WO2006104504A2

WO2006104504A2 - Integrated fermentation product recycling

Info

Publication number: WO2006104504A2
Application number: PCT/US2005/022853
Authority: WO
Inventors: Eugene M. Peters, Jr.; Suhas K. Mehra; Eugene J. Fox; Ting L. Carlson; Aharon M. Eyal; Donald L. Shandera, Jr.; Ki Park; Rod R. Fisher
Original assignee: Cargill Inc
Current assignee: Cargill Inc
Priority date: 2004-06-24
Filing date: 2005-06-24
Publication date: 2006-10-05
Anticipated expiration: 2006-12-24
Also published as: WO2006104504A3

Abstract

Methods for treating starch-containing materials are described. The methods include processing the starch-containing material to form a fermentation material, and fermenting at least a portion of the fermentable material for form a fermentation broth including a fermentation product. At least a portion of the fermentation broth is then used in processing a starch-containing material.

Description

Integrated Fermentation Product Recycling

TECHNICAL FIELD This invention relates to processing starch-containing materials, and more particularly to processing cereal material.

BACKGROUND

Grains such as corn, sorghum, wheat, and rice may be processed using numerous methods including wet milling, dry milling, and extrusion. A wet milling process is a type of wet processing, and may include steeping grains to soften the kernels for separation of various components. Following steeping, the grain is subjected to grinding and high speed centrifugation and/or filtration to separate various components such as germ, protein, fiber and starch. Usually, the germ is subsequently processed to vegetable oil, the protein and fiber are used for animal feed, and the starch is used for sweetener or alcohol production. The operational costs of wet milling are high due to the energy requirements of dehydrating excess water, operating high speed centrifuges to separate and concentrate desirable matter, and providing fresh water.

Dry milling requires a smaller operating cost and a smaller capital investment than wet milling and is therefore often used as an alternative to wet milling. However, the low costs of dry milling are offset by generally poor recovery of germ and poor protein-starch separation. Conventional dry milling involves tempering with water to soften the kernels for cracking. The major products of this process include grits, meal, and flour. Dry milling may also be used for the production of alcohol because the separation of protein and starch may not be necessary for fermentation. In the United States today, the majority of corn processing is carried out using the wet milling process, and significant efforts in the corn milling industry since the 1970's have been directed towards increasing the production of sweeteners and/or alcohol. For sweetener production, generally the milled corn starch product is gelatinized and then hydrolyzed by amylases. During this process, corn protein is separated from the starch to form hydrolyzed syrup necessary for the production of high fructose syrup. Due to a necessity to obtain relatively pure high dextrose syrup, the more efficiently the protein is removed from the process, the better the result. Thus, wet milling is often the milling process of choice for sweetener manufacturers. Alcohol production does not require as efficient of a separation, and thus, dry milling has been a more common processing approach for alcohol production because of cost considerations. Alcohol may be produced from corn by liquefying and saccharifying corn endosperm or purified corn starch to provide fermentable sugars that can be converted to alcohol. The most commonly produced alcohol is ethanol for use in the beverage industry, as an industrial solvent, as a petrol additive, and as gasohol. Due to this variation, carbohydrates for sweetener and alcohol production are often manufactured separately for sale to various industries.

More efficient and lower cost grain milling processes are increasingly desirable as the sweetener, vegetable oil, and alcohol industries become more competitive.

SUMMARY In one aspect, a method for treating a starch-containing material is described. The method includes wet processing the starch-containing material to produce a fermentable material, fermenting at least a portion of the fermentable material using a fermentation organism to produce a fermentation broth including a fermentation product, and using at least a portion of the fermentation product in wet processing the starch-containing material. In another aspect, an integrated process for treating starch-containing material is described. This process includes processing a first starch-containing material to produce a first stream including a first fermentable material, and fermenting the first fermentable material using a first fermentation organism to produce a first fermentation broth including a first fermentation product. It also includes processing a second starch-containing material to produce a second stream including a second fermentable material, and introducing at least a portion of the first fermentation broth into processing the second starch-containing material, wherein the process of producing a first stream is integrated with a process of producing a second stream.

In another aspect, a process for treating cereal material is described. The process includes wet processing a first cereal material to produce a first stream including a first starch and fermenting the first starch using a first fermentation organism to produce a first fermentation broth including a first fermentation product. It also includes wet processing a second cereal material to produce a second stream including a second starch, and fermenting the second starch using a second fermentation organism to produce a second fermentation broth including a second fermentation product. It further includes introducing at least a portion of the first fermentation broth into wet processing the second cereal material and introducing at least a portion of the second fermentation broth into wet processing the first cereal material.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a process flow diagram of a wet processing of corn integrated with a lactic acid fermentation, with a product depleted stream from the purification of lactic acid, containing residual lactic acid, being recycled to the steeping phase of the corn wet milling process. FIG 2 is a process flow diagram of a wet milling of corn integrated with a protease production fermentation, with a product depleted stream from the purification of the protease, containing residual protease, being recycled to the milling phase of the corn wet milling process.

FIG. 3 is a process flow diagram of a wet milling of corn integrated with a cellulase production fermentation, with a product depleted stream from the purification of the cellulase, containing residual cellulase, being recycled to the milling phase of the corn wet milling process.

DETAILED DESCRIPTION

A starch-containing material may be processed to form a fermentable material. The fermentable material may be fermented with a suitable organism to form a fermentation product in a fermentation broth. The broth may be separated to provide at least one product-enriched stream including a fermentation product and at least one product-depleted stream, which has a reduced concentration of fermentation product. At least one component of the broth may be recycled for use in processing the starch-containing material into fermentable material. Terms and Definitions

As used herein, "processing" and "processed" refers to a chemical and/or physical alteration of starch-containing material by one or more operations. These operations may facilitate penetration of reagents or degradation of the cereal material into its component parts. Examples of processing operations may include, without limitation, flaking, steeping, tempering, degerming, dewatering, fiber removal, liquefaction, saccharification, oil extraction, loosening protein coating of starch granules in the cereal material, comminuting, degrading, fractionating and/or any operation that facilitates protein separation and/or penetration of at least one agent into the cereal material. The processing operations may be assisted with fermentation product and chemical and/or biological reagents, including operations for hull degradation, liquefaction, saccharification and/or protein degradation of the coating and/or matrix which holds together the starch granules of the cereal material, and fractionating protein, fiber, starch and germ.

As used herein, "starch-containing material" refers to any material containing a starch including potato, soybean, sweet potato, sago, palm, cassava, and cereal materials. As used herein, "cereal material" refers to whole grain, parts, components, or products derived thereof. Thus, cereal material may include whole grain kernels or portions of kernel at least partially separated by known processes such as dry-milling, wet-milling and/or extrusion. For example, cereal material may include a typical com kernel having a hull, germ, and endosperm. Cereal material may also include "grits," which refers to material remaining after whole grain kernels have been comminuted and then separated to remove the bulk of the germ material and fibrous outer layer of the kernel. Cereal material may also include separated components of grains including endosperm, pericarp, germ, or derived components such as starch, protein, fiber, oil, etc. Cereal material may also be present in production streams such as a starch-containing stream, a protein-containing stream, a fiber containing stream, and a germ stream. Common examples of cereal materials include, but are not limited to, corn (maize), sorghum, rice, barley, oats, rye and wheat.

As used herein, "wet processing" refers to processing a starch-containing material wherein an amount of water exceeding the amount of water that can be absorbed by the starch- containing material is used to enhance separation of the components of the grain. Wet processing is distinguished from dry processing and subsequent water addition and hydrolysis of starch in a dry processing stream in that the addition of excess water occurs before the starch hydrolysis stage to enhance component separations.

As used herein, "wet milling" refers to a method of wet processing a starch-containing material wherein an amount of water exceeding the amount that can be absorbed by the grain is used to steep the starch-containing material and then mill the starch-containing material. Therefore, wet milling is one example of a type of wet processing.

As used herein, the term "lactic acid" refers to lactic acid, lactate salt(s) of lactic acid, or a combination of lactic acid and lactate salt(s).

As used herein, the term "fermentation broth" refers to a mixture including at least one fermentation product and additional components. Examples of additional components which maybe present include water, nutrients, and non-fermented materials.

A fermentation product may be produced from a starch-containing material by a method that includes processing a starch-containing material to provide a fermentable material, addition of a fermentable material to a fermentation media, fermenting at least a portion of the fermentable material with a fermentation organism to provide a fermentation broth including a fermentation product, and recycling at least a portion of the fermentation broth for use in processing the starch-containing material.

Processing a starch-containing material to produce a fermentable material may be a simple or complex process. In general, the starch-containing material provided may be processed in any conventional manner. For example, processing the starch-containing material may involve only comminuting the starch-containing material to provide fermentable material for subsequent fermentation. Usually, however, processing a starch-containing material to produce a fermentable material involves more complex and numerous processing steps. For example, producing a fermentation feedstock may be accomplished by hydrolyzing the starch slurry of the starch-containing material product of wet milling or wet processing. Any processes, including those typically used in wet processing or wet milling may be utilized in processing a starch-containing material.

The starch-containing material may be provided in whole or in part. The starch- containing material may be a cereal material or another source. In various embodiments, the starch-containing material includes whole grains, milled grains, a dry milled grain product, and purchased processed material. Typically, the starch-containing material is provided as a whole grain.

Processing may include separation of the starch-containing material, and components thereof, into various streams or stages. Examples of streams that may be separated include a fiber stream, an oil-containing stream (e.g., germ stream), a protein-rich stream (e.g., gluten stream), a starch stream, a solubles rich stream, or any combination thereof. Each of these streams may contain other components, but is enriched in at least one component compared with the starting starch-containing material. These streams may be directed to downstream operations for further processing (e.g., production of fermentation feedstock), directed to one or more sidestreams (e.g., oil extraction), or used or sold directly (e.g., production of animal feed or of a fermentation feedstock). In various combinations, the streams may be removed from the process, dried and sold as a product.

Processing a starch-containing material may include hydrolysis of one or more streams. If hydrolyzed, the product may be hydrolyzed to any extent. For example, a starch stream may be hydrolyzed to form a hydrolyzed starch, including hydrolyzing to dextrose. Starch slurry may be hydrolyzed by any manner, including by acid hydrolysis, enzymatic digestion of the starch, or by another method. Suitable acids for acid hydrolysis include inorganic acids such as hydrochloric acid and the like. However, acid hydrolysis maybe limited in the extent of starch hydrolysis possible. Therefore, it maybe desirable to use enzymes or other means of hydrolysis to exceed a certain hydrolysis level. In general, elevating the temperature increases the rate of hydrolysis, and the temperature may therefore vary, depending on the degree of hydrolysis desired.

In some cases, chemical or biological reagents may be used to facilitate processing of the material. Examples of chemical or biological reagents that may be used include water, gelling agents, reducing agents, chelating agents, surfactants, liquefaction agents, saccharification agents, agents to assist in loosening protein coating of starch granules, oil extractants and/or protein extractants. These operations and the use of reagents may be a variation of a wet-milling, dry-milling, extrusion process and/or other treatments to generate fermentable materials. At least one of the streams formed in the processing of cereal material provides a fermentable material. The fermentable material may include carbon sources, nitrogen sources, or both.

Typically, processing a starch-containing material will include subjecting provided whole grains to wet processing. Depending upon the starch-containing material being processed, the steps of wet processing may vary. For example, in wet processing a whole grain such as corn, wet processing will generally include steeping the whole grain, a coarse grind step, density separation of the germ, one or more fine-grind steps, screening the fiber, separating starch and protein streams, and hydrolyzing the starch stream. In the processing of other starch-containing material, all of these steps may not be necessary or conducted, while other steps not listed may be utilized.

At least a portion of the fermentable material may be fermented to provide a fermentation broth. During fermentation, a suitable organism converts the fermentable material to one or more fermentation products. The fermentation broth includes a fermentation product that has been fermented by the fermentation organism, as well as any unconsumed nutrients or byproducts of the fermentation organism. According to Brock and Madigan (Biology of Microorganisms, 5th ed, p. 125, Prentice-Hall, Lie, Englewood Cliffs, NJ, 1988), fermentation may be described as an internally balanced oxidation and/or reduction of organic compounds with the release of energy by a suitable organism. The fermentable material stream may be used directly, or may be subjected to further processing before fermenting. The fermentable material may include media for fermentation having sufficient nutrients and water. Alternatively, a fermentation media may be added with the fermentable material prior to fermentation.

The fermentable material and a fermentation media are supplied to form a fermenting mixture. A fermentation media may include a carbon source (such as starch or hydrolyzed starch), water, and one or more of nitrogen, vitamins, minerals, and other trace components as required that will support the growth and/or production of a fermentation product. Other components may be added to the fermentation media including flocculants such as polyamines, reducing agents, and surfactants, hi certain processes the fermentation media may be present as part of a process stream including fermentable material. Additional reagent components may also be added to the broth or fermentation media prior to fermentation, during fermentation, or after fermentation to assist in isolating the fermentation product or to enhance fermentation conditions.

The fermentation media may already include the fermenting organism, or the fermenting organism may be added for fermentation. Fermentation organisms that may be utilized include any fermentation organism that will produce the fermentation product using the appropriate carbon source, typically starch or hydrolyzed starch. The organism may vary depending on the desired fermentation product. The organism may be an aerobic or anaerobic fermenter. Examples of suitable organisms include, but are not limited to, prokaryotic (e.g., species of Lactobacillus, Acetobacter, Acetobacterium, Propionibacterium, Clostridia, Streptococcus,

Bacillus, Corynebacterium, Brevibacterium, Psuedomonas, Serratia, Zymomonas, Streptomyces, Actinomycetes, Micromonospora, Actinobacillus), eukaryotic (e.g., yeast or fungi including Saccharonmyces, Kluyveromyces, Candida, Eremohecium, and Ashbya, Rhizopus and Aspergillus), or a genetically engineered strain of bacteria or eukaryotic cells. Genetically engineered strains of bacteria or eukorytic cells may be created to produce fermentation products which are non-native to that organism. For example, Agrobacterium and Rhizobium have been transformed with E. coli genes to produce biotin. Such recombination and transformation will be apparent to one skilled in the art. Examples of commonly used fermenting eukaryotic organisms include yeasts (e.g., Saccharonmyces, Kluyveromyces, Candida, Eremohecium, and Ashbya) and fungi (e.g., Rhizopus and Aspergillus). Further information regarding suitable fermentation organisms can be found in A.L. Demain, Biotech. Adv. 18:499-514 (2000).

The organisms ferment the fermentable material and in the process provide a fermentation broth which includes fermentation product. A variety of fermentation products may be formed depending on the organism and desired products. The fermentation product is a product produced in a fermentation process by the fermentation organism. The fermentation product may a volatile or non-volatile compound. Examples of fermentation products include, but are not limited to:

• organic acids (e.g., carboxylic acids such as lactic acid, citric acid, malic acid, gluconic acid, itaconic acid, tartaric acid, succinic acid, butyric acid, acetic acid, and propionic acid);

• solvents (e.g., ethanol, glycerol, butanol, and acetone

• enzymes, including hydrolytic and proteolytic enzymes (e.g., cellulases, hemicellulases, xylanases, proteases, peptidases, amylases, glycoamylases, phytases, phosphatases, or any combination thereof); • amino acids (e.g., either the L or D forms of aspartic acid, lysine, threonine, isoleucine, tryptophan, phenylalanine, and glutamic acid);

• nucleotides (e.g., 5 '-inosinic acid (5'-IMP) and 5'-guanylic acid (5 '-GMP));

• vitamins (e.g., Bi₂, C, riboflavin, and biotin);

• polysaccharides; • antibiotics (e.g., cephalosporins, penicillins, erythromycins, and tetracycline);

• hypocholesterolemic agents (e.g, statins);

• immunosuppressants (e.g., cyclosporin);

• plant growth enhancers (e.g., gibberellins);

• antioxidants and carotenoids (e.g. ascorbic acid, erythorbic acid, astaxanthin, zeaxanthin and lutein);

• high-intensity sweeteners;

• insecticides;

• bacteriocins; and

• antihelrnintics. For example, various mutants of Corynebacterium can overproduce lysine. Bacillus subtilis mutants may be producers of inosine and guanosine. Strains of S. marcescens can produce biotin. Aspergillus species may be used as a citric acid producer, while Lactobacillus, Rhizopus and Acetobacter species are known for producing lactic acid during fermentation. Rhizopus oryae, for example, produces a stereochemical^ pure L-(+)- lactic acid. Actinobacillus and Succinogenes species are known to produce other organic acids. Saccharomyces, Kluyveromyces, Candida, Clostridia, and Zymomonas are known for producing ethanol. Corynebacterium and Brevibacterium species produce amino acids. Other species and fermentation products will be apparent to those skilled in the art. The fermentation product commonly has a value in its pure form, but does not necessarily have to have its own market. Further, the fermentation product desirably has characteristics or activity which facilitates processing starch-containing material.

At least a portion of the fermentation broth may be recycled for use in processing the starch-containing material. Typically, this will involve recycling at least some amount of the fermentation product. For example, the recycled component may include at least a portion of the fermentation broth, the product-enriched stream, the product-depleted stream, or any combination thereof. Generally, the recycled component acts to facilitate the processing and will not have a deleterious effect on the method of processing a starch-containing material. Preferably, the recycled component includes fermentation product, but may also include at least one of the additional components. In general, the recycled stream will include more than a trace amount of fermentation product. Thus, the recycled stream may include at least 0.1% by mass of fermentation product as a percentage of the final mass in the fermentation broth. Desirably, the recycled stream will include at least 0.25% by mass, 0.5% by mass, 0.75% by mass, 1% by mass, 2% by mass, 5% by mass, or more of fermentation product as a percentage of the final product concentration in the fermentation broth.

The location selected for introducing the recycled component into the processing step will vary, depending upon factors such as the fermentation product, the location of maximum benefit or impact for recycling the fermentation product, and where the additional components present in the recycled stream may be useful in processing. The fermentation product may be used as a reagent in processing the starch-containing material. For example, the fermentation product may be recycled for use in the initial processing stage to facilitate steeping, tempering, or loosening the protein coating of starch granules in cereal material. The fermentation product may also be used in gelatinization, liquefaction, or any other operation that facilitates penetration of reagents or degradation of the starch-containing material into its component parts for forming fermentable material. A stream containing at least a portion of the fermentation product may be recycled to any stage of processing, including but not limited to, steeping, grinding, degerming, starch washing, hydrolyzation, liquefaction, and saccharification.

The recycled component may also be recycled to multiple processing steps or to streams formed during processing. For example, at least a portion of the recycled component may be recycled to a stream to produce fermentable material and at least a portion of the recycled component may also be recycled for use in a protein stream or germ stream. In addition, a portion of the recycled component may be recycled to one stage in processing, and another recycled component may be recycled to a different processing stage or stream. The fermentation broth may be treated prior to recycling. For example, the fermentation broth may be treated to remove non-fermentable compounds. In addition, any product-enriched stream or product-depleted stream which is recycled for use in processing may also be treated to remove non-fermentable compounds prior to recycling.

The recycling step may include one or more separations. Separation of the fermentation broth to form a product-enriched stream and a product depleted stream may be achieved by any suitable means. For example, separation may be carried out by extraction, distillation, filtration, reverse osmosis, precipitation, or the like. For example, the broth may be initially separated to provide a product-enriched stream of substantially pure fermentation product and a product depleted stream. Typically, the product depleted stream will also include unfermented material. The product-enriched stream can be removed from the process for specialized use, used for commercial sale if the fermentation product has independent value, or recycled for use in further processing of the starch-containing material. The remaining product depleted stream may often include fermentation product which has not been efficiently separated during the initial separation. The product depleted stream may then be further separated to provide a second product-enriched stream and a second product depleted stream. Again, this second product- enriched stream may be removed for specialized use, used for commercial sale, or recycled for use in processing cereal material. This second product depleted stream, like the first, may be recycled for use in processing the cereal material.

The product-enriched stream commonly has a greater ratio of fermentation product to impurities (e.g., ancillary components) than the product-depleted stream, and also a greater fraction of the fermentation product. This may be measured, for example, by mass %, weight percent per volume, or by material content. Conversely, the product-depleted stream generally has a higher ratio of impurities and a smaller fraction of the fermentation product.

Ih some cases, it may be desirable to separate the broth in a manner which provides less than a substantially pure fermentation product. If desired, the broth may be separated to provide a cruder product-enriched stream, which has a ratio of fermentation product to impurities higher than that in the product-depleted stream, but that also includes a significant proportion of additional components other than the fermentation product. Examples of additional components that may be present include non-fermented carbohydrates, peptides, water, organism cells, fermentation by-products, or other components.

Generally, the product-enriched stream has at least 50% of the fermentation product present in the solution from which the product-stream derived. More desirably, the product- enriched stream has at least about 60% of the fermentation product from the fermentation broth, and even more desirably at least about 85% of the fermentation product from the fermentation broth. In general, separation of substantially pure fermentation product from the fermentation broth becomes increasingly less efficient as the relative concentration of fermentation product within the fermentation broth decreases. As a result, a first product-enriched stream of fermentation product may be removed from the broth at relatively low cost per unit of product, leaving the product-depleted stream for either additional processing or for recycling within the system.

In one embodiment, fermentation product may be recycled for use in initial stage processing when the fermentation product is an organic acid. Examples of possible organic acids include carboxylic acids, lactic acid, citric acid, succinic acid, butyric acid, acetic acid, and propionic acid, and may be produced by an acid producing organism such as species of Lactobacillus, Aspergillus, Candida, Actinobacillus, Rhizopus and Acetobacter. A component of the broth containing the organic acid fermentation product may be recycled for use in steeping the starch-containing material to facilitate degradation of starch-containing material into its component parts. The organic acid is recycled together with additional components from the fermentation broth including water, which reduces the need to provide additional process water to the system. This acts to reduce the evaporation load of the overall process. This type of component recycling is beneficial because it facilitates processing, reduces costs associated with providing new materials and eliminating waste volume, reduces the cost of product recovery, decreases waste generation, and reduces costs related to waste treatment.

In other embodiments, the recycled component may be provided to other processing operations including starch and/or protein degradation. For example, the broth may include fermentation product in the form of enzymes such as phytase or amylase. These enzymes may be produced by genetically engineered microbes. The broth may also contain other enzymes as by-products of the fermentation process. The enzymes may have activity to degrade carbohydrates, phytic acid, and protein, to provide carbon, nitrogen, phosphates, and other valuable nutrients in a form that is most suitable for uptake by the organism and to permit active fermentation. The broth components may be recycled for use in hydrolization, saccharification, or liquefaction of the starch-containing material to provide the fermentable material for forming the fermentation product. When an enzyme product is recycled, it is the desired enzyme product of the fermentation that is considered in calculations and possible recycle locations. The myriad of natural enzymes already present in starch-containing materials or the trace enzyme byproducts produced by all fermentation organisms are not considered in calculating recycle rates or conversion percentages or capabilities.

Alternatively, the fermentation product may be recycled into a proteinaceous stream to provide available nutrients for a source of fermentation feedstock, and assist in the fermentation of fermentable material.

Other embodiments of the present process may include recycling multiple portions of the broth within the system. For example, a portion of the broth may be recycled for use in processing the starch-containing material and another portion of broth recycled for use in fermenting the fermentable material. In another example, at least a fraction of a product-enriched stream is recycled to one operation in processing (e.g., hydrolization) and at least a fraction of a product depleted stream is recycled to another operation of processing (e.g., steeping). There are a large number of possible locations for recycling of the fermentation product. ples only, possible recycling of fermentation products include:

• a lactic acid fermentation product might be recycled to a mill water stream for steeping, or directly into a steeping stream to improve corn material separation, improves sulfite absorption in steeping, or to prevent deleterious contamination;

• a lactic acid fermentation product might be recycled to an aqueous wet processing stream to inhibit contamination, improve sulfite chemistry, or to eventually enter steeping;

• an ethanol fermentation product might be recycled to an aqueous wet milling stream to alter separation characteristics of the mill streams;

• an ethanol fermentation product might be recycled to a heavy gluten stream to assist in the extraction of zein components;

• a protease fermentation product might be recycled to mill water to steep or directly to steeping to improve starch release; • a protease fermentation product might be recycled to an aqueous milling stream to improve starch release during milling;

• a protease fermentation product might be recycled to steeping to reduce subsequent steep water evaporator fouling or to change nutritional characteristics of the steepwater; • a cellulase fermentation product might be recycled to mill water or directly to steeping to improve starch release;

• a cellulase fermentation product might be recycled to an aqueous milling stream to improve starch release during milling;

• an amylase fermentation product might be recycled to processing streams containing primarily germ, fiber, or gluten to assist in destarching the stream;

• a phytase fermentation product might be recycled to steeping to digest phytate, reducing phytate scaling and increase free phosphorus; • a phytase fermentation product may be added to processing streams containing primarily germ, fiber, or gluten to lower the phytate content of the final product of those stream; and

• other combinations of possible recycling products and processing steps. Also provided is an integrated method for processing a cereal material. Desirably, cereal material is processed to a carbohydrate material such as dextrose, fermentation feedstock, fermentation product, germ and animal feed, through at least two operational stages.

The first operation includes providing and processing a cereal material. Typically, the cereal material will be steeped in a liquid to provide steeped cereal material and a steeping water stream. The steeped cereal material may then be substantially separated from the steeping water stream, and then may be further separated. For example, the separation may provide a germ stream, a fiber stream, a protein stream, a starch stream, or combinations thereof. Generally, a germ stream may be used for production of vegetable oil. A fiber stream, protein stream and at least a portion of the steep water may be used as ingredients for animal feed. A starch stream is generally directed to further processing, including liquefaction and saccharification to a carbohydrate material or high dextrose equivalent material by methods commonly known by those skilled in the art. The steep water may also be concentrated to form a steeping liquor stream, which may be combined with other streams. The cereal material is processed to form at least one fermentable material. In addition, non-fermentable material may be formed. The fermentable material and non-fermentable material may be separated, with the fermentable material directed to a fermentation stage for fermenting a fermentation product. Alternatively, both the fermentable material and non-fermentable material may be directed to the fermentation stage.

A second operation includes fermenting the fermentable material using a suitable organism to form a broth. The broth may then be separated to form a stream containing a volatile or non- volatile fermentation product and at least one product-depleted stream. The product-depleted stream may, but not necessarily, include an amount of fermentation product.

A third operation may include fermenting the fermentable material with a suitable organism, which may be the same or different organism of that in the second operation, to form a second broth, which may also be separated to provide a stream containing a fermentation product and at least one product-depleted stream.

The process may be integrated in that at least one stream formed in one of the operations is used in one of the other operations. For example, a fermentation product may be recycled for use in the steeping stage of the first operation. Alternatively, a fermentation product may be used in the processing of at least one stream produced by separating the cereal material. The fermentation product may include organic acids, enzymes, alcohol or other fermentation products capable of being used in one of the operations.

These processes may be integrated. For example, the fermentation product from the first operation may be directly conveyed via piping to the second operation. The fermentation product from the first operation may be immediately used in the second operation.

Thus, the method may be used in producing a fermentation product from a starch- containing material by processing the starch-containing material with the aid of the fermentation product from the first operation to form a fermentable material. In a second operation, the fermentable material may be fermented to form a broth including the fermentation product. The broth may then be separated to provide a product-enriched stream, which includes at least a portion of the fermentation product, and a product depleted stream which includes non- fermented carbohydrate material. At least a portion of the fermentation product may then be recycled to the first operation to aid in forming the fermentable material. The fermentation product may be recycled from the product-enriched stream or product depleted stream for use in forming the fermentable material. The fermentation product may be an organic acid, enzyme, vitamin, amino acid, nucleotide, solvent, or any other fermentation product known by those skilled in the art.

The starch-containing material may be processed by any number of methods known by those skilled in the art including dry-milling, wet-milling, and extrusion. Each of these methods has processing steps, the fermentation product may be recycled to aid in the processing of the cereal material at any step used in the processing method. Preferably, a wet-milling process will be used. Wet-milling traditionally includes at least one of a soaking stage, a starch separation stage, and a starch degradation stage, and therefore the cereal material may be contacted with the fermentation product at the soaking stage, the starch separation stage, the starch degradation stage, or any combination thereof. Additionally, the fermentation product may be solubilized in solution or concentrated before being used to contact the cereal material.

In other embodiments, the fermentable material may be provided to a first fermentation stream and a second fermentation stream. In the separate streams, the fermentable material may be fermented to provide broths with different fermentation products. For example, the fermentable material in the first fermentation stream may be fermented to provide a carboxylic acid. The fermentable material in the second fermentation stream may be fermented to provide an alcohol. Pre-processing might also be done as part of an integrated production path.

Another embodiment includes an integrated process including two processing trains. A first train includes a first starch-containing material which is processed and fermented to form a first fermentation product. A second train includes a second starch-containing material which is processed and fermented to form a second fermentation product. The first fermentation product is used in processing the second starch-containing material, while the second fermentation product is used in processing the first starch-containing material. The starch-containing material used in the two trains may be the same or different.

In other embodiments, the first fermentation product is used in processing the first and the second starch-containing materials. Alternatively, the second fermentation product is used in processing the first and the second starch-containing materials.

One common fermentation product is an alcohol, such as ethanol and butanol. Alcohol is common because of its wide commercial need as an industrial solvent and as a consumable (e.g., for use in the beverage industry). Alcohol may also be recycled for use in processing starch- containing material, such as cereals. As alcohol is produced from fermentable material, the broth may be de-watered by readily known methods including reverse-osmosis and distillation to form a product-enriched stream having a higher concentration of alcohol than the remaining product- depleted stream. The product-enriched stream may then be recycled for use in processing. Alternatively, the product-depleted stream may be de- watered if a portion of the alcohol was separated as a product-stream for other uses, and the product-depleted stream recycled for use in processing.

In one embodiment, at least a portion of the alcohol product-enriched stream may be used to extract protein from a stream produced from processing the starch-containing material. For example, in processing a cereal, the gluten overflow stream and steep water both contain many soluble and insoluble proteins, which may be separated from solution with the assistance of the alcohol fermentation product. The extracted protein-alcohol mixture may then be further processed to remove the alcohol from the protein to provide a highly concentrated protein fraction. Protein extraction may be effected by factors such as changing the water content of the solution, and changing the solution pH. For example, a base (e.g., NaOH, Ca(OH)₂, KOH, and NH₄OH) can be added to increase solubility of protein in a solution. The protein fraction may then be used as a nutritional supplement for fermentation (e.g., as fermentation feedstock) or for use in alternative products such as animal feed. The product-depleted fraction may also be recycled for use in processing to conserve nutrients and water within the system, or a portion may also be used to add nutritional value to alternative products such as animal feed.

The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.

EXAMPLES

Concentrations of the constituents of the fermentation media and fermentation broth, in the examples, were determined by high pressure liquid chromatography (HPLC). Ih carrying out the HPLC, the following equipment and conditions were employed: a Waters HPLC with a Biorad HPX-87H Organic Acid column were utilized; the mobile phase for the column was 0.01 N sulfuric acid with 0.00001% sodium azide, and the flow rate was 0.5ml/min; detection was achieved by refractive index (RI); and quantitation was performed using Millennium software, comparing peak areas in the sample against peak areas of known solutions.

Example 1. Corn Wet Milling Corn kernels are cleaned using a series of perforated screens of a size suitable to retain the corn and to allow removal of dust and debris. Cleaned corn is steeped in an aqueous solution originating from process water used in the mill containing 1800 ppm of SO2, at 49°C (120°F) for 30 hours in a 10 tank steep battery connected in series with a counter-current flow of the aqueous solution to the age of the steeping corn, with the aqueous solution first contacting the corn having the longest residence time in the battery. Approximately, 1.2 rn³ of the aqueous solution is used per metric ton of corn (8 gallons of aqueous solution/bushel of corn) being steeped. After 30 hours of steeping, the corn and the aqueous solution, now enriched in corn solubles and metabolic products of naturally occurring lactic acid fermentation in the steep battery, are recovered as the steeped corn and light steep water product of steeping, respectively.

Then, the steeped corn product is ground in the presence of mill process water. Grinding of the steeped corn is performed in three stages. The first stage ("first grind") releases most of the germ from the steeped corn using a 91 cm (36 inch) grind mill fitted with Devil's toothed plates operating at 900 rpm. The slurry discharge from the first grind mill is pressure feed at approximately is 6.2 bars (90 psi) through a two-pass hydrocyclone battery consisting of 15.24 cm (6 inch) hydrocyclones to separate the germ. The separated germ is washed with mill process water and dried in a rotary drum drier to yield a dried germ product.

The remaining slurry from which most germ has been separated is milled again, coarsely ground using a second 91 cm (36 inch) grind mill (" second grind") fitted with Devil's toothed plates operating at 900 rpm to detach remaining germ from ground corn in the slurry. Freed germ present in the second grind discharge slurry is separated and recovered using hydrocyclones as described above.

After the removal of germ, the remaining corn material is passed over 50 micron screen ("third grind dewatering screen"). The filtrate containing starch-protein moves forward, while the corn material retained as overs by the screen is fine ground using a 36 inch grind mill ("third grind") fitted with Devil's toothed plates operating at 1800 rpm. The fiber component in the slurry of the third grind discharge is removed by a 7 stage screen separation system arranged such that the fiber is washed in a counter current flow of fiber to mill process water, where the cleanest fiber is washed with the mill process water added to the screen system. Washed fiber is discharged at the seventh and last stage, while starch and protein containing slurry is discharged at the first stage. The screen opening on the first fiber wash stage is 50 micron, followed by 75 micron on the second through sixth stage and 150 micron of the last stage. The washed fiber is dewatered using screw presses, and dried using a rotary drier, resulting in the dried fiber product. The discharge from the third grind dewatering screen and first stage fiber wash are combined, creating a slurry with a density of approximately 8 Baume. This slurry is thickened with a Merco H36 centrifuge. This centrifuge operates at 2600 rpm and is fitted with No. 24 size nozzle. The overflow from the centrifuge is used as process water for steeping (also known as mill water), while the underflow slurry, having a Baume of 12, is fed to a second H36 centrifuge (referred to as primary centrifuge). The starch-protein in the fed slurry is separated by the primary centrifuge. The primary centrifuge operates at 2200 rpm and is fitted with No. 24 nozzle to yield an underflow and overflow slurry.

The overflow slurry is protein-enriched, containing approximately 60% (db) protein, while the underflow slurry is starch enriched. The protein enriched overflow slurry from this centrifugation is then further dewatered by centrifugation with a third Merco H36 centrifuge operating at 2600 rpm, dewatered on a rotary drum filter and dried using a flash drier. This results in the dried protein rich product, also known as corn gluten meal. The starch enriched slurry originating from the underflow of the second Merco H36 centrifuge described above is passed through a 12 stage Dorr-Oliver clam shell hydrocyclone starch wash battery. The starch wash battery is designed such that a counter-current flow between the starch enriched stream entering the first stage of the battery and potable water entering at the twelfth stage of the battery is achieved. Each stage starch wash stage has several 10 mm hydroclones arranged in parallel fashion. Typical feed pressure to each starch wash stage, except the twelfth stage, is 6.2 bar (90 psi); the feed pressure on the twelfth stage is 8.27 (120 psi). Purified starch with a slurry density of 23 Baume is recovered as underflow from the twelfth stage of the starch wash battery, also known as starch slurry or starch product of corn wet milling.

Further information regarding the wet milling of corn is found in Technology of Corn Wet Milling and Associated Processes p. 69-125, Paul H. Blanchard, Elsevier Science Publishers B. V. Amsterdam.

Example 2. Wheat Wet Processing

Wheat kernels are cleaned using a series of perforated screens of a size suitable to retain the wheat and to allow removal of dust and debris. The cleaned wheat is passed through a series of breaking and sifting steps, breaking accomplished with break roller mills with grinding rolls that are corrugated followed by sifting and repeated as necessary. The product is then introduced into a purifier to separate the wheat material into a bran stream, a purified endosperm stream, and a stream of particles that are a composite of bran and endosperm. The various streams are then separated for further processing.

The purified endosperm stream is size reduced with a roller mill with smooth rolls and sifted to produce wheat flour. The flour is delivered to a water jacked continuous dough mixer by a vibrating single deck feeder, temperature of the continuous dough mixer is maintained at 25°C, and water is delivered. The resulting dough is fed directly to a 2 stage washing unit. In the first stage drum washer, wash water and dilute starch slurry from the second stage washer are combined with the dough and flow concurrently through the drum of the washer, to form an insoluble gluten mass and a aqueous slurry of starch. The gluten from the first stage drum washer is separated from the starch slurry using a 200 micron stainless steel vibratory screen. The resulting starch slurry contains 5.5 Baume starch. The separated gluten is then passed through a second stage drum washer in combination with fresh water to further purify the gluten from the entrained starch. The gluten from the second stage drum washer is separated from the dilute starch slurry using a 200 micron stainless steel vibratory screen, the dilute starch slurry is then recycled to the first stage drum washer to wash the dough. The wet gluten is then pumped to a gluten dryer to form the gluten product and the starch slurry is recovered.

The starch slurry resulting from the wet processing of wheat may be passed through a 90 micron Dorr-Oliver DSM sieve. This sieve retains impurities and passes the desired starch slurry, and processing includes washing the material retained on the screens with water sprays to free entrained starch. The purified wheat starch slurry is recovered.

The purified wheat starch slurry maybe passed through a Merco nozzle bowl disc-type centrifuge modified to allow for introduction of fresh wash water into the bowl. The introduction of fresh water acts to displace dissolved solids, B starches, and pentosans. Awash water ratio of 0.1 part wash water to 1 part purified wheat starch slurry is desirable, and concentrates the starch to 20 Baumέ. The concentrated, purified wheat starch slurry is then recovered.

Further information regarding the wet processing of wheat may be found in Starch Production Technology, p. 161-176, J. A. Radley, ed., 1976. Example 3. Starch Hydrolysis a. Starch hydrolysis by acid hydrolysis:

A starch slurry with a 23 Baume is provided. The pH of the slurry is then adjusted to 1.8 using 22 Baume hydrochloric acid. The pH 1.8 slurry is then introduced into a Dedert continuous acid conversion system (Olympia Fields, Illinois, USA) at 146°C ( 295⁰F) for 18 minutes. After the treatment in the conversion system, the starch is hydrolyzed to 85 dextrose equivalents (DE). The pH of the converted starch is then adjusted to pH 4.8 using 10% soda ash and cooled. b. Starch hydrolysis by enzyme/enzyme hydrolysis:

Enzyme hydrolysis of starch is performed by liquefaction and saccharification. The liquefaction step is conducted by adding water to the starch to adjust the dry solid content of the mixture to 35%. The pH of slurry is then adjusted to pH 5.5 using sodium hydroxide solution. Calcium chloride is added to the slurry to reach a minimum of at least 5 ppm free calcium. TERMAMYL SUPRA® enzyme, (an amylase available from Novozymes North America, Inc) is added to the slurry in the amount of 0.4 liter per metric ton of starch dry solids. The mixture is then heated in a continuous jet cooker to 108⁰C (226.4°F) and held for 5 minutes in a pressurized vessel. After heating, the cooked mixture is cooled to 95⁰C (203⁰F) and held for 100 minutes. A starch hydrolyzate with a DE of 8 to 12 is produced. The saccharification step is performed by obtaining the starch hydrolyzate from the liquefaction step and cooling the hydrolysate to 60⁰C. In addition, the dry solid content is adjusted to 32 % by adding water. The pH of this diluted hydrolyzate is adjusted to ph 4.1 - 4.3 using sulfuric acid. DEXTROZYME E® enzyme (a mixture of amyloglucosidase and pullunase, available from Novozymes North America, Inc) is added in the amount of 0.7 liters per metric ton of dry solids. The mixture is then held for 40 hours. After 40 hours, a dextrose content of 95-97%, on a dry solid basis, is achieved.

Further information regarding starch hydrolysis is found in Technology of Corn Wet Milling and Associated Processes p. 217-266, Paul H. Blanchard, Elsevier Science Publishers B. V. Amsterdam. Example 4. Recycling acid fermentation product to steeping

A IL batch fermentation was carried out in a water jacketed glass vessel with an impeller using as the fermentation organism a lactic acid producing strain of Lactobacillus sp. isolated from the steeping phase of the corn wet milling process. The fermentation media contained 3.04% dry basis light steep water solids obtained from a corn wet milling process, lOg/L yeast extract, 80g/L dextrose (a hydrolyzed corn starch), 2g/L K₂HPO₄, 2g/L ammonium citrate, 0.2g/L MgSO₄, 0.05g/L MnSO₄, and 33g/L CaCO₃. Fermentation was carried out at 48⁰C with agitation. After 46 hours the fermentation broth was drained from the fermenter and analyzed. The resulting fermentation broth contained 72.7 g/L lactic acid, and had a pH of 4.21.

A 400ml portion of the fermentation broth was separated, brought to pH 6.46 using dry calcium hydroxide, and then concentrated by rotoevaporation at 60⁰C until a 4x concentrate was achieved. The concentrate at 60⁰C was then centrifuged to remove precipitate in the sample. The sample was then cooled to room temperature with 2 subsequent centrifugations removing the crystallized calcium lactate fermentation product from the fermentation broth. A total of 24.64g of lactate was precipitated from the broth, leaving 4.44g of lactic acid in the residual broth which was then diluted to 50ml. The precipitated fermentation broth was then acidified to pH 3.2 with concentrated sulfuric acid and the resulting precipitate removed by centrifugation. Two laboratory scale batch steeps were set up in 250ml glass jars with lids placed in a water bath to control temperature, containing lOOg of corn and 150ml of steep water of the following compositions:

Control steep: 150ml of mill water obtained from a conventional corn wet mill was laced with sodium bisulfite to achieve O.llg SO₂ in the mill water/100g corn steeped. Lactic acid steep: 125ml of mill water was combined with 25ml of the precipitated acidified fermentation broth to deliver 15g/L lactic acid, and sodium bisulfite was added to achieve O.llg SO₂ in the mill water + precipitated acidified fermentation broth/10Og corn steeped.

After 24 hours of steeping at 48°C, the control steep was inoculated with the steep bacteria harvested from 100ml of freshly sampled light steep water from a conventional corn wet milling process. This was done to allow for natural production of lactic acid from the steep solids leeching from the corn kernels, as occurs in the industrial corn steeping process. This provided a better control to compare the lactic acid steep sample against, as lactic acid was introduced in the lactic acid steep without requiring natural production. After the addition, both the control steep and the lactic acid steep were steeped for a further 24 hours. At 48 hours the steep water from both of the steeps was drawn and analyzed by

HPLC. The steeps had the composition as shown in Table 1.

Both the steep waters resulting from the control steep and the lactic acid steep can be used as the steep solids component of the fermentation broth of the initial lactic acid fermentation. However, the lactic acid steep water has higher concentrations of dextrose and fructose. The desirable effect of having higher concentrations of dextrose and fructose in the steep water is that it reduces the amount of supplemental dextrose required in the fermentation media.

Example s.

The procedure of Example 4 was followed with the modification that the lactic acid steeping was conducted at 56⁰C. The steep water product from this steep has the composition shown in Table 1.

Table 1 : Composition of steep water products from laboratory steeps at 48⁰C g/L in the sample Steep Dextrose Fructose Lactic acid

Control steep 0.08 0.52 17.88

Ex. 4 Lactic acid steep 11.39 7.67 19.44

Ex. 5 Lactic acid steep 11.04 8.77 18.96

Example 6.

The process of Example 1 is followed with some modifications. A protease enzyme is produced by a protease producing Bacillus sp. in a fermentation media comprising hydrolyzed starch derived from the corn wet milling process of Example 1. However, the process of Example 1 is also modified, in that the fermentation broth, after separation of a portion of the protease product, is introduced to the coarse grind step of corn wet milling.

Example 7.

The process of Example 1 is followed with some modifications. A cellulase enzyme is produced by a cellulase producing strain of Thrichoderma sp. in a fermentation media comprising hydrolyzed starch derived from the corn wet milling process of Example 1. However, the process of Example 1 is also modified, in that the fermentation broth, after separation of a portion of the cellulase product, is introduced to the coarse grind step of corn wet milling.

Example 8.

The process of Example 2 is followed with some modifications. A cellulase enzyme is produced by a cellulase producing strain of Thrichoderma sp. in a fermentation media comprising hydrolyzed starch derived from the wheat wet milling process of Example 2. However, the process of Example 1 is also modified, in that the fermentation broth, after separation of a portion of the cellulase fermentation product, is introduced to the first stage drum washer of the process for wet processing of wheat.

Example 9.

A corn processing process including recycling of acid fermentation product to the soaking stage of corn is illustrated schematically in the flow chart included as FIG 1, and is accomplished by the following: After corn kernels are soaked 8-24 hours in a lactic acid/SO₂ solution (as provided below), the corn is milled using conventional wet milling techniques. Corn starch purified by this process is converted to dextrose by means typical to the industry and incorporated into a fermentation media. The fermentation media contains 24-36% soak water from the com acid/Sθ2 corn soaking step, 5g/L yeast extract, 50-100g/L dextrose, 2g/LK.₂HPO₄, lg/L Tween80, 2g/L ammonium citrate, 0.2g/L MgSCM, 0.05g/L MnSO₄, and 20-40g/L CaCO₃. This fermentation media is then fermented using a Lactobacillus sp. lactic acid producing microorganism. The lactic acid fermentation product produced by the Lactobacillus sp. is preferably enriched for one isomer of lactic acid, and the fermentation product is ideally 90% or greater L(+) or D(-) lactic acid. The fermentation is allowed to continue for up to 48 hours, achieving at least about 75g/L lactic acid at a pH of no more than 4.5 in the fermentation broth. The lactic acid is then precipitated as a calcium lactate salt with calcium hydroxide, with a resulting pH of at about 6.0 in the fermentation broth. The resulting calcium lactate precipitate is separated from the fermentation broth. The calcium lactate may then be purified and used to produce lactic acid. The broth remaining after precipitation, including about 8-10g/L lactic acid

(approximately 10% of the lactic acid of the original fermentation broth), is then acidified with a mineral acid, such as sulfuric acid and/or sulfurous acid, to an appropriate pH for corn soaking, such as about 3.0-4.5 pH. Any resulting calcium sulfate is then removed by filtration. The clarified, acidified, precipitated, product-depleted, aqueous solution is then made up to about 1000-3000 ppm sulfur dioxide through addition of either gaseous SO₂ or BSS (bisodium disulfite, also known as sodium bisulfite). The depleted aqueous solution is then used to soak fresh corn. This soaking replaces the traditional steeping phase of the corn wet milling process, in which lactic acid producing bacteria ferment free sugars leeching from the corn kernels into roughly a racemic mixture of lactic acid. The corn is soaked in this aqueous solution from 8 to 24 hours. The resulting soak water includes water soluble nutrients that have leeched/perfused out of the corn. The soak water is added to the fermentation as a nitrogen and nutrient source, in the proportions described above.

Example 10 Another corn processing process follows the same process as Example 9, with a few changes. First, the corn is pretreated with gaseous sulfur dioxide before soaking. Second, sulfurous acid/SO₂ is not added to the precipitated, acidified fermentation broth during recycling. Thus, the pretreated corn is soaked in the acidified, filtered, product-depleted stream for up to 12 hours. As in Example 9, because the typical lactic acid fermentation of steeping has been replaced, the lactic acid in the soak water is enriched for the isomer of lactic acid prevalent in the downstream lactic acid fermentation. In addition, the required soak time for the corn kernels is greatly reduced through the combination of gaseous SO₂ pretreatment application, and soaking in an aqueous solution including lactic acid, as there is no delay caused by waiting for lactic acid production.

Example 11

Another corn processing process follows the same process as Example 9, with a few changes. In this process, the steeping temperature is maintained at or above 55°C, inhibiting the Lactobacillus sp. typically present in steeping. In addition, lactic acid production during the soaking stage is further reduced due to the use of the elevated temperature during steeping.

Example 12 A corn processing process including recycling of protease enzyme to an upstream stage to facilitate starch separation is illustrated schematically in the flow chart included as FIG. 2, and is accomplished by the following:

Corn is steeped and milled as typical to the wet milling industry. Corn starch is purified as known to those in the industry and is converted to dextrose and incorporated into the fermentation media. The media (derived from Adinarayana, et. al., AAPS PharmSciTech 2003; 4(4) Article 56) contains 5.0g/L dextrose, 7.5g/L peptone, and 5% vol/vol of a salt solution containing 0.5% MgSO₄*7H₂O (wt/vol), 0.05% KH₂PO₄, 0.5% wt/vol, and 0.01% FeSO₄*7H₂O wt/vol. The media is then inoculated with a strain of Bacillus subtilis, which secretes protease. Once fermentation is complete, the protease is purified by means typical to the industry, generating a partially purified or purified protease product-enriched stream and a spent fermentation broth stream which contains residual protease activity, but less protease activity than the purified stream. The spent fermentation broth with residual protease activity is recycled to the steeping phase. Starch-gluten separation is facilitated by the protease such that SO₂ addition to steeping would be reduced to 500ppm or less in mill water vs. 2000ppm (0.2%) typical to the industry. Example 13

A corn processing process including recycling of protease enzyme is accomplished according to the process described in Example 12, expect the protease enzyme is recycled to a post-grind step in the mill, where starch-gluten complexes are more readily accessible to protease action. In this process, starch-gluten separation would also be facilitated by the protease such that SO₂ addition to steeping would be reduced to 500ppm or less in mill water vs. 2000ppm (0.2%) typical to the industry.

Example 14

A corn processing process including recycling of protease enzyme is accomplished according to the process described in Example 12, except the process is modified such that corn is ground prior to a soaking step (in place of steeping of whole kernels of corn), and protease is recycled into this soaking step to facilitate breakdown of starch-gluten complexes to liberate starch. In this process also, starch-gluten separation would be facilitated by the protease such that SO₂ addition to steeping would be reduced to 500ρpm or less in mill water vs. 2000ppm (0.2%) typical to the industry.

Example 15 A corn processing process including recycling of protease enzyme is accomplished according to the process described in Example 12, except that corn insoluble protein is purified from a wet milling process by means typical to the industry. The corn insoluble protein is then incorporated into the fermentation media, substituting in part or completely for the peptone.

Example 16

A corn processing process including recycling cellulase enzyme to an upstream stage to facilitate starch separation and reduce steep time is accomplished by the following:

Corn is steeped and milled using conventional wet milling techniques. Corn starch purified by this process is converted to dextrose by means typical to the industry and incorporated into cellulase growth and production media. A growth media as shown in Table 2 (described in Turker & Mavituna, Enzyme and Microb. Technol. 9:739-742), and having pH 4.8 is used in fermentation. The growth media is inoculated with Thrichoderma sp. and incubated at 30⁰C until reaching late long/early stationary phase, as determined by cell mass, then inoculated into production media (also described in Turker & Mavituna, Enzyme and Microb. Technol. 9:739-742), as shown in Table 3, and having pH 4.8. This mixture is then incubated for 5 days at 30⁰C. Cellulases are purified from the fermentation broth as typical to the industry, generating a partially purified or purified cellulase product-enriched stream and a spent fermentation broth stream which contains residual cellulase activity, but less cellulase activity than the purified stream. The spent fermentation broth with residual cellulase activity is recycled into the first grind tank after corn from steeping is course ground. The activity of the cellulase reduces starch in fiber.

Table 2. Trichoderma Growth Media

Dextrose, 25g/L Manganese sulfate 10.3mg/L Ammonium sulfate 7g/L Iron sulfate 25mg/L

Potassium phosphate monobasic lOg/L Urea, 1.5g/L

Magnesium sulfate, 1.5g/L Zinc sulfate, 7mg/L

Calcium chloride, 2g/L Cobalt chloride, 18.3mg/L

Table 3. Trichoderma Production Media

Dextrose, lg/L Cobalt chloride, 18.3mg/L

Cellulose, 20g/L Zinc sulfate, 7mg/L

Ammonium sulfate 7g/L Manganese sulfate 10.3mg/L

Potassium phosphate monobasic lOg/L Urea, 1.5g/L Magnesium sulfate, 1.5 g/L Iron sulfate 25mg/L

Calcium chloride, 2g/L Example 17

A corn processing process including recycling of celhilase enzyme is accomplished according to the process described in Example 16, except the spent fermentation broth with residual cellulase activity is recycled to the steeping phase of the corn wet milling process rather than recycling into the first grind tank.

Example 18

A first grain processing train includes corn provided to a conventional corn wet milling process to produce starch, fiber, germ, and gluten co-product streams, along with corn steep water. The corn steep water is further concentrated about 4-fold to corn steep liquor by evaporation. The starch is liquefied and saccarifϊed to form a dextrose solution. The dextrose solution is combined with the corn steep liquor product of corn wet milling in forming a first fermentation growth media. The first fermentation growth media is adapted from Chan et al., Applied Biochem. and Biotech. 45/46: 531-544, and has the composition as shown in Table 4.

Table 4. Yeast Growth Media

Cora steep liquor, 2% Dextrose, 7%

The first growth media is inoculated with Saccharomyces cerevisiae and incubated at

30⁰C in a shaker with agitation until reaching late long/early stationary phase, as determined by optical density, then inoculated into a production media. The production media is adapted from Chan et al., Applied Biochem. and Biotech. 45/46: 531-544, and has the composition as shown in Table 5. After inoculation, the mixture is incubated for 2 days at 30⁰C. The dextrose in the first fermentation media is fermented by the yeast to yield a first fermentation broth containing ethanol. Ethanol is then distilled from the first fermentation broth, to yield a product reduced broth and an ethanol rich product-enriched stream. Table 5. Yeast Production Media

Corn steep liquor, 1% Dextrose, 18%

A second grain processing train includes wheat provided to a wheat wet processing process to produce starch and gluten streams. The starch is liquefied and saccarified to form a dextrose solution. The dextrose solution is combined with corn steep liquor from the first train in forming a second fermentation media. The second fermentation growth media is adapted from Chan et al., Applied Biochem. and Biotech. 45/46: 531-544, and has the composition as shown in Table 6. The second fermentation media is inoculated with a Lactobacillus species, and the pH maintained constant at pH 6.0 with the automatic addition of calcium hydroxide solution. Fermentation is carried out at 48°C with agitation for 2 days.

Table 6. Growth Media

3.04% dry basis corn steep liquor 2g/L ammonium citrate

1 Og/L yeast extract 0.2g/L MgSO4

80g/L dextrose 0.5g/L MnSO4

2g/L K2HPO4

The second fermentation broth is then separated into a product-reduced broth and a calcium lactate product-enriched stream as described in Example 1. The produce reduced broth is then acidified with sulfuric acid to pH 3.2, the resulting precipitate removed by centrifugation, and the broth introduced into the steeping phase of the corn wet milling process of the first grain processing train.

Example 19

A first grain processing train includes wheat provided to a wheat wet processing process to yield a starch stream and a gluten stream. The starch is then liquefied and saccarified to form a dextrose solution. A second grain processing train includes corn provided to a corn wet- milling process to produce a starch, fiber, germ, and gluten co-product streams stream along with corn steep. La part, the corn steep is further concentrated about 4-fold to corn steep liquor by evaporation. The dextrose solution from the first train is combined with the corn steep liquor formed in the second grain processing train to form a first fermentation growth media. The combined first media has the composition as shown on Table 6. The first media is inoculated with a Lactobacillus species, and the pH maintained constant at pH 6.0 with the automatic addition of calcium hydroxide solution. Fermentation is carried out at 48°C with agitation for 2 days. The combined first fermentation broth is then separated into a product-reduced broth and a calcium lactate product-enriched stream as described in Example 4. The product reduced broth is then acidified with sulfuric acid to pH 3.2, the resulting precipitate removed by centrifugation, and the remaining broth introduced into the steeping phase of the corn wet milling process of the second grain processing train. The starch stream from the second train is liquefied and saccarified to form a dextrose solution, which is combined with the steep water product of corn wet milling to form a second fermentation growth media. The second media has the composition as shown in Table 2. The second growth media is inoculated with Thrichoderma sp. and incubated at 3O⁰C until reaching late long/early stationary phase as determined by cell mass, and then inoculated into production media having the composition as shown in Table 3. The mixture is incubated for 5 days at 30⁰C, while being maintained at pH 4.8 by base addition (sodium hydroxide, 20% w/w, available from Sigma Chemical).

Cellulases are purified from the second fermentation broth as typical to the industry, generating a partially purified or purified cellulase product-enriched stream and a spent fermentation broth stream which contains residual cellulase activity, but less cellulase activity than the purified stream. The spent fermentation broth with residual cellulase activity is recycled into the hydration process of wheat wet milling, where the activity of the cellulase reduces intact fiber. Example 20

A corn processing process including recycling of cellulase enzyme is accomplished according to the process described in Example 16, where additionally cellulose is purified from a fiber containing stream from the corn wet milling process and incorporated into the production media. The process is illustrated schematically in the flow chart included as FIG. 3.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for treating a starch-containing material, comprising: wet processing the starch-containing material to produce a fermentable material; fermenting at least a portion of the fermentable material using a fermentation organism to produce a fermentation broth comprising a fermentation product; and using at least a portion of the fermentation broth in wet processing the starch-containing material.

2. The method of claim 1 , wherein wet processing comprises wet milling.

3. The method of claim 1 , wherein the fermentable material comprises starch.

4. The method of claim 3, wherein the fermentable material comprises hydrolyzed starch.

5. The method of claim 1 , further comprising separating at least a portion of the fermentation product from the fermentation broth to form a product-enriched stream and a product-depleted stream.

6. The method of claim 5, wherein using at least a portion of the fermentation broth comprises introducing into the wet processing at least a portion of the fermentation broth, the product-enriched stream, the product-depleted stream, or mixtures thereof.

7. The method of claim 1, wherein the starch-containing material comprises cereal material.

8. The method of claim 7, wherein the cereal material comprises corn, wheat, sorghum, or parts thereof.

9. The method of claim 7, wherein wet-processing the starch-containing material comprises: grinding the cereal material; and at least one of a soaking stage, a starch separation stage, and a starch hydrolysis stage.

10. The method of claim 9, further comprising contacting the cereal material with solubilized fermentation product to form the fermentable material.

11. The method of claim 10, wherein contacting the cereal material with solubilized fermentation product occurs at a soaking stage, at a starch separation stage, or at a starch degradation stage.

12. The method of claim 1, wherein using at least a portion of the fermentation broth comprises recycling at least a portion of the fermentation broth to a hydrolyzation stage.

13. The method of claim 1, wherein the fermentation product comprises a carboxylic acid, an enzyme, an alcohol, or a combination thereof.

14. The method of claim 13, wherein the fermentation product comprises lactic acid.

15. The method of claim 13, wherein the fermentation product comprises a protease, amylase, phytase, peptidase, phosphatase, cellulase, or hemicellulase.

16. The method of claim 13, wherein the fermentation product comprises ethanol.

17. The method of claim 1, wherein the fermentation organism comprises an anaerobic or aerobic prokaryote or eukaryote.

18. The method of claim 1, wherein the fermentation organism comprises Lactobacillus, Acetobacter, Acetobacterium, Propionibacterium, Clostridia, Streptococcus, Bacillus, Corynebacterium, Brevibacteήum, Psuedomonas, Serratia, Zymomonas, Streptomyces, Actinomycetes, Micromonospora, Actinobacillus, Saccharonmyces, Kluyveromyces, Candida, Eremohecium, Ashbya, Rhizopus, or Aspergillus.

19. The process of claim 1, wherein using at least a portion of the fermentation product comprises using a stream having at least 0.1% by mass of fermentation product as a percentage of the final mass in the fermentation broth.

20. The process of claim 19, wherein using at least a portion of the fermentation product comprises using a stream having at least 1% by mass of fermentation product as a percentage of the final mass in the fermentation broth.

21. The method of claim 1 , further comprising: separating the fermentation broth into a product-enriched stream and product-depleted stream, wherein using at least a portion of the fermentation product comprises: recycling at least a portion of the product-enriched stream into wet processing the starch-containing material; and recycling at least a portion of the product-depleted stream into wet processing the starch-containing material.

22. An integrated process for treating starch-containing material, comprising: processing a first starch-containing material to produce a first stream comprising a first fermentable material; fermenting the first fermentable material using a first fermentation organism to produce a first fermentation broth comprising a first fermentation product; processing a second starch-containing material to produce a second stream comprising a second fermentable material; and introducing at least a portion of the first fermentation broth into processing the second starch-containing material, wherein the process of producing a first stream is integrated with a process for producing a second stream.

23. The process of claim 22, further comprising fermenting the second fermentable material using a second fermentation organism to produce a second fermentation broth comprising a second fermentation product.

24. The process of claim 22, wherein introducing at least a portion of the first fermentation broth comprises introducing at least a portion of the first fermentation product into processing a second cereal material.

25. The process of claim 22, further comprising introducing at least a portion of the second fermentation broth into processing a first starch-containing material.

26. The process of claim 25, wherein introducing at least a portion of the second fermentation broth comprises introducing at least a portion of the second fermentation product into processing a first starch-containing material.

27. The process of claim 22, wherein processing a first starch-containing material comprises wet processing.

28. The process of claim 22, wherein processing a second starch-containing material comprises wet processing.

29. The process of claim 22, wherein the first starch-containing material and the second starch-containing material each comprise a cereal material.

30. The process of claim 29, wherein the first starch-containing material and the second starch-containing material each comprise the same cereal material.

31. A process for treating cereal material, comprising: wet processing a first cereal material to produce a first stream comprising a first starch; fermenting the first starch using a first fermentation organism to produce a first fermentation broth comprising a first fermentation product; wet processing a second cereal material to produce a second stream comprising a second starch; 5 fermenting the second starch using a second fermentation organism to produce a second fermentation broth comprising a second fermentation product; introducing at least a portion of the first fermentation broth into wet processing the second cereal material; and introducing at least a portion of the second fermentation broth into wet processing the o first cereal material.

32. The process of claim 31, wherein wet processing a first cereal material is integrated with wet processing a second cereal material.

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