for contacting an enzyme, or microorganism producing an enzyme, with a composition of the invention to achieve partial or complete degradation of a target product (e.g., a toxin, a fiber, a protein).

[0061] In some embodiments, removing water-soluble material (e.g., pH-sensitive macromolecular complexes) and / or enzymatic degradation products from composition comprises centrifuging the composition and heating the resulting supernatant. The supernatant may be heated to a temperature of from about 100oF to about 200oF (e.g., about 110oF to about 200oF, about 110oF to about 150oF, about 130oF to about 150oF, etc.). In some embodiments, the supernatant is heated to about 140oF. In some embodiments, removing water-soluble material from composition further comprises adjusting the pH of the supernatant. In some embodiments, the pH of the supernatant is adjusted to a pH of from about 7.0 to about 8.0 (e.g., about 7.1 to about 8.0, about 7.2 to about 8.0, about 7.3 to about 8.0, about 7.4 to about 8.0, about 7.5 to about 8.0, about 7.5 to about 7.9, about 7.5 to about 7.8, or about 7.5 to about 7.7). In some embodiments, the pH of the supernatant is adjusted to about pH 7.6. Adjusting the pH of the supernatant precipitates the water-soluble material, and purification methods (e.g., chromatography, centrifugation or sedimentation) are employed to filter the supernatant and thereby remove the water-soluble material (e.g., pH-sensitive macromolecular complex) and / or enzymatic degradation products from the supernatant. - 14 - 4151998.v16109.1001001

[0062] In some embodiments, the processed CFP has a viscosity of from about 100 mPa·s to about 5000 mPa·s (e.g., about 100 mPa·s to about 1000 mPa·s, about 100 mPa·s to about 900 mPa·s, about 100 mPa·s to about 800 mPa·s, about 100 mPa·s to about 700 mPa·s, about 100 mPa·s to about 600 mPa·s, about 100 mPa·s to about 500 mPa·s, about 100 mPa·s to about 400 mPa·s, about 100 mPa·s to about 300 mPa·s, about 100 mPa·s to about 200 mPa·s, about 100 mPa·s, etc.). In some embodiments, the processed CFP has a viscosity of from about 100 mPa·s to about 500 mPa·s.

[0063] In some embodiments, methods of the present disclosure further comprise drying the processed CFP. Drying the processed CFP may be facilitated by the processed CFP’s reduced viscosity. Drying the processed CFP can be performed, for example, with (e.g., ring drying, drum drying, spray drying) or without heat exposure (e.g., freeze drying). Other non- limiting examples of drying include hot-air drying, vacuum freeze-drying, drying due to high- frequency heating and microwave drying. In some embodiments, the processed CFP is dried using spray drying (e.g., when the processed CFP has a viscosity of about 1000 mPa·s or less, such as about 500 mPa·s or less). In some embodiments, the CFP is dried to a moisture content of less than about 10 wt% (e.g., less than about 9 wt%, less than about 8 wt%, less than about 5 wt% etc.). For example, the moisture content of the dried processed CFP is from about 0 wt% to about 10 wt%, about 0 wt% to about 9 wt%, about 0 wt% to about 8 wt%, or about 0 wt% to about 5 wt%.

[0064] In some embodiments, the composition is contacted with an effective amount of single cell organism that produces the enzyme. The single cell organism may be a yeast (e.g., Lactococcus lactis, Komagataella phaffii) or a bacterium (e.g., Trichosporon mycotoxinivorans), Sphingopyxis sp.), or a combination thereof.

[0065] Processed CFP (e.g., dried processed CFP) may be incorporated into extruded or pelleted feed. In some embodiments, provided herein are animal feeds (e.g., mixed feeds, single-ingredient feeds) that comprise processed CFP obtained using methods of the present disclosure. In some embodiments, processed CFP is an ingredient in complete feeds (e.g., extruded and non-extruded feeds) that are fed to terrestrial and / or aquatic animals. In some embodiments, the complete feeds comprise processed CFP and feed ingredients from animal, plant, mineral and synthetic sources.

[0066] Technical and Financial Limitations on the Current Use of Fiber Degrading or Proteolytic Enzymes by Ethanol Plants to Produce Valuable Corn-based Protein Enriched Co-Products. - 15 - 4151998.v16109.1001001

[0067] In present day ethanol plants, the addition of enhanced fiber-degrading enzymes during fermentation combined with mechanical separation technologies have improved both the yields and protein content of mechanically separated high protein corn co-products such as CFP. However, there are significant technical drawbacks and cost limitations of this “up front” combination of enzymatic-and mechanical separation methodologies. Several of these limitations and / or drawbacks are listed below.

[0068] The “up front” (during fermentation of corn sugars) addition of fiber and toxin degrading enzymes requires large quantities of these enzymes to be added to large volume fermentation tanks. As a result, these enzymes must degrade their fibrinous or protein substrates at relatively dilute concentrations within such large tanks designed to accommodate sugar fermentation by yeasts. This large-scale operational format makes the addition of any enzyme very expensive on a tank volume basis alone.

[0069] The operational parameters (temperature, pH, etc.) within these fermentation tanks are rigidly established to maximize ethanol fermentation efficiency and yield. As a result, the optimal conditions to maximize the activity of native (produced by natural sources) fiber- degrading or proteolytic enzymes do not correspond to those of anaerobic fermentation despite the fact that this interval commonly lasts for 48-60 hr. Thereafter, these enzymes are inactivated by the high temperatures in the subsequent distillation step and therefore cannot be recycled.

[0070] As a result of these problems, multiple companies have created a class of specialized fiber degrading enzymes using protein engineering of native enzymes so as to improve their operational efficiency under such fermentation conditions. However, the cost and availability of these specialized enzymes are expensive and, as a result, have limited their use in ethanol plants to produce high protein / low fiber ethanol co-products. Additionally, the present policy of ethanol plant operators is that all of the expenditures related to the use of these enzymes are included in the cost of manufacturing and, as a result, must be recovered within the pricing of this single protein product. Since, at the present time, dried CFP comprises only ~5-6% of the total mass of corn flour feedstock, this cost assignment policy when applied to enzymes used in large fermentation tanks greatly reduces a profit margin that might be achievable for dried CFP.

[0071] Another limitation on the use of fiber degrading and / or proteolytic enzymes during ethanol fermentation is the fact that the degree of fiber degradation and / or protein digestion produced by these enzymes must be balanced with the particle size, viscosity and - 16 - 4151998.v16109.1001001 flow requirements of the downstream mechanical separation processes responsible for efficient separation and sequestration of whole stillage components to create high-protein corn co-products. More complete digestion of various corn fibers and / or protein components by these enzymes in corn mash will cause valuable protein components to escape capture by mechanical separation processes and, as smaller particles, be carried to the more liquid components of stillage fractions that possess lower value and contain other less desirable components. As a result of these changes, the target product, dried CFP, will be reduced in quality and value.

[0072] The “up front” addition of enzymes prior fermentation lacks any co-product application specificity. If the biological or financial benefit is limited to only a single or selected set of co-products, up-front addition of enzyme(s) will result in the treatment of all other co-products present in whole stillage. In many cases, the expense of these enzymes does not justify their cost to gain marginally valuable improvements in various other co- products.

[0073] Another limitation of this “up-front” use of enzymes to produce protein-rich co- products is the fact that the operational changes necessary to create such value-added co- products can often only be performed by changing the operational characteristics of an entire ethanol plant rather than possessing the ability to limit such changes to those processes immediately surrounding the manufacture of the specific co-product. Hence, new high- protein corn co-products can often be produced in a series production format composed to sequential “campaigns”. By contrast, a more efficient format would be to produce such products in parallel using the methods of the present disclosure.

[0074] Limitations on the Use of Enzymes and / or Specialized Yeast Strains to Reduce or Eliminate Fungal Toxins Present in Corn Feedstock Used by Ethanol Plants in Order to Produce Toxin-Free Protein Enriched Co-Products.

[0075] Aflatoxins and other mycotoxins such as fumonisin are produced by several fungal species and molds that grow under certain environmental conditions which occur on a periodic basis in corn production (19). These fungal toxins are extremely toxic to monogastric animals including poultry, swine, fish and various pet species that consume these corn protein products (20-24). By contrast, ruminants that consume DDGs are much less sensitive to such toxins (19). As a result, feed ingredient specifications include certification standards that demand such toxins are present in only negligible concentrations (25,26). Moreover, for some toxins like fumonisin, the toxin is actually concentrated in the - 17 - 4151998.v16109.1001001 protein-rich co-product as compared to intact corn (27) and, due to its heat stability, is believed to be recycled within thin stillage of ethanol plants. Thus, in order to prevent the production of dried CFP that contains elevated levels of such mycotoxins, ethanol plants have few options at the present time. One choice is the screening and rejection of incoming corn deliveries that possess elevated toxin contents. This costly process involves the testing of corn lots as well as the dried CFP and its possible rejection / loss based on toxin content (28). This multi-step process adds considerable expense to the overall costs of production of the dried CFP product. A second option more recently introduced is to inactivate such toxins using specialized enzymes developed as either processing aid or feed additive for complete feeds (27,29,30). However, use of this enzyme for toxin inactivation is subject to many of the same limitations as those described for fiber-degrading and / or proteolytic enzymes.

[0076] Limitations of Present-Day Technology and Infrastructure to Dry CFP Slurry to Produce a Corn Fermented Protein Possessing a Low Moisture Content.

[0077] In order to produce a dried CFP sold as product that does not mold during its storage and shipment, CFP slurry must be dried from a moisture content of >70% to a final dried product moisture of <10%. This drying process must keep pace with the plant’s production of CFP slurry and the resulting overall water balance of the entire plant. Due to the inter-relationship between the drying process and the plant’s water balance, CFP slurry is dried using high temperature ring or drum dryers that recycle partially dried CFP product in an effort to accelerate the drying of incoming slurry (31,32). This arrangement creates a spectrum of particle sizes within the final dried CFP product where different particle sizes each experience different degrees of high temperature exposure. This process results in undesirable Maillard reactions within the partially dried CFP slurry (32-34). The flocculation or aggregation of smaller protein-fiber components during this this drying process creates more complications since larger particles require more heat exposure to achieve the required moisture content due to their reduced surface area to volume ratios.

[0078] In order to address the biological, operational and financial limitations described above, the present disclosure provides exemplification as to the utility of further modifying the existing ethanol production format illustrated in Panels A and B of FIG.1. As shown in Panel C of FIG.1, one or more additional tanks can be utilized for multiple purposes allowing for the selective treatment(s) of valuable protein-rich ethanol co-products without alteration in other ethanol plant production side streams. Such selective treatments may be performed singly or in parallel at a significantly reduced cost so as to enzymatically digest - 18 - 4151998.v16109.1001001 CFP slurry as well as provide a culture environment for the propagation of valuable or desired organisms such as specialized yeasts or bacteria possessing specific enzymatic activities that will, in turn, degrade or inactivate undesired or toxic components of the corn co-product.

[0079] In addition, the discovery of the pH sensitivity of soluble smaller components within the CFP slurry allows for the efficient separation of these entities from the remaining CFP components in order to increase the protein content and increase the abilities of enzymes to remove fiber while decreasing the viscosity so as to improve the drying process for CFP slurry. After removing such entities, the processed CFP may be spray dried rather than ring or drum dried so as to increase the value and quality of the resulting processed CFP product.

[0080] An overall advantage achieved by practicing embodiments of the present disclosure is derived from the ability to perform such specialized treatments or modifications of CFP slurry in a highly efficient and cost-effective manner. As a result, valuable properties of this processed CFP product are enhanced, including but not limited to, reducing its water holding capacity, viscosity, fiber, anti-nutritional and toxin contents while increasing or improving its protein, digestibility, palatability and drying characteristics in a highly cost- effective manner. In addition, embodiments of the present disclosure can be used to isolate a macromolecular complex via alterations in CFP slurry pH so as to improve the overall value of the resulting processed CFP product.

[0081] Present day alternative methods attempt to achieve improvements in this protein- rich ethanol co-product by the “up front” addition of enzymes or other modifications prior to fermentation and / or distillation steps as is utilized in the present state of ethanol production as shown in Panels A and B of FIG.1. These up-front methods generate an existing CFP slurry possessing a moderate protein content (48-55% crude protein) while achieving a yield of ~3.5-5.0 lb. of corn fermented protein / bushel for use as wet cake feedstock starting material. In this regard, any such improvements in the protein content or yield of the CFP slurry that is achieved by other “front end” processes will be additive to the benefits provided by the present disclosure. By contrast, since embodiments of the present disclosure reduce the tank volume and necessary reactive components such as enzymes by >95% vs standard fermentation tanks, there are corresponding savings in costs of such enzymes that potentially increase the profitability of the final product. Moreover, the increased treatment flexibility and reliability provided by the present disclosure allow for creation of multiple new specialized unique products as well as eliminate the need for ethanol plants to engage in - 19 - 4151998.v16109.1001001 costly intake testing to produce protein-enriched co-products with little or no mycotoxins, fumonisin or plant-based anti-nutritionals such as phytate or oxalate.

[0082] Prior to providing exemplification of specific aspects of the present disclosure, the text below summarizes non-limiting examples of treatments of CFP slurry that can be achieved by the present disclosure and their benefits to create additional value for the product and overall production in the ethanol production plant.

[0083] 1. Reduction in cellulose and non-starch polysaccharide (hemicellulose or xylan) content - The protein-rich CFP product produced by the current ethanol production format described above contains ~35% fiber content (NDF) as shown in Table 1. Due to this abundant fiber, both the water holding capacity and viscosity of this co-product are large and represent challenges to its efficient large scale drying as a commercial product as well as its downstream handling in processes like complete feed manufacture and, ultimately, its value as a biological feed ingredient. Application of standard fiber-degrading enzymes that are commercially available under defined conditions as part of the present disclosure without the constraints imposed by mechanical separation processes enable a reduction in neutral detergent fiber content of ~37% and resulting reductions of ~40% in product viscosity and ~16% in its water holding capacity.

[0084] 2. Increase in crude protein content – The use of fiber-degrading enzymes to reduce both the cellulose and hemicellulose content of the protein-rich co-product as part of the present disclosure provides for the ability to increase the protein content of the resulting processed CFP co-product to achieve a value of ~62% crude protein on a dry matter basis with enzymes (e.g., natural enzymes). This represents an increase in crude protein content of ~12% as compared to the starting material (~55% protein dry matter basis (DMB)). Removal of a pH-sensitive complex from mechanically separated CFP provides further increases the protein content of processed CFP. Moreover, pH-sensitive complex removal increases the capabilities of fiber degrading enzymes to remove fiber such that increases in protein content by 14% from an original value of 59.8% to achieve a value of 73.8% DMB in processed CFP.

[0085] 3. Reduction in CFP fiber content by isolating soluble entities from CFP slurry via pH changes to improve its drying and protein content. Comparison of the fiber content of dried CFP with and without heat exposure shows that fiber (measured as NDF and ADF) is created by heat exposure and pH changes. To reduce the fiber content of CFP, various combinations of the soluble portion of CFP slurry can be separated from larger insoluble components and the pH changed in order to isolate a macromolecular complex that can be - 20 - 4151998.v16109.1001001 easily separated from other soluble components. The isolation and removal of this complex and / or its component parts from the CFP slurry has multiple benefits including increasing the protein content of the final processed CFP product, improving the abilities of commercial fiber degrading enzymes to further increase its protein content, facilitating CFP drying (e.g., by significantly reducing the viscosity of the processed CFP, e.g., to about 500 mPass), and improving overall water balance for the ethanol plant by preventing the recycling of these soluble components that will form the macromolecular complex during subsequent separations and drying cycles.

[0086] 4. Expose the CFP co-product to specialized enzymes to alter its overall protein characteristics or reduce its content of mycotoxins or plant-based anti-nutritionals in the final dried processed CFP product. The addition of specialized enzymes either singly or in various combinations on a highly cost-effective basis can be achieved by the present disclosure due to the smaller volume of its reaction tank as compared to using identical digestions prior to mechanical sorting and collection of materials from whole stillage. A variety of enzymes can be used for this purpose and several are listed below: a. Proteolytic Enzymes – In vitro digestion of protein constituents has been shown to be associated with improvements in the digestibility of proteins in vivo (6). Proteolytic digestion of larger proteins can be accomplished under conditions provided by the present disclosure to provide for a more digestible feed ingredient without constraints of mechanical separation requirements. b. Toxin Degrading Enzymes – Mycotoxins such as zearalenone and fumonisin display significant toxicity to multiple important farmed species and are produced by fungi associated with damaged corn. Since mechanically separated CFP is used as a feed ingredient for multiple sensitive monogastric species the monitoring of zearalenone and fumonisin levels in intake corn as well as the final protein-rich co-product represents a significant challenge and expense to ethanol production companies. Enzymes that alter the molecular structures of zearalenone and fumonisin and deactivate them have recently been introduced (30). Each of these enzymes act in a highly temperature and pH dependent manner (27,29,30). Application of these mycotoxin inactivating enzymes under the conditions of the present disclosure to CFP slurry will provide for multiple biological and financial advantages as compared to their application either “upstream” within the - 21 - 4151998.v16109.1001001 fermenters of ethanol plants or post production applications as a component of the complete feeds for various animals. Utilization of the flexible production conditions of the present disclosure to reduce or possibly eliminate zearalenone and fumonisin levels in the final protein-rich co-product together with the possible capability of the present disclosure to recycle these enzymes in order to treat multiple batchers of high protein product prior to its drying would greatly reduce the cost impact and risk of this corn mycotoxin to ethanol plants. c. Enzymatic Digestions to Reduce the Content of Anti-nutritional Components Contained within the CPF Co-product that Affect Various Monogastric Commercially Important Species – Corn contains certain anti-nutritional compounds that either reduce the ability of monogastric animals to digest nutritional components within the mechanically separated CFP product such as amino acids and minerals or, alternatively, are readily absorbed and pose problems for the animal to excrete or metabolize. For example, phytate is a phosphorylated inositol compound contained in corn that reduces the intestinal absorption of nutrients by the binding of these nutrients in complexes unavailable to monogastric animals (35). Phytate is effectively degraded by phytase (36) that can be added to mechanically separated CFP under conditions of the present disclosure to selectively reduce or eliminate the phytate content of the processed CFP product. In a similar manner, the organic acid oxalate is contained in mechanically separated CFP and is readily absorbed during the digestive process (37,38). Subsequently, this absorbed oxalate is excreted via the kidneys and urine (38). However, for certain animals particularly domesticated housecats and some dogs, ingestion of meals containing high contents of oxalate pose a particular hazard since excretion of urine containing high oxalate contents causes the formation of kidney and urinary bladder stones that compromise the health of the animal (39-41). At the present time, various low oxalate diets are designed and manufactured to prevent kidney stones within these susceptible animals at considerable expense using selective ingredients possessing low oxalate contents. Treatment of mechanically separated CFP slurry under conditions of the present disclosure using oxalate degrading enzymes such as oxalate - 22 - 4151998.v16109.1001001 decarboxylase (42,43) that results in a valuable processed CFP feed ingredient containing low or absent oxalate together with low calcium and phosphorus contents that can then be readily utilized as a component in such specialized diets to maintain the health of these animals and prevent renal or bladder stone formation.

[0087] 5. Utilize the sugar and protein liberated via enzymatic digestion of mechanically separated CFP as a growth medium to propagate specialized yeasts and / or bacterial strains possessing desired characteristics. Digestion of fiber components present in the mechanically separated CFP slurry by fiber degrading enzymes generates various sugar components that can be utilized together with existing nitrogen sources to create a nutrient medium for growth of specialized yeasts or bacteria within the same reaction tank used for enzymatic digestion. Specialized yeast strains might include those isolated by traditional isolation methods or genetically engineered to produce valuable components that would further contribute to the overall value of the processed CFP co-product after its processing by the present disclosure. For example, a non-pathogenic yeast Trichosporon mycotoxinivorans has been used to detoxify zearalenone and ochratoxin as a commercial product added to animal feeds (44). A fumonisin esterase gene derived from the bacterium, Sphingopyxis sp. has been engineered into the nonpathogenic yeast, Komagataella phaffii, and available for use as a feed additive (27). Similarly, recombinant Lactococcus lactis bacteria expressing the oxalate degrading enzymes, oxalate decarboxylase and oxalate oxidase (42) could be used under the conditions of the present disclosure to reduce or remove oxalate from a mechanically separated CFP corn co-product. Each of these organisms is being utilized as feed additive in various animals and thus their use as a processing aid for the present disclosure would be simplified.

[0088] 6. Estimate of reduction in costs associated with production of processed CFP protein product from mechanically separated CFP using the present disclosure in ethanol plants. While the exact cost reduction provided by the present disclosure will vary depending on the engineering and production formats of individual ethanol plants, it is possible to estimate several significant reductions based on the exemplification as described below. These areas are: a. Enzyme Cost Reduction – As described above, at the present time it is required that “up-front” enzyme treatments are performed in large fermentation tanks under conditions to optimize ethanol fermentation. Commonly, such single fermenter tanks are 80,000 gallons (302,400 liters) in volume and are loaded at a solid’s loading rate - 23 - 4151998.v16109.1001001 of 34%. Hence, this production format has required large quantities of expensive enzymes “customized” to perform under fermentation conditions where all tank components are treated with enzymes. However, the use of such enzymes is designed principally to modulate the characteristics of mechanically separated CFP slurry components that actually constitutes only 5-6% of the tank’s total solids load. By contrast, practice of the present disclosure uses mechanically separated CFP slurry as its feedstock and therefore requires a treatment tank possessing a volume of only 5,000 gallons that is loaded at an identical solid’s loading rate to accommodate an identical quantity of CFP slurry. As a result of this 93.8% reduction in volume, enzyme costs required to treat the mechanically separated CFP product using the same enzymes is reduced greater than 90% to produce the processed CFP product as described in the present disclosure. b. Ability to Optimize Conditions for Enzymatic Digestion Instead of Fermentation – Use of “front end” enzyme treatments require trade-offs between fermentation, mechanical separation and process conditions. By contrast, the conditions of the present disclosure are not impacted by these parameters and can be optimized for temperature, pH, etc. of specific or multiple enzymes (in parallel or series intervals) to achieve maximal performance. In this regard, all of the exemplification of the present disclosure was performed with naturally occurring fiber degrading enzymes that can be obtained a much lower cost vs. specialized “customized” enzymes currently required by ethanol plants. Lastly, the present disclosure allows for the use of specialized enzymes possessing operational characteristics that could not function under standard fermentation conditions. c. Reduction or Elimination of Mycotoxin Screening Programs Required by Ethanol Plants to Produce a processed CFP Protein Product Possessing an Acceptably Low Toxin Content – Ethanol plants currently screen incoming corn feedstock for toxins and reject corn with an elevated content to produce mechanically separated CFP product with low toxin content. These screening programs are costly in both labor and screening assays expenses. Using the present disclosure, either enzymatic or single cell toxin removal methods can be targeted specifically to mechanically separated CFP slurry and optimized for maximum toxin removal. This will allow the ethanol company to certify its processed CFP product has a low or no toxin content. Moreover, this “back-end” toxin removal process can be started or stopped based on incoming toxin loads in feedstock corn with minimal - 24 - 4151998.v16109.1001001 disruption of ethanol plant overall production. In addition, it will allow ethanol plants to purchase corn possessing an elevated mycotoxin or fumonisin content when corn prices are low with the confidence that the present disclosure could reduce or eliminate such toxins during the manufacture of a processed CFP corn co-product at a later date. d. Ability to Create Multiple Value-Added processed CFP Products from a Single Mechanically Separated CFP Slurry Stream in the Ethanol Plant That is Focused on Achieving Maximal Yields from Whole Stillage – The manufacture of multiple mechanically separated CFP products using the existing production format as illustrated schematically in FIG.1- Panel B, has significant limitations due to the impacts that such production changes have on other ethanol plant products and the plant’s overall water balance. Thus, the manufacture of multiple value-added products from mechanically separated CFP using this existing format can only be achieved by a series of “campaigns” in which production of the entire plant must be adjusted each time a different value-added product is produced. By contrast, use of the present disclosure as shown in FIG.1 – Panel C enables multiple value-added processed CFP co-products to be produced using multiple tanks where each tank accepts mechanically separated CFP slurry as feedstock after its mechanical separation in Panel B and then alters this product in different ways to create multiple processed CFP products each of which can be sold as a specialized product where each has different desirable and valuable characteristics.

[0089] 7. Further incorporation of processed CFP slurry produced by methods of the present disclosure into feed mill mash by mixing with other dried ingredients and allowing for enzymes and / or organisms present in processed CFP slurry to digest other hydrated feed ingredients prior to their extrusion and processing as unique pelleted complete feeds.

[0090] As disclosed above, enzymes with the ability to degrade indigestible fiber components as well as plant anti-nutritional compounds have been incorporated into animal feeds for the purposes of improving the nutrient content of complete feeds after their consumption by monogastric animals (6). There has also been the commercial development of extruded feed manufacturing techniques that utilize emulsification and specialized pumping equipment to incorporate animal meat or plant-based slurries into extruders for the manufacture of pelleted feeds (45). Thus, it is possible to incorporate these commercially available methods and know-how together with the know-how of the present disclosure for a - 25 - 4151998.v16109.1001001 novel application that further extends the commercial value of the present disclosure. In this new application, the processed CFP slurry resulting from its treatment by the present disclosure shown in FIG.1 – Panel C is mixed with dry ingredients to create a mash suitable for feed mill extrusion. However, since the enzymes and selected bacteria are still active under such conditions (no heat exposure and liquid conditions prior to extrusion) these active enzymes will alter components of newly added feed ingredients in a manner similar to that occurring in the original processed CFP slurry. As a result, the undesirable fiber, toxins or anti-nutritional components of various non-ethanol plant derived feed ingredients that are mixed together with the processed CFP slurry will be reduced prior to their extrusion into pellets. The result of these actions will be the production of new value-added complete feeds. EXAMPLES

[0091] For the purposes of exemplification, the source of protein-rich co-product mechanically separated from corn as illustrated in FIG.1 was corn fermented protein (CFP) slurry that was isolated using the Fluid Quip Process (U.S. Patent No.11103811) and dried with either no heat exposure (freeze dried) or heat exposure (ring dried or in selected cases, oven dried) to create the dried feed ingredient.

[0092] Example 1: General Properties and Overall Composition of Mechanically Separated CFP and its Dried Derivative Product.

[0093] FIG.2 shows the appearance and general properties of both “wet” and ring dried mechanically separated CFP as produced from whole stillage (Patent #11103811; 10800944; 11603507; 1427784; 20220243143). It is noteworthy that the water activity of these mechanically separated CFP products (both wet and rehydrated dried) is significant since the undried mechanically separated CFP slurry maintains a consistency similar to that of crumbly cookie dough despite its 70% moisture content. By contrast, when mechanically separated CFP is subjected to conditions of the present disclosure to create processed CFP, its fiber content is reduced by ~35% via addition of fiber degrading enzymes (see below) and there is a visible change in its characteristics. As shown in Panel B of FIG.3, the crumbly characteristic of the mechanically separated untreated CFP is altered so that the processed CFP product is significantly more deformable (FIG.3 - Panel B) and displays a lighter coloration after drying (FIG.3 - Panel D). - 26 - 4151998.v16109.1001001

[0094] Example 2: Compositional and water holding capacity analysis

[0095] Compositional and water holding capacity analysis shows that mechanically separated CFP contains 30-40% fiber which is likely what provides it with the ability to retain high amounts of water. Treatment of mechanically separated CFP slurry under conditions of the present disclosure reduces its water holding capacity.

[0096] Table I shows a comparison of the composition of undried and ring dried mechanically separated CFP expressed on an “as received” (as Rec.) and dry weight (Dry Weight) basis. These data reveal that the neutral detergent fiber content (NDF) of mechanically separated CFP is 34-40% on a dry matter basis and is increased by ~20% in the dried mechanically separated CFP sample vs. its wet counterpart. A component of NDF measured by acid detergent fiber (ADF) represents ~20% of mechanically separated CFP DMB content and consists of cellulose and lignin. The remainder of NDF is composed of hemicellulose and non-starch polysaccharides (NSP) components. Previous work has shown that plant cellulose and hemicellulose retain between 3.5 and 10 times their dry weight in absorbed water (46). In this regard, the high hydrated fiber content of mechanically separated CFP is problematic to achieve efficient drying of the product due to its unusual water retention capabilities.

[0097] Table II compares the water activity using methods described by Brachet et al. (47) for freeze dried mechanically separated CFP before and after its treatment under the conditions of the present disclosure. When compared to other feed ingredients, an average water holding capacity or WHC value of 7.55 for dried mechanically separated CFP is very large (47,48). However, when subject to conditions of the present disclosure, this value for the processed CFP is decreased by 16%. Thus, these data in Tables I and II support the conclusion that undried or “wet” mechanically separated CFP displays unusual water retention capabilities due to its 33-40% fiber content. This large water holding capacity is retained after drying and rehydration of dried mechanically separated CFP. However, as shown in Table II, treatment of CFP under conditions of the present disclosure reduces the large water holding capacity of the final processed CFP product. - 27 - 4151998.v16109.1001001 Table I: Comparison of compositional analysis of undried and ring dried Mechanically Separated CFP product. “Wet”-Undried Ring Dried Protein

[0098] Table II: Measurement of water holding capacity of freeze-dried CFP before and after treatment under conditions of the present disclosure. Water Holding Capacity (gm H20 / gm of CFP DMB) Mean S.E. NUndigested CFP* 7.55 0.52 6Digested CFP* 6.33 0.27 6*Samples Freeze Dried Before Analysis

[0099] Example 3. Liquid extracts from never-dried and rehydrated dried mechanically separated CFP slurry display high viscosity values that are reduced after treatment by the present disclosure yielding processed CFP.

[0100] The large hydrated fiber content of mechanically separated CFP is problematic not only to achieve efficient drying of the wet mechanically separated CFP product due to its large water holding capacity but also since its fiber content increases the viscosity of water solutions that are in contact with CFP. Such viscosity increases affect not only mechanically separated CFP drying to form its corresponding dried protein product but also with - 28 - 4151998.v16109.1001001 interactions within the gastrointestinal tract during digestion of feeds containing mechanically separated CFP protein by multiple monogastric animals as described below. Table III: Measurements of the viscosity of water exposed to either undried or dried Mechanically Separated CFP before or after being subjected to treatment by the present disclosure to produce processed CFP. Flat Plate Cone Viscometer Viscosity Measurements (cP)#Average S.E. N Water 0.94 0.1 6Wet Undigested CFP 24.3 6.1 11Wet Digested CFP* 6.6 2.4 8Dried Undigested CFP^ 4.6 0.9 4Dried Digested CFP*^ 2.7 1.3 4Dried Pea Protein Concentrate 13.5 1.8 6Dried Fishmeal 1.5 0.6 4^Freeze Dried Samples #Viscosity measured at 19.9oC at 2RPM expressed as centipoise (cP). *Digestion Conditions Correspond to Digest 4 in Table IV.

[0101] Table III shows data obtained using a flat-plate cone viscometer measuring the viscosity of liquid obtained after the mixing of various mechanically separated as well as processed CFP products with an equal volume of water followed by centrifugation to remove any particulate matter. Note that water mixed with either undried or dried mechanically separated CFP not subject to digestion under the conditions of the present disclosure displayed increased viscosities that were 24 and 4.6 times that of water. As described in text accompanying FIG.s 17-19, this increased viscosity is likely due to the presence of soluble components of the pH sensitive macromolecular complex present in mechanically separated CFP slurry (see below). By contrast, after digestion with fiber-degrading enzymes under conditions of the present disclosure to produce processed CFP, these viscosities were reduced significantly to values corresponding to ~27% and 59% of their original values. By comparison, water obtained from freeze dried undigested mechanically separated CFP displays viscosity values that are intermediate between viscosity values obtained for another plant-based feed ingredient (pea protein concentrate) and an animal meal (fishmeal). Each of these plant and animal meals are used in extruded fish aquafeeds. - 29 - 4151998.v16109.1001001

[0102] In most monogastric animals, the ability to degrade and digest cellulose and NSP components is limited or non-existent and so these components remain indigestible (49). However, both the contents and origin of these fiber components, particularly NSPs, have multiple effects on the physiology, function, morphology and efficiency of the gastrointestinal tract of both terrestrial and aquatic animal species (50-55). Such changes include alterations in the viscosity of intestinal contents which have been shown to have variable effects depending on the species and NSP source (51,56-58). For example, variable results on growth, overall performance as well as feed utilization have been obtained in studies with various species of fish after the addition of dietary NSPs to feeds. In some species, diets rich in NSPs appear to have little impact on growth performance (59-62) whereas in others NSP additions have produced either a deleterious (63,64) or beneficial effects (65,66). The effects of NSP and other fiber components on intestinal functions and overall digestive and metabolic processes have been explored on a molecular basis (67-70).

[0103] NSP and fiber contents of aquatic feeds alter the characteristics of fecal waste material from fish (71,72) and other species (73) that impact the downstream technologies designed to provide their efficient removal from waste and recycled water (74). These dynamics have a significant commercial impact and must be accounted for in any fish production facility (75). In summary, the ability to alter the total content of cellulose and NSP fibers in protein-rich co-products produced by ethanol plants has significant commercial value in that changes in this fiber fraction of feed ingredients can then be “customized” to the species requirements for which the feed ingredient is intended including the conditions under which the fish is reared.

[0104] Fiber degrading enzymes are added to complete feeds to counteract or modulate the effects of cellulose / NSP components in animal diets and improve animal performance and health (6,7,12,35,36). These enzymes are designed to become active after ingestion of the feed and during its digestion as chyme within the gastrointestinal tract (76,77). However, in this regard, addition of fiber degrading enzymes to the mash in feed ingredients used to produce extruded feeds is problematic since these enzymes will not survive high temperature extrusion conditions (78). Moreover, addition of fiber degrading enzymes to complete feeds with their activation within the animal’s gastrointestinal tract exposes the intestinal mucosa to fiber degradation products produced by the actions of these enzymes regardless of whether these are beneficial or detrimental (8). - 30 - 4151998.v16109.1001001

[0105] As an alternative to these existing commercial solutions described above, the present disclosure allows for the controlled treatment of mechanically separated CFP slurry with fiber degrading enzymes to reduce both the cellulose and NSP fiber content of its corresponding dried processed CFP product together with the subsequent removal of these enzymatic degradation products before drying or processing into a complete feed.

[0106] Example 4: Demonstration of the ability to degrade cellulose and NSP fiber fractions using standard enzymes obtained from natural sources under conditions of the present disclosure.

[0107] Extended treatment of mechanically separated CFP under present disclosure conditions results in significant increases in the protein content of resulting processed CFP.

[0108] To demonstrate the ability of the present disclosure to reduce the fiber components in mechanically separated CFP and its dried product, as shown in Table I, various combinations of cellulase and hemicellulase enzymes were added to mechanically separated CFP slurry (see General Considerations and Methods section below). Table IV shows the addition of various combinations of cellulase / hemicellulase enzymes added to mechanically separated CFP slurry at 86oF (30oC) for an interval of 4 hours of digestion. In an effort to define digestion parameters under the conditions of the present disclosure, both the quantities and ratios of cellulase and hemicellulase were varied while the digestion interval was kept constant. Moreover, in order to demonstrate the utility of this inventive process, only enzymes isolated from natural sources and obtained via standard laboratory products were utilized. Such enzymes from natural sources are unsuitable for use under standard fermentation conditions in ethanol plants. Table IV: Summary of various ratios of cellulase and hemicellulase enzymes added to 10kg of mechanically separated CFP slurry for interval of 4 hours of digestion. Gm Added to Mix Units / kg of CFP for Digestion Activity Ratio Cellulase Hemicellulase Cellulase Hemicellulase Cellulase / Hemicellulase Digest 1 1 4 6120 600 10.2Digest 2 8 40 48960 6000 8.16Digest 3 4 40 24480 6000 4.08Digest 4 8 60 44509 8182 5.44

[0109] For the studies shown in Table IV, the activity of each fiber degrading enzyme as well as the ratio of cellulase to hemicellulase was varied while the quantity of mechanically separated CFP substrate (10 kg) was held constant. - 31 - 4151998.v16109.1001001 Table V: Summary of changes in protein, acid detergent and neutral detergent fiber content in Mechanically Separated CFP slurry subjected to digestions under conditions of Table IV for an interval of 4 hours and freeze drying prior to analyses. Dry Matter Basis - All Values Moisture Acid Det. Neutral Det.SAMPLE Protein Fiber Fiber CONTROL - No Digest 72.1 54.8 19 42Digest 1 75.6 54.1 25.8 42.6Digest 2 75.7 54.5 27.2 40.8Digest 3 77.5 54.5 20.4 39.9Digest 4 76.1 55.6 6.14 26.3

[0110] Table V shows changes in protein, acid detergent fiber (ADF) and neutral detergent fiber (NDF) resulting from digestions shown in Table IV. The processed CFP protein content increased while the content of both acid detergent and neutral detergent fiber decreased. These data are shown graphically in FIG.4. Taken together, these data suggest that Digest #4 conditions provided a small increase (1.5%) in protein content while significantly reducing the acid detergent (68%) and neutral detergent (37%) contents of the digested processed CFP. Table VI: Summary of changes in viscosity of undried digested or rehydrated processed CFP subjected to conditions described in Tables IV and V and FIG.4. Flat Plate Cone Viscometer Viscosity Measurements (cP)#All Four Hour Digests Time 0^ End Digest^ End Wet Product&Digest 1 4.4 2.6 5.4Digest 2 4.1 11.2 2.6Digest 3 4.5 0.9 1.5Digest 4 4.2 1.8 1.6#Viscosity measured at 19.9oC at 2RPM in centipoise (cP). ^Viscosity of Supernant in Digestion Reaction &Digestion Supernant Removed and Viscosity of Liquid After an Equal Volume (wt / wt) Addition of Water Followed by Centrifugation

[0111] Table VI shows viscosity values obtained from undried undigested mechanically separated CFP and digested processed CFP as well as the corresponding digested processed CFP products after their freeze drying and rehydration before measurement. While the control undigested mechanically separated CFP products from Digests 1-4 display very similar values, the viscosities measured at the end of each 4-hour digest are different. - 32 - 4151998.v16109.1001001 Similarly, after drying the resulting digested processed CFP products, values for the liquid obtained after their rehydration also change based on their digestion parameters. It is noteworthy that changes in such viscosity measurements may result from differences in average fiber length produced by the combined action of cellulase and hemicellulase on CFP cellulose and NSP components during the interval of digestion (see FIG.6 below).

[0112] To further validate changes in the CFP slurry that are produced by the present disclosure, additional digestions using the same cellulase and hemicellulase enzymes at the same ratio (1: 7.6 - cellulase: hemicellulase) but with 5X (Digestion B) and 10X (Digestion C) increases in enzyme concentrations as compared to the concentration of these enzymes used for Digest 4 as described previously in Tables IV-VI. These are designated as Digests A-C in Table VII. Table VII: Summary of cellulase and hemicellulase enzyme additions to Mechanically Separated CFP slurry where Digestion A corresponds to conditions described in Run 4 Table IV. Cellulase Hemicell. Cellulase Hemicell.gm / kg X 10-3 gm / kgDigest A 0.06 0.454 44.08 8175.27Digest B 0.3 2.27 220.41 40876.35Digest C 0.6 4.54 440.82 81752.70

[0113] FIG.5 shows the results of changes in the protein content of undried mechanically separated CFP incubated for an interval of 16 hours with Digest A-C or Control (no enzymes added) additions. These data show that increasing the interval of enzymatic digestion to 16 hr. vs.4 hr. produces an increase in DMB protein content from 55% (see FIG.4) to 62% at the same or greater enzyme concentrations.

[0114] Example 5: The combined action of cellulase and hemicellulase under conditions of the present disclosure degrades mechanically separated CFP slurry carbohydrate fiber content to produce a mixture of sugars, oligo and polysaccharides.

[0115] To assess the combined actions of the cellulase and hemicellulase enzymes on carbohydrate fibers present in mechanically separated CFP slurry subject to the present disclosure conditions to produce processed CFP, slurries from Control as well as Digests A, B and C were obtained at 6 hours and centrifuged (1200 X G for 5 min.) to remove solids and the supernatants were subjected to HPLC fractionation that separated sugars (DP1-2), oligosaccharides (DP 3-9) and polysaccharides (DP>10) as shown in FIG.6. The - 33 - 4151998.v16109.1001001 concentrations of these DP1 – DP10 and fructose were normalized to the individual corresponding concentrations present in the no enzyme Control sample. As show in FIG.6, a combination of sugars, oligo and polysaccharides are generated via enzymatic digestion under these conditions.

[0116] In summary, these data provide exemplification for the present disclosure where fiber-rich high protein corn co-products like mechanically separated CFP are subjected to enzymatic digestion with fiber degrading enzymes thereby changing their physical and compositional properties. As a result of these changes, the liquid supernatant obtained after this digestion contains multiple species of sugars, oligosaccharides and polysaccharides together with protein that could serve as media for the growth of various yeasts and bacteria during and after the digestion process. For example, inoculation of the corn co-product material under conditions of the present disclosure would enable these organisms to propagate and alter the composition of the digest by, for example, reducing its ochratoxin, fumonisin or oxalate content and increase its value.

[0117] In addition, the pH of the mechanically separated CFP slurry may also be altered to form and separate a large macromolecular complex providing for further improvements in the drying of processed CFP to produce an improved and value added product (see below).

[0118] Example 6: Treatment of undried mechanically separated CFP aqueous slurries with Proteinase K, a proteolytic enzyme results in digestion of the mechanically separated CFP protein components.

[0119] To demonstrate that mechanically separated CFP protein components can be modified by the use of the present disclosure, aqueous slurries of mechanically separated CFP were exposed to Proteinase K (EC 3.4.21.64), a broad-spectrum serine protease produced by the mold Tritirachium album (70) and then processed for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). As shown in FIG.7, exposure of mechanically separated CFP slurry to Proteinase K for an interval of 30 minutes at both low and higher enzyme concentrations produced changes in the intensity of bands present in SDS- PAGE. Notably, there is a decrease in the staining intensity of protein bands in the 45-75 kDa region of the gel (indicated by the leftward facing bracket) and increase in intensity of a smaller band in the 10-15 kDa region of the gel (larger leftward pointing arrow). These changes are consistent with proteolysis of larger proteins and appearance of smaller molecular weight fragments that appear near the bottom of the gel. In this regard, proteolysis - 34 - 4151998.v16109.1001001 of larger proteins by proteolytic enzymes has been demonstrated to increase the digestibility of feed ingredients in multiple species (6).

[0120] Example 7: Treatment of mechanically separated CFP slurry with fumonisin esterase to reduce total fumonisin content of resulting Processed wet decanter cake and dried processed CFP Protein product.

[0121] Toxins such as fumonisin are variably present in incoming corn to ethanol plants and represent a major challenge in the production of co-products like mechanically separated CFP. In order to prevent fumonisin accumulation in mechanically separated CFP, significant costs are incurred performing the screening of corn upon its receipt as well as downstream testing and possible rejection of the final product if its fumonisin content exceeds levels deemed safe by regulatory guidelines (19).

[0122] In contrast to the addition of toxin degrading enzymes to large anaerobic fermentation tanks where these are constrained by the requirements of ethanol fermentation, addition of toxin degrading enzymes in the present disclosure is confined to smaller downstream tanks. Thus, the present disclosure has major advantages as compared to toxin removal or inactivation in large tanks that include digestions at higher substrate concentrations, the ability to tailor reaction conditions to the enzymatic process and major reductions in total tank volumes. Taken together, the present disclosure accomplishes the treatment of large quantities of mechanically separated CFP slurry to produce processed CFP at a rate that matches the throughput of large plants.

[0123] To demonstrate the utility of the present disclosure in this respect, mechanically separated CFP slurry was mixed with an equal amount of water and heated to 120oF as shown in FIG.8. After adjusting slurry pH to 6.0 by addition of NaHCO3, the slurry was divided into equal portions where one served as the control while the other received fumonisin esterase enzyme (79-81) at a 6 x 10-8gm enzyme / gm CFP ratio (wt. / wt.). After incubation for 30 minutes, both portions were heated to 203oF to inactivate the enzyme, centrifuged and the total fumonisin content of resulting pellets measured using gas chromatography-mass spectroscopy (GC-Mass Spec.).

[0124] Table VIII shows representative data from a single experiment performed a total of 6 times. Incubation of wet decanter cake composed of mechanically separated CFP under the conditions of the present disclosure resulted in a 99.8% reduction in total fumonisin content to concentrations less than 8 ppb as analyzed. After removal of 70% water content, these values would correspond to a reduction from 11 parts per million (ppm) to 0.026 ppm. - 35 - 4151998.v16109.1001001 Table VIII: Comparison of fumonisin content of wet decanter cake before and after treatment with fumonisin esterase under the conditions of the present disclosure.

[0125] The highly efficient and rapid removal of total fumonisin from mechanically separated CFP slurry prior to drying is anticipated to add significant value to the resulting processed CFP protein product. Multiple feed ingredient markets are highly sensitive to the fumonisin content of their products include the pet food sector (25). Moreover, it is also anticipated that large cost savings can be achieved by the ethanol producer since the stringent requirements for fumonisin screening and incoming corn can be relaxed and likely eliminated.

[0126] Utilization of fumonisin esterase enzyme that is added to mechanically separated CFP slurry as part of the present disclosure can be used to further reduce complete feed fumonisin content via addition of treated processed CFP slurry to dry ingredients followed by mixing and addition of this mash to the preconditioning cylinder of extruded feed mills.

[0127] The application of fumonisin esterase in this facet of the present disclosure is distinct from its current use as commercial feed and water additives for poultry and swine feeds (79-81). The present disclosure utilizes the residual enzyme activity present in processed CFP slurry to inactivate any fumonisin present in other ingredients from other sources and allow for the production of complete feeds with ultralow fumonisin content.

[0128] In summary, these data demonstrate that treatment of mechanically separated CFP slurry with various enzymes including proteolytic or toxin removing enzymes modify the properties and / or composition of multiple components present in the slurry and can include other enzymes like either oxalate decarboxylase or phytase.

[0129] Example 8: Comparison of the performance of processed product that has been modified by the present disclosure and either freeze dried or ring dried as a feed ingredient into extruded aquafeeds and fed to rainbow trout.

[0130] Significant fiber content of mechanically separated CFP provides challenges to both its commercial scale drying process as well as its utilization by monogastric animals such as fish. In this regard, the addition of fiber degrading enzymes into the complete feeds - 36 - 4151998.v16109.1001001 ingested by some terrestrial animal species has provided improvements in their performance (6,7,9,35,36). By contrast, similar experiments in salmonid fish have not shown any benefits (8,76). To test whether enzymatic digestion under the conditions of the present disclosure (Digest #4 – Tables IV-VI and FIG.4 above) would improve the growth and feed utilization in rainbow trout as well as whether such fish performance is affected by heat exposure to CFP slurry during its drying by ring dryers, a fish performance trial of 8 weeks (56 days) duration was performed. Briefly, domesticated rainbow trout (average wt.216 gm at trial inception) were reared in freshwater tanks (0.45 M3or 120 gallons / tank volume) at 13oC where 15 fish / tank (starting density of 7.1 kg / M3) were tested in replicates of four tanks per treatment. Trout were exposed to a 15-hour light / 9-hour dark photoperiod regimen and fed to satiation a total of twice per day. Feed fed to individual tanks of fish was carefully determined and recorded on a daily basis. Fish were graded and weighed at the start of the trial to ensure as little variation in weight as possible and then weighed as a group on the same day at every 2 weeks (14 day) intervals and individually at the end of the trial. There were no mortalities in any of the groups during the trial.

[0131] Preparation of Mechanically Separated and Processed tested in Aqua Trial – To provide for direct comparisons in trial testing, mechanically separated CFP slurry or its corresponding ring dried (heat exposed) CFP protein product was collected from a single ethanol plant within an interval of 30 minutes, processed and used for the trial. A portion of the wet decanter cake containing mechanically separated CFP was frozen and freeze dried under conditions where immediately before freezing of the slurry an identical quantity of fiber degrading enzymes (cellulase and hemicellulase) used the present disclosure was added to the slurry. This addition was performed so that the results of the present disclosure could be compared directly to data where such enzymes have been added to feed ingredients prior to their extrusion in feed manufacturing (8,76). Alternatively, a portion of the mechanically separated CFP in wet decanter cake was subjected to digestion using the present disclosure to create processed CFP and then freeze dried.

[0132] Table IX compares the composition of these test CFP feed ingredients used in the trial. Several compositional differences are notable between these CFP ingredients. As compared to mechanically separated CFP slurry that is immediately freeze dried (Table IX middle column), heat exposed ring dried mechanically separated CFP product possesses an apparent 8% increase in ash content as well as ~23-24% increase in both acid and neutral detergent fiber and a reduction in total dietary fiber. By contrast, treatment of CFP slurry - 37 - 4151998.v16109.1001001 with the present disclosure prior to its freeze drying to create processed CFP has modestly reduced its ADF and NDF but increased its dietary fiber components.

[0133] Table IX also shows differences in both lysine and zinc content of these test products. Heat exposure via ring drying produces a 20% decrease in lysine content in mechanically separated CFP where its treatment with the present disclosure to produce Process CFP results in an 8% increase in lysine when compared to freeze dried untreated CFP (middle column). By contrast, freeze dried processed CFP subjected to the present disclosure shows an ~25% reduction in zinc content as compared to freeze dried untreated mechanically separated CFP. Heat exposure via ring drying also reduces the zinc content of CFP by ~8%.

[0134] Test feed formulation and energy content - Commercial trout feed was sourced from Skretting (Classic Trout) – 40% Crude Protein; 12% Fat. Experimental test feeds were custom formulated and included CFP at a 10% inclusion rate (wt. / wt.) of the total test ingredients shown in Table IX. All feeds were then extruded on a Brabender ExtMB device equipped with a rotating cutter to create floating feed pellets suitable for the trial. Table X shows the energy content of the feeds used in the trial and are each designated based on their respective principal test feed ingredients. Note that both the Commercial and the three test feeds displayed similar total energy contents.

[0135] Trial results show that both heat exposure (ring drying) and prior enzyme treatment with the present disclosure affect fish performance, tissues and intestinal viscosity. FIG.9 shows the increases in average weights of each of the groups of trout during the 8- week trial. Note that all 3 test groups of fish fed feeds containing regardless of its processing (+ / - enzyme and heat exposure during drying) displayed larger increases in average weight after 8 weeks of growth as compared to the commercial control. - 38 - 4151998.v16109.1001001 Table IX: Comparison of the compositions of either mechanically separated CFP slurry that have been either ring dried, freeze dried or previously subject to treatment by the present disclosure to reduce its fiber content creating processed CFP.- 39 - 4151998.v16109.1001001 Table X: Comparison of the energy content of feeds used in the trout trial. Individual energy content was determined by standard bomb calorimetry.

[0136] The performance parameters of these trout fed the four test diets are shown in Table XI. There are several differences that are notable between these groups of fish. These are: • The specific growth rates (SGR) of fish fed the commercial or test diet containing heat exposed ring dried CFP slurry are less than fish fed diets containing CFP slurry that was freeze dried without heat exposure. The largest total biomass gain occurred in the fish receiving a diet containing processed CFP slurry digested using the present disclosure and freeze dried. • The feed conversion ratio (FCR) was lowest (meaning highest feed efficiency) in the fish group receiving diets containing CFP slurry that was freeze dried and not exposed to heat. However, the FCRs were not different between fish reared on diets containing CFP regardless of whether CFP had been digested by the present disclosure. • The feeding rates (feed consumed as % body weight / day) of all four test groups of fish were similar regardless of CFP treatment. Table XI: Performance summary of fish shown in FIG.9. Groups possessing different capital alphabet designations are statisically different vs. others (p<0.05). See text for details. SGR Stat. FCR Stat. Feed % Stat. Biomass Gained Stat.Commercial 1.82 C 1.24 C 2.31 NS 177.0% CCFP 10% RD 1.96 A 1.18 A 2.31 NS 199.6% ACFP 10% E 2.13 B 1.02 B 2.17 NS 223.0% ABCFP 10% ED 2.10 B 1.06 B 2.23 NS 225.0% B- 40 - 4151998.v16109.1001001

[0137] FIG.10 and Table XII compares the body length vs. body weight relationship in the four groups of fish tested in the trial. The body weight / length relationship (condition factor or K factor) for fish fed the commercial diet was different from those that were fed each of the three diets containing CFP regardless of its treatment. Table XII: Comparison of average K factors for each test fish group in the trial. Groups possessing different capital alphabet designations are statisically different vs. others (p<0.05).

[0138] Table XIII shows data obtained from comparisons of the average ratio of either the liver weight (HSI) or weight of abdominal fat (Ab. Fat Index) vs. body weight for each of the four test groups of fish. Note that the HSI of the fish fed a diet containing CFP slurry processed without heat exposure where enzymes were added immediately before freezing was smaller vs. all other test groups. By contrast, the abdominal fat content of trout reared on test diets containing CFP not exposed to heat during drying (regardless of enzyme digestion) were both larger vs. either a diet containing heat exposed ring dried CFP or fish fed a commercial diet. Table XIII: Comparison of hepatosomatic index (HSI) and abdominal fat index (Ab. Fat Index) in test fish fed one of four different diets. Groups possessing different capital alphabet designations are statisically different vs. others (p<0.05). See text for details.

[0139] Trout fed a test diet containing 10% freeze dried processed CFP slurry that had been enzymatically digested by the present disclosure possesses intestinal contents that displayed a reduced viscosity as compared to other fish in the trial. To determine if diets containing dried processed CFP slurry that had been enzymatically treated by the present disclosure result in changes in the intestinal viscosity of the fish ingesting these diets, - 41 - 4151998.v16109.1001001 intestinal viscosity measurements on fish intestinal contents were performed on the 4 test groups. Before performing these studies, it was necessary to determine if there were any time dependent changes in intestinal viscosity as a result of feeding a standard diet. As shown in Table XIV, serial measurements of intestinal viscosity over an interval of 8 hours of time showed no significant changes in intestinal viscosity as compared to the unfed state.

[0140] Table XV shows intestinal viscosity data obtained from trout during an interval of 3-7.5 hours post feeding of each of the four test diets. These data show that the viscosities of the contents of the anterior and posterior intestines of trout fed a test diet containing 10% dried processed CFP that was enzymatically digested by the present disclosure were reduced as compared to any of the other 3 test diets. Table XIV: Comparison of the intestinal viscosity of unfed trout vs. fed fish where viscosity was measured at intervals of 2 hours for as long as 8 hours. See text for details. Table XV: Comparison of viscosity of intestinal contents collected from either the anterior or posterior intestine of trout fed one of four different test feeds. See text for details.

[0141] In summary, these data in Tables XI-XIV and FIG.s 9 and 10 demonstrate that both heat exposure via ring drying and enzymatic digestion of mechanically separated CFP slurry with the present disclosure to reduce its fiber content alters the dried processed CFP. When these different dried CFP slurries are incorporated into extruded diets as a feed - 42 - 4151998.v16109.1001001 ingredient at a 10% inclusion rate and fed to trout, these respective feeds produce significant changes in trout performance and / or trout body composition.

[0142] Example 9: Heat exposure to mechanically separated CFP slurry via ring drying produces a darkening of the resulting mechanically separated CFP product together with a loss of its lysine content and an increase in its apparent fiber content.

[0143] Heat exposure to mechanically separated CFP slurry via a ring dryer causes multiple changes in the resulting dried mechanically separated CFP protein product other than the moisture content. These deleterious changes include: a) an 8% increase in ash content; b) ~24% increase in fiber content as measured by NDF and ADF and c) reduction in zinc content by ~8%. When compared to freeze dried mechanically separated CFP, the corresponding ring dried mechanically separated CFP product shows reduced performance as a feed ingredient when assayed in growth trial using rainbow trout.

[0144] To characterize these changes further, mechanically separated CFP slurry was collected from 2 different ethanol plants and freeze dried as well as a corresponding sample of ring dried heat exposed mechanically separated CFP product obtained at the same time. These data are shown in FIG.11 together with a flow chart summarizing the processing of these samples.

[0145] FIG.12 compares changes in various parameters produced by heat exposure to mechanically separated CFP slurry. While there is ~1% reduction in protein content, the fiber content of ring dried mechanically separated CFP product as measured by ADF and NDF increased by 34-41% and 28-34% respectively. At the same time, the CFP product displayed a 9-19% reduction in its lysine content.

[0146] FIG.13 shows data obtained from Hunter Color Analyses of the same samples that are shown in FIG.s 11 and 12. These Hunter Color analyses show that heat exposure via ring drying darkens the mechanically separated CFP product (reduced L value), increases both its red and yellow coloration (increases in A and B values). This combination of changes causes the ring dried mechanically separated CFP product to display a darker brown (red + yellow = orange / brown) coloration. In this regard, it is important to note that the coloration of corn-derived protein co-products produced by ethanol plants is an important determinant in consumer preference. A lighter colored yellow product is preferred since it is known to one skilled in the art that darker brown coloration is associated with the deleterious changes shown in FIG.11. - 43 - 4151998.v16109.1001001

[0147] In agreement with published reports (82), decreases in Hunter Color L score and increases in Hunter Color A score of dried CFP slurry are both correlated with loss of lysine content after heat exposure of CFP slurry via ring drying (FIG.14).

[0148] Example 10: Ring drying of mechanically separated CFP slurry produces particles displaying a wide range of sizes where larger articles are darker, more difficult to dry and contain a macromolecular complex not present in smaller particles.

[0149] Commercial ring dryers rely on particle recycling in order to efficiently remove moisture from incoming slurries while limiting the heat exposure encountered by particles that are already sufficiently dried to possess a <10% moisture content (31-33). As a result, larger particles within mechanically separated CFP slurry require more heat exposure to dry sufficiency and thus are recycled more times vs. smaller particles. As a result of the ring dryer’s operation and heterogenous particle sizes within CFP slurry, the final dried mechanically separated CFP product contains a wide spectrum of particle sizes.

[0150] Differences in the amounts of different size particles contained within dried mechanically separated CFP products can be quantified and compared by fractionating the ring dried CFP product using mesh screens possessing different porosity sizes as shown in FIG.15. Several features are notable when comparing the quantities and characteristics of particles from dried mechanically separated CFP obtained from the two ethanol plants. Ring dried mechanically separated CFP from Plant #1 displays the lightest and most yellow coloration where 34% of its particles are smaller -#70-#80 size (180-212 microns) and only 16% are larger - >#35 mesh (>500 microns). By contrast, mechanically separated CFP protein product from Plant #2 is darker in color and possesses 30% of its particles as larger >#35 mesh size and only 18% of smaller #70-#80 size particles. Hunter color analyses of the particle fractions from Plant #2 indicate that the L score of the larger particles is less than that value of the total mechanically separated CFP product (43 vs.53) whereas particles of smaller sizes display L values greater than the value for the composite ring dried CFP product. Thus, the preponderance of larger darker particles contained in the Plant #2 mechanically separated CFP results in its overall darker coloration. By contrast, the corresponding mechanically separated CFP from Plant #1 is lighter in color and possess fewer larger darker particles in its composite makeup.

[0151] To understand components that might reduce the drying efficiency of larger particles and necessitate their excessive recycling in ring dryers, samples of the largest and - 44 - 4151998.v16109.1001001 smallest particles were water extracted and the contents of these supernatants were compared after removal of insoluble material.

[0152] The smaller figure (bottom left) shows Hunter Color values for each fraction as compared to the total for Plant #2. Hunter color L (black circles – left Y axis), A values (triangles – right Y axis) and B values (green circles – left Y axis) are shown. In the larger photograph, downward pointing arrows show >#20 and #80-#100 retained particles that were water extracted, centrifuged to remove insoluble materials and analyzed by size exclusion chromatography. See text for details.

[0153] FIG.16 shows the results of size exclusion chromatography to fractionate the supernatants from the water extraction component of either larger or smaller particles shown in FIG.15. The downward pointing arrows in FIG.16 show the position of two macromolecular components that are present in the larger (mesh #20) particles and minimally present in the smaller particles (#80-#100) where both are derived from the same sample of Plant #2 mechanically separated CFP as shown in FIG.15. Analyses of these same samples using chromatographic separation of organic acids and sugars revealed no significant differences.

[0154] In summary, analyses using Hunter Color, fiber analysis and mesh fractionation of ring dried mechanically separated CFP slurry reveals that it is a composite of multiple size particles where larger particles are darker in color vs. smaller particles. These larger particles encounter a larger degree of heat exposure due to their recycling in ring dryers. Heat exposure increases the fiber and ash content of mechanically separated CFP product and reduces its lysine content. Water extraction of larger but not smaller particles reveals the presence of a macromolecular complex as detected by size exclusion chromatography that likely hampers the efficient drying of these particles in ring dryers.

[0155] Example 11: Changing the pH of mechanically separated CFP slurry produces color changes and reversible formation of a macromolecular complex similar to that isolated from larger particles of ring dried mechanically separated CFP wet decanter cake.

[0156] It is not presently known by a person that is skilled in the art of ethanol plant operations that pH adjustments of mechanically separated CFP slurry immediately before it is dried by ring dryer heat exposure would significantly change its characteristics. To demonstrate this discovery by the present disclosure, the pH of mechanically separated CFP slurry maintained at a temperature of 140oF (operating temperature of ethanol plant MSCTM- 45 - 4151998.v16109.1001001 process flow – See FIG.1) was first increased from pH 4.82 to 7.6 using additions of NaOH. This pH change resulted in an alteration in the color of the slurry as shown in FIG.17. After dividing this mechanically separated CFP slurry in equal portions, the pH of one half of this CFP slurry was then decreased to 5.7 using HCl titration. This pH change resulted in a secondary change in color to return the mechanically separated CFP slurry to a coloration similar to its original appearance (See FIG.17).

[0157] After titrations of each portion of mechanically separated CFP slurry, supernatants obtained from each of these portions were collected (see right lower panel of FIG.17). These supernatants were freeze dried, resolubilized to yield a concentrated sample that was then analyzed using size exclusion chromatography in a manner identical to that shown in FIG.16. These data (FIG.18) reveal the appearance of macromolecular components with the same chromatographic elution profiles as shown in the pH 7.6 supernatant of FIG.18 which are also greatly diminished or absent from the pH 5.7 supernatant. No differences were observed when these supernatants were analyzed for differences in sugars or organic acids.

[0158] Taken together, these data indicate that macromolecular components similar to that present in larger ring dried mechanically separated CFP particles can be reversibly formed in mechanically separated CFP slurry when the pH is increased (pH 7.6) but then disassembled or removed when the pH of the mechanically separated CFP slurry is readjusted to pH 5.7. It is likely that this macromolecular complex that is present in the larger particles in mechanically separated CFP slurry contribute to the challenge of drying these CFP larger particles. By contrast, the absence or minimal presence of this complex in smaller particles of the same slurry is correlated with the ability of ring dryers to dry these smaller particles to a low moisture content with less heat exposure.

[0159] Furthermore, the reversible pH-sensitive macromolecular complex can be isolated from CFP slurry and separated from its bulk protein fraction prior to drying by heat exposure.

[0160] To isolate this pH sensitive macromolecular complex, mechanically separated CFP slurry (pH 4.9) was first centrifuged without any pH adjustment and the resulting supernatant collected and heated to 140oF. After dividing this supernatant into equal portions, the pH of one portion was increased to pH 7.6 with NaOH whereas the pH of the other corresponding portion remained at pH 4.9. As shown in FIG.19, there was the visible formation of a “spider web” like complex in the pH 7.6 supernatant within 5 minutes whereas the pH 4.9 supernatant remained opalescent. Over an interval of 30 minutes, the presence of the macromolecular complex increased in the pH 7.6 supernatant where it appeared to - 46 - 4151998.v16109.1001001 possess a density greater than water. When incubated at room temperature for a longer interval, the complex formation was accompanied by a color change.

[0161] Example 12: Characterization of pH Sensitive Complex from Mechanically Separated CFP.

[0162] Example 11 demonstrates that the pH-sensitive complex can be isolated from the liquid supernatant obtained after centrifugation of mechanically separated CFP slurry by increasing its pH from 4.9 to 7.6. Sufficient pH-sensitive complex was isolated and analyzed to compare it to that of mechanically separated CFP. As shown in FIG.20, the overall appearance of the pH sensitive complex is different than mechanically separated CFP and possesses a fine fibrous appearance.

[0163] Table XVI shows a comparison of differences in the composition of the isolated pH sensitive complex vs. mechanically separated CFP. Notable differences between them include that the pH sensitive complex has a reduced protein and acid detergent fiber content while possessing significantly higher contents of fat, ash and multiple inorganic minerals particularly divalent cations (Ca2+, Mg2+, Mn2+and Zn2+). However, these content increases appear selective since other divalent cations (Fe2+and Cu2+) are reduced not increased. Table XVI: Comparison of the proximate and mineral compositions of freeze-dried CFP vs. isolated and freeze-dried pH sensitive complex. See text for details. All Values are DMB Basis Shenandoah CFP pH SensitiveContent Change Component Freeze Dried Complex UnitsCompared to CFP Protein 60.6 41.2 % -0.32Fat 5.9 21.4 % 2.62Fiber (ADF) 18.6 10.1 -0.46Ash 2.78 19.7 % 6.09Sulfur 0.68 0.75 % 0.10Phosphorus 0.69 4.23 % 5.13Magnesium 0.19 2.52 % 12.26Calcium 0.02 1.78 % 88.00Sodium 0.05 1.3 % 25.00Iron 118 202 ppm 0.71Manganese 10.8 431 ppm 38.91Copper 10.2 14.7 ppm 0.44Zinc 84.8 1260 ppm 13.86- 47 - 4151998.v16109.1001001

[0164] Example 13: Removal of the pH Sensitive Complex from Mechanically Separated CFP Increases Its Protein Concentration and Significantly Facilitates the Abilities of Cellulase and Hemicellulase to Reduce its Fiber Content and Further Increase Its Protein Content.

[0165] Processing of mechanically separated CFP slurry as described in Example 11 to remove the pH sensitive complex present in the supernatant after its centrifugation yields a pellet containing processed CFP. Table XVII compares the differences between slurries prepared from a single sample of mechanically separated CFP before and after removal of the pH sensitive complex to yield processed CFP. As compared to the protein content of the starting sample of mechanically separated CFP (59.8% DMB), mechanically separated CFP displays a slightly increased protein content (62.9% DMB) after its preparation as a slurry. By contrast, removal of the pH sensitive complex from mechanically separated CFP slurry to yield processed CFP produces a significant further increase in its protein content (67.1% DMB). These data are consistent with Table XVI showing that the protein content of the pH sensitive complex is significantly reduced as compared to mechanically separated CFP and its removal is expect to boost the protein content of the remaining material that is now termed processed CFP. Table XVII: Comparison of the protein contents (all values are % DMB) and digestibility of mechanically separated CFP vs. processed CFP where its pH sensitive complex has been removed. See text and methods for details. Original Mechanically Separated CFP 59.8% Slurries Prepared Before and AfterRemoval of pH Sensitive Complex1 Hours of Digestion 0 2 4 8 Increase in ProteinContent After 8 Hours Mechanically Separated CFP No Enzyme Addition 62.9% 62.0% 61.4% 61.8%Cellulase + Hemicellulase2,3 62.9% 62.1% 63.6% 64.9% 3.1%

[0166] Aliquots of the two corresponding slurries were subjected to incubation at 140oF for 8 hours with or without the addition of identical quantities of commercially available cellulase and hemicellulase enzymes in a manner identical to that described in Example 4 - 48 - 4151998.v16109.1001001 Tables IV and VII. Note that the protein content of the mechanically separated CFP increased by 3.1% while that of the processed CFP increased by 7.2%. Overall, the protein content of mechanically separated CFP increased by 5.1% (59.8% to 64.9% DMB) whereas the protein content of processed CFP after removal of the pH sensitive complex and identical digestion conditions using fiber degrading enzymes increased by 14.0% (59.8% to 73.8% DMB). This overall greater increase in the protein content of processed CFP is due to the combination of removal of the pH-sensitive complex together with the increased ability of fiber degrading enzymes to digest fiber components present in the processed CFP.

[0167] Taken together, these data demonstrate that the soluble components of this macromolecular complex present in mechanically separated CFP slurry can be isolated from larger insoluble proteinaceous particles and then assembled by pH adjustment. Removal and / or manipulation of this complex can be accomplished which results in an increase in the protein content of the processed CFP that can increased still further via an improved ability for fiber degrading enzymes to digest the larger insoluble proteinaceous particles. These improvements are combined with other viscosity and compositional data showing these changes facilitate the final processing and drying of the processed CFP.

[0168] Example 14: Removal of the pH Sensitive Complex from Mechanically Separated CFP followed by Enzyme Digestion Results in Low Viscosity Processed CFP that can be Dried Using Spray Drying Methods.

[0169] Data shown in Table III contains measurements of liquid extracted from mechanically separated CFP using a flat plate viscometer where, in selected cases, it was digested with a combination of cellulase and hemicellulase. These studies show that liquid extracts of mechanically separated CFP are highly viscous but this viscosity can be reduced by digestion with fiber degrading enzymes. However, these data are subject to the inability of the flat plate viscometer method to measure more viscous high particulate slurries including those prepared from either mechanically separated or processed CFP.

[0170] Table XVIII shows data obtained using a rotational viscometer capable of quantifying much larger viscosities that are present in high solids CFP slurries. As shown, mechanically separated CFP is very viscous but this viscosity is reduced by ~86% after digestion with commercial cellulase at 140oF for 12 hours. By contrast, removal of the pH sensitive complex from mechanically separated CFP as described in Example 11 reduces the viscosity of the resulting processed CFP by ~55% as compared to the original slurry (Table XVIII). Digestion of this processed CFP slurry under identical conditions used for - 49 - 4151998.v16109.1001001 mechanically separated CFP still further reduces slurry viscosity by ~96% to <500 mPa·s (millipascal-secs or cP). Thus, this cellulase digestion of processed CFP represents an overall reduction in viscosity of ~98% as compared to mechanically separated CFP. Table XVIII: Comparison of viscosity measurements of mechanically separated CFP vs. processed CFP before and after digestion with cellulase at 140oF for 12 Hours. Viscosity2After 12 Hour Initial Viscosity2Cellulase Digestion1Mechanically Separated CFP Slurry*24,970 3565Processed CFP Slurry*11,200 490*Slurry composed of 50% Total Solids 1Digestion at 140oF 2Viscosity Values shown in mpass or millipascals-sec.

[0171] Dewatered slurries of mechanically separated CFP are presently dried using drum or ring dryers due to their high viscosity and water activity. As described in Example 9, the drying of mechanically separated CFP is accompanied by changes in the coloration and composition of the resulting dried mechanically separated CFP. Other drying methods particularly spray drying technology has not been previously possible to apply to mechanically separated CFP protein due to its very high viscosity and water activity. However, application of spray drying methods to digested processed CFP is now possible since its viscosity is <500 mPa·s that are required by most commercial large-scale spray dryers (https_dedert_com / spray-dryers / guide / ). Spray drying methods typically yield superior dried feed ingredient products possessing increased quality, smaller particle sizes and higher digestibility as compared to those dried by ring or drum dryers. As a result, the combination of the Back End Process producing processed CFP with a higher protein content that can be spray dried is anticipated to add significant value to the resulting end product(s).

[0172] General Considerations and Methods

[0173] Financial considerations of the methods and applications of the present disclosure to existing ethanol plants and reduction in costs of enzymes serve as a non-limiting demonstration of the utility of the present disclosure.

[0174] This section outlines non-limiting general considerations on the methods of present disclosure in existing ethanol plants and highlight some examples of its commercial value. - 50 - 4151998.v16109.1001001

[0175] FIG.21 is a schematic diagram that contrasts the operation of the present disclosure as compared to present day operations in use in existing ethanol plants based on operational tank footprints.

[0176] Fiber degrading or other enzymes are presently added to large fermentation tanks (80,000 gallons or 304 M3) which are commonly loaded with ~45,600 bushels of corn at 34% solids loading rate. Thus, enzymatic digestion of the mixture is controlled by fermentation parameters listed in FIG.21 and results in the targeted production of 79.9 tons of dried mechanically separated CFP (239 tons of CFP slurry containing 70% moisture) after mechanical separation of the resulting whole stillage. By contrast, collecting the same 239 tons of mechanically separated CFP slurry allows for its suspension at the same solids loading rate in a tank of only 5,000 gallons or 18.9 M3. The incubation conditions in this “back-end” tank can be established for optimal enzymatic digestion without regard to other parameters. As a result, the identical quantity of mechanically separated CFP slurry can be processed using the present disclosure and the same enzymes. Importantly, since enzymatic dosing of tanks is directed by both substrate content (identical in the case of FIG.21) and volume, this will result in a ~94% reduction in enzyme costs via use of the present disclosure.

[0177] It is recognized by one skilled in the art that these basic conditions described in FIG.21 can be scaled up to involve larger stirred tank bioreactors (500-10,000 liters) equipped with conventional stirring mechanisms and corresponding inlets and outlets designed for the pumping and processing of slurried ingredients. Moreover, while such tanks are completely separate from equipment involved in mechanical separation, they could be integrated with existing Maximal Stillage Co-productTMprocess equipment so as to provide for the loading of a measured quantity of mechanically separated CFP decanter centrifuge contents into tanks for further batch processing as shown in FIG.22. Back-End Processing Tanks practicing the present disclosure could receive mechanically separated CFP slurry from MSCTMprotein decanters and, after processing, the treated processed CFP slurry dried using a spray dryer due to the reduced viscosity of the product or, if desired, using an existing ringer dryer. The X symbol shown in FIG.22 indicates that the Back-End Process of the present disclosure could be integrated with the existing MSCTMprocess in such a manner that processed CFP slurry could be sent to a spray dryer rather than sent to the ring dryer (path indicated by X) so as to create specialty value added products. Alternatively, it is also possible to design for a continuous flow apparatus for continuous treatment of mechanically - 51 - 4151998.v16109.1001001 separated CFP slurry by enzymes under conditions defined by the present disclosure to produce processed CFP that could then be dried via spray drying.

[0178] As shown in FIG.22, the Back-End Process of the present disclosure can also be used to remove a macromolecular complex from mechanically separated CFP slurry via alterations in pH as described above. Removal of this complex will both facilitate drying of the processed CFP slurry by a spray dryer as well as prevent recycling of the complex via backset to be used for slurrying incoming corn. In each of these pathways, it is anticipated that operational cost reductions will be achieved in addition to improving the quality of the final product.

[0179] Description of methods used for digestions of the present disclosure.

[0180] To perform digestions, a sample of mechanically separated CFP slurry material was obtained from decanter centrifuges under normal operating conditions using the Maximal Stillage Co-productTMprocess of Fluid Quip Technologies. This material is shown in Panel A of FIG.s 2 & 3. After determining its weight, an equal weight of water was added to this decanter cake and stirred vigorously to create a thick slurry that was suspended in a tank (1- 30 liters) equipped with a continuous stirring apparatus and heating jacket to maintain the slurry at a constant temperature of 86oF throughout the digestion interval. The pH of the slurry remained stable at a value of 4.4. Prior to the addition of any enzymes, control (Time 0) samples were obtained and processed as described below.

[0181] After the required interval of digestion of mechanically separated CFP slurry by the enzyme(s), the resulting mixture was centrifuged at 700 x g for a total of 1 minute followed by deceleration with the brake applied. This produced a thick pellet and corresponding liquid supernatant that were each subject to further analyses.

[0182] A total of 5 enzymes were used for exemplification purposes. These included: • Cellulase from Trichoderma reesei Laboratory Grade 61,200 U / gm with pH and temperature optima of 5.0 and 55oC respectively obtained from www.carolina.com • Hemicellulase from Aspergillus niger 0.3-3.0 U / mg (assumed average of 1,500 U / gm) using locust bean gum as a substrate at pH 4.5 • Proteinase K from Tritirachium album >30 U / gm using denatured hemoglobin as a substrate. Digestion conditions were performed as described in reference #82. • Fumonisin Esterase from Komagataella phaffii with a specified minimum content of 3,000 units of esterase / gm was used for digestions where the mechanically separated CFP slurry was adjusted to pH to 6.0 using NaHCO3, heated to 120oF after incubation - 52 - 4151998.v16109.1001001 for 30 minutes, heated to 203oF to inactivate the enzyme, centrifuged and the total fumonisin content determined. • Commercial Cellulase and Hemicellulase was obtained from readily available sources.

[0183] Rainbow trout growth trial and analyses. • Test feeds were formulated using CONCEPT 5 software and internal feed ingredient database assembled after third party analyses of feed ingredients used. Feeds were manufactured using a Brabender ExtMB instrument at the Green Plains Omaha Innovation Center and the trial conducted at the Shenandoah Optimal Aquafeed Aquatic Laboratory both of which are described at www_optimalaquafeed_com. • Rainbow trout were reared from eyed eggs (Riverence) using standard commercial feeds or Optimal Aquafeed trout production feed until trial testing. Fish from a single egg group were graded prior to testing using standard methods and housed in recirculating aquaculture tanks as described at www_optimalaquafeed_com. Weighing, body measurements, tissue sampling and analyses of fish performance and composition were performed using standard methods (62,74).

[0184] Miscellaneous methods.

[0185] Flat Plate Viscometry – Viscosity measurements of CFP slurry, slurry supernatants and intestinal contents of fish were performed using a Brookfield LVDV-I Prime CP-Cone Plate viscometer equipped with a CPE-42 cone spindle. Viscosity measurements of trout intestinal contents were performed using methods and analyses as described previously (56).

[0186] Rotational Viscometry – Viscosity measurement of both mechanically separated and processed CFP prior to and after enzymatic digestions were performed on a NDJ -9S Digital Viscometer using a #2 spindle at multiple rotation speeds yielding values within an angle of 15-80%.

[0187] Hunter Color Measurements on various freeze and ring dried mechanically separated and processed CFP slurry samples were performed with a Colorflex EZ spectrophotometer according to the instrument manual.

[0188] Freeze Drying – was performed using a Labconco 4.5 liter benchtop freeze dryer equipped with 12 port system. - 53 - 4151998.v16109.1001001

[0189] Sieve Fractionation of Ring Dried Mechanically Separated CFP Protein Samples – was performed using 8-inch diameter ASTM Round All Brass Test Sieves (Gilson Inc.) using standard methods.

[0190] Fumonisin Content of Mechanically Separated and processed CFP Slurry Fractions – was performed using liquid chromatography-mass spectrophotometry (LC-MS) with modifications of the method as described in reference #84 by the Green Plains Analytical Laboratory at the Omaha Innovation Center (www_gpreinc_com / location / omaha- innovation-center / )

[0191] Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) - was preformed using standard methods (84).

[0192] Chromatography of Mechanically Separated and processed CFP Slurry Supernatants – was performed using both size exclusion and calcium HPLC columns using standard methodology.

[0193] Analyses of Multiple Samples of Mechanically Separated and processed CFP Slurries and Extracts as well as Fish Feeds Containing Dried Mechanically Separated and Processed Feed Ingredients - were performed by third party analysis (www_midwestlabs_com). - 54 - 4151998.v16109.1001001 DEFINITIONS

[0194] It is to be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting. 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 to which the disclosure pertains.

[0195] Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present disclosure, exemplary materials and methods are described herein.

[0196] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.”

[0197] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0198] Unless the context requires otherwise, throughout the specification and claims that follow, the word “comprise” and synonyms and variants thereof such as “have” and “include”, as well as variations thereof, such as “comprises” and “comprising”, are to be construed in an open, inclusive sense, e.g., “including, but not limited to.” The transitional terms “comprising,” “consisting essentially of,” and “consisting of” are intended to connote their generally accepted meanings in the patent vernacular; that is, (i) “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open- ended and does not exclude additional, unrecited elements or method steps; (ii) “consisting of” excludes any element or step not specified in the claim; and (iii) “consisting essentially - 55 - 4151998.v16109.1001001 of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed disclosure and disclosure. Embodiments described in terms of the phrase “comprising” (or its equivalents) also provide as embodiments those independently described in terms of “consisting of” and “consisting essentially of.”

[0199] “About” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the disclosure, claims, result or embodiment, “about” means within one standard deviation per the practice in the art, or can mean a range of ± 20%, ± 10%, ± 5%, ±4, ±3, ±2 or ± 1% of a given value. It is to be understood that the term “about” can precede any particular value specified herein, except for particular values used in the Examples.

[0200] All percents are intended to be weight percent unless otherwise specified. The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, other various embodiments of and modifications to the present disclosure, in addition to those described herein, will be apparent to those of ordinary skill in the art from the foregoing description and accompanying drawings. Thus, such other embodiments and modifications are intended to fall within the scope of the present disclosure. Further, although the present disclosure has been described herein in the context of a particular implementation in a particular environment for a particular purpose, those of ordinary skill in the art will recognize that its usefulness is not limited thereto and that the present disclosure may be beneficially implemented in any number of environments for any number of purposes. Accordingly, the claims set forth below should be construed in view of the full breadth and spirit of the present disclosure as described herein.

[0201] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are intended to be encompassed in the scope of the claims that follow the examples below. - 56 - 4151998.v16109.1001001 REFERENCES 1. Johnson, C. K. Moriarty, T Alleman and D. Santini (2021). History of ethanol fuel adoption in the United States: Policy, Economics and Logistics. NREL / TP-5400- 76260. 2. Kim, Y., NS Mosier, R Hendrickson, T. Ezeji, H Blaschek, B Dien, M Cotta, B Dale and MR Ladish. (2008) Composition of corn dry-grind ethanol by-products: DDGs, wet cake and thin stillage. Bioresource Tech.99:5165-5176. 3. Gibson, L. (2020) Masters of the Market. Ethanol Producer Magazine July 15 issue. 4. Kang, X, A Kirui, MC Windanage, F Mentink-Vigier, DJ Cosgrove and T. 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[0202] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0203] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments. - 64 - 4151998.v1

Claims

6109.1001001 CLAIMS What is claimed is:

1. A method of obtaining processed corn fermented protein (CFP), comprising: a) providing a composition that comprises mechanically separated CFP, wherein the mechanically separated CFP comprises water-soluble material and one or more of a fiber and an animal toxin; b) removing the water-soluble material from the composition; c) contacting the composition with an effective amount of an enzyme, a single cell organism that produces an enzyme, or a combination thereof, wherein the enzyme is at least one of a proteolytic enzyme, a fiber-degrading enzyme, or a toxin-degrading enzyme, thereby producing enzymatically-treated CFP comprising enzymatic degradation products; and d) removing the enzymatic degradation products from the enzymatically-treated CFP, thereby producing processed CFP.

2. The method of claim 1, further comprising drying the processed CFP.

3. The method of claim 1 or 2, wherein the water-soluble material comprises a pH- sensitive macromolecular complex.

4. The method of any one of claims 1-3, wherein b) comprises centrifuging the composition, collecting resulting supernatant, and heating the supernatant.

5. The method of claim 4, wherein the supernatant is heated to a temperature of from about 130oF to about 150oF.

6. The method of claim 4 or 5, wherein b) further comprises adjusting the pH of the supernatant to a pH of from about 7.0 to about 8.

0.

7. The method of claim 6, wherein the pH of the supernatant is about 7.

6.

8. The method of claims 6 or 7, wherein b) further comprises filtering the supernatant.

9. The method of any one of claims 1-8, wherein c) comprises adjusting the pH of the composition. - 65 - 4151998.v16109.1001001 10. The method of claim 9, wherein the pH of the composition is adjusted to a pH of from about 5.0 to about 7.

5.

11. The method of any one of claims 1-10, wherein in c), the enzyme, the single cell organism, or the combination thereof is in contact with the composition for about 0.5 hours to about 20 hours.

12. The method of any one of claims 1-11, wherein the fiber-degrading enzyme is cellulase or hemicellulase, or a combination thereof.

13. The method of any one of claims 1-12, wherein in c), the composition is contacted with an effective amount of cellulase and hemicellulose, wherein the ratio of cellulase to hemicellulase in the composition is from about 1:7 to about 1:

8.

14. The method of any one of claims 1-13, wherein the proteolytic enzyme is proteinase K.

15. The method of any one of claims 1-14, wherein the toxin-degrading enzyme is phytase, oxalate decarboxylase, or fumonisin esterase, or a combination thereof.

16. The method of any one of claims 1-15, wherein d) comprises filtering out the enzymatic degradation products.

17. The method of any one of claims 2-16, wherein drying the processed CFP is performed by spray drying, freeze drying, drum drying, ring drying, or a combination thereof.

18. The method of any one of claims 2-17, wherein drying the processed CFP is performed by spray drying.

19. The method of any one of claims 2-18, wherein the processed CFP is dried to a moisture content of less than about 10 wt%.

20. The method of any one of claims 1-19, wherein in c), the composition is contacted with an effective amount of a single cell organism that produces the enzyme.

21. The method of claim 20, wherein the single cell organism is a yeast. - 66 - 4151998.v16109.1001001 22. The method of claim 21, wherein the single cell organism is a bacterium.

23. The method of claim 20, wherein the single cell organism is Lactococcus lactis, Trichosporon mycotoxinivorans, Sphingopyxis sp. or Komagataella phaffii, or a combination thereof.

24. The method of any one of claims 1-23, further comprising incorporating the processed CFP into an animal feed.

25. The method of claim 24, wherein the animal feed is an extruded or pelleted feed.

26. The method of claim 24 or 25, wherein the animal feed is a mixed feed or a single ingredient feed.

27. The method of any one of claims 1-26, wherein b) further comprises contacting the composition with one or more additional enzymes or single cell organisms.

28. The method of any one of claims 1-27, wherein the composition is a slurry, a wet cake, or a powder.

29. The method of any one of claims 1-28, wherein the CFP is obtained as a co-product of ethanol production.

30. The method of any one of claims 1-29, wherein the processed CFP has a viscosity of about 500 mPa·s or less.

31. An animal feed comprising processed CFP obtained using the method of any one of claims 1-30. - 67 - 4151998.v1

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