WO2009129320A2 - Protein concentrate from starch containing grains: composition, method of making, and uses thereof - Google Patents
Protein concentrate from starch containing grains: composition, method of making, and uses thereof Download PDFInfo
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- WO2009129320A2 WO2009129320A2 PCT/US2009/040687 US2009040687W WO2009129320A2 WO 2009129320 A2 WO2009129320 A2 WO 2009129320A2 US 2009040687 W US2009040687 W US 2009040687W WO 2009129320 A2 WO2009129320 A2 WO 2009129320A2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
- Y02A40/818—Alternative feeds for fish, e.g. in aquacultures
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Definitions
- Embodiments of the invention relate to processes for producing a protein concentrate from a starch containing grain or oil seed and uses thereof.
- the protein concentrate produced using the disclosed processes comprises a high quality, highly digestible protein that is suitable for use as an aquaculture feed.
- Protein is an essential component of the diet of all domestic animals and is necessary for growth, body maintenance, the production of young and the output of meat and non-meat products e.g., milk, eggs and wool. Thus, for productive animal agriculture, protein is essential.
- Aquaculture is the fastest growing food production sector in the world. Thus, addressing the needs of aquaculture practices promises to improve food and nutrition for the future of civilization. The biggest need in aquaculture is to provide feeds which supply sufficient high quality protein in a palatable form.
- Aquaculture feeds typically comprise fishmeal as a source of protein. Unfortunately however, in recent years, the cost of fishmeal has increased, concerns have been raised about the residues of toxic pollutants e.g., dioxin, in fishmeal, regulation of nutrients in hatchery effluents has intensified, and questions regarding the sustainability of fishmeal as an aquafeed ingredient have arisen.
- toxic pollutants e.g., dioxin
- soy contains anti-nutritional compounds e.g., phytate, and/or undigestable carbohydrates e.g., oligosaccharides, and these anti-nutritional compounds limit the use of soy derived proteins in aquaculture feeds. This is a particular issue with feeds for species such as trout and salmon ⁇ see e.g., Knudsen, D., et al. (2007) Journal of Agriculture and Food Chemistry 55: 2261-2267; Knudsen, D., et al. (2006). Journal of Agriculture and Food Chemistry 54:6428-6435.
- the invention provides a process for producing a protein concentrate from a starch containing grain or oil seed, wherein the method comprises: dehulling the grain or using a hulless variety, grinding the starch containing grain to produce a ground starch containing grain; slurrying the ground starch containing grain with water; solublizing starch and glucans with enzymes; adding a fermentation organism to the slurry; fermenting the slurry comprising the fermentation organism until fermentation is complete, thereby producing a fermented slurry; separating the fermented slurry into solid and liquid fractions; recovering the solid and liquid fractions; drying the recovered solid fraction at a temperature below that which would denature or damage proteins; thereby producing a protein concentrate.
- solublizing the starch and glucans with enzymes produces glucose.
- the process is a no-cook process wherein raw ungelatinized granular starch is hydrolyzed to glucose.
- the process is a cooking process wherein a ground starch slurry is heated to a temperature which gelatinizes the starch granules.
- the process further comprises: distilling the recovered liquid fraction to recover a fermentation product.
- the fermentation product is ethanol.
- the starch containing grain is a member selected from the group consisting of barley, wheat, oats, corn, rye, tritcale, and sorghum, or a combination thereof.
- the starch containing grain is a starch containing oil seed e.g., soybeans, flax, camelina.
- the starch containing grain is a processed grain e.g., an oilseed meal e.g., soybean meal.
- the starch containing grain is barley.
- the barley is hulless.
- the barley is mechanically de- hulled.
- the invention provides a protein concentrate made according to a process for producing a protein concentrate from a starch containing grain, wherein the process comprises: grinding the starch containing grain to produce a ground starch containing grain; slurrying the ground starch containing grain with water; solublizing starch and glucans with enzymes; adding a fermentation organism to the slurry; fermenting the slurry comprising the fermentation organism until fermentation is complete, thereby producing a fermented slurry; separating the fermented slurry into solid and liquid fractions: recovering the solid and liquid fractions; drying the recovered solid fraction at a temperature below that which would denature or damage proteins: thereby producing a protein concentrate.
- the protein concentrate comprises protein in an amount that is in a range that is between about 30% protein to about 65% protein on a dry basis.
- the invention provides an aquaculture feed comprising a plant protein made according to a process for producing a protein concentrate from a starch containing grain, wherein the process comprises: grinding the starch containing grain to produce a ground starch containing grain; slurrying the ground starch containing grain with water; solublizing starch and glucans with enzymes; adding a fermentation organism to the slurry; fermenting the slurry comprising the fermentation organism until fermentation is complete, thereby producing a fermented slurry; separating the fermented slurry into solid and liquid fractions: recovering the solid and liquid fractions; drying the recovered solid fraction at a temperature below that which would denature or damage proteins; thereby producing a protein concentrate.
- the invention provides a process for producing a protein concentrate with an increased protein concentration from a starch containing grain or oil seed, wherein the method comprises: dehulling the grain or using a hulless variety, grinding the starch containing grain to produce a ground starch containing grain; slurrying the ground starch containing grain with water; solublizing starch and glucans with enzymes; adding a fermentation organism to the slurry; fermenting the slurry comprising the fermentation organism until fermentation is complete, thereby producing a fermented slurry; separating the fermented slurry into solid and liquid fractions; recovering the solid and liquid fractions; culturing an appropriate microbe to provide a cell mass in a culture medium comprising the recovered liquid fraction recovered, recovering the cell mass with the grain solids, and drying the recovered solid fraction at a temperature below that which would denature or damage proteins; thereby producing a protein concentrate.
- the appropriate microbes is a member selected from the group consisting of Rhizopus ory ⁇ ae and Rhizopus microsporia.
- the invention provides a method for producing a protein concentrate with an increased protein concentration from a starch containing grain, the method comprising: grinding the starch containing grain to produce a ground starch containing grain; slurrying the ground starch containing grain with water; solublizing starch and glucans with enzymes; adding an appropriate organism selected for cell mass production to the slurry; fermenting the slurry comprising the organism until fermentation is complete, thereby producing a slurry in which soluble sugars have been converted to cell mass; separating the slurry into solid and liquid fractions; recovering the solid and liquid fractions; recovering the cell mass with the grain solids, and drying the recovered solid fraction at a temperature below that which would denature or damage proteins; thereby producing a protein concentrate with an increased protein concentration.
- the appropriate microbe is a member selected from the group consisting of Rhizopus ot ⁇ zae and Rhizopns microsporia.
- the invention provides a protein concentrate made by the method.
- the protein concentrate made by the method is a soy protein concentrate with reduced or eliminated phytate and reduced or eliminated anti- nutritional factors.
- FIG. 1. Is a flow diagram illustrating on the left side, ethanol production using standard commercial dry mill processes.
- the left side of the flow diagram illustrates the conventional cooking process used in converting starch to fermentable sugars.
- the right side illustrates the process for conventional a "no cook” process.
- FIG. 2 Is a flow diagram illustrating on the left side, the production of a protein concentrate and ethanol production using the disclosed processes.
- the left side of the flow diagram illustrates the cooking process.
- the right side illustrates the process for conventional a "no cook” process.
- FIG. 3 Shows the effect of substituting fish meal or soy protein concentrate with barley protein concentrate.
- protein concentrate refers to proteins that are isolated from their native source, e.g., proteins isolated from a starch containing grain or oil seed. Typically, the isolated proteins are in a more concentrated form than they are in their native state.
- starch containing grain refers to grains that provide sources of carbohydrate, typically in the form of starch.
- Exemplary starch containing grains include, but are not limited to cereal grains and seeds or "pseudocereals".
- cereal grain is used conventionally herein and refers generally to the edible grain of the members of the grass family.
- Exemplary cereal grains from grass family plants include but are not limited to e.g., oats, barley, wheat, corn, flax, hops, rice, rye, sorghum, millet, triticale etc.
- Exemplary "starch containing grains” from seeds or “pseudocereals” include, but are not limited to e.g., quinona, buckwheat, amaranth, etc.
- Exemplary "starch containing grains” from oilseeds include, but are not limited to e.g., soybeans, flax, sunflower, cottonseed, camelina, etc. Indeed, soybeans and soybean meal prepared using methods disclosed herein provide a high quality soy protein wherein anti-nutritional oligosacharides and phytate are reduced or eliminated by comparison to soy protein meal prepared by mechanical methods.
- the term "oilseed” as used herein, refers to seeds comprising a relatively high oil content such that they can readily be processed to provide vegetable oil.
- oilseeds e.g., soybeans, canola, sunflower, flax, etc, are commonly used in the production of vegetable oils, oilseeds also comprise carbohydrates in the form of starch or simple sugars and thus are a "starch containing grain" as defined herein. As such they are suitable for use in preparing a plant protein concentrate.
- starch refers to a carbohydrate compound having the formula (CeHi 0 Os) n , where the subscript "n” denotes the total number of glucose monomer units.
- starches comprise the polysaccharides amylose and amylopectin.
- the amylose polysaccharide is comprised primarily of glucose monomer units joined to one another in Z -1,4 linkages.
- Amylose is typically considered a linear molecule, however some minor branching sometimes is found.
- amylose polymers range in length from between about 500 to about 20,000 glucose monomer units, but any length is possible.
- Amylopectin is also comprised of glucose monomer units, but is not usually considered to be a strictly linear molecule.
- the polysaccharide comprises Z -1,4 linked glucose monomers interspersed at intervals with branches formed by glucose monomers in Z-1, 6 linkage ⁇ see e.g., Advances in Food and Nutrition Research, Vol. 41 : Starch: Basic Science to Biotechnology, Mirta Noemi Sivak and Jack Preiss eds. Academic Press (1998) which is incorporated herein by reference in its entirety).
- amylose comprises about 20% to about 25% to about 30% of the starch, but may be present in higher concentrations as well.
- "high-amylose corn starch” comprises at least about 40% amylose, and in some embodiments comprises about 50%, about 55%, about 60% , about 65%, about 70%, about 75% amylose, and in other embodiments comprises about 80% amylose or about 85% amylose.
- Amylopectin typically comprises about 70% to about 75%, to about 80% of the starch, but may occur in higher proportions or lower proportions as well, e.g., waxy con? starch may comprise more than 99% amylopectin, and HACS may comprise as little as 15% amylopectin or less.
- Starch is found in nearly every type of plant tissue including, but not limited to the fruit, seeds, stems, leaves, rhizomes and/or tubers. Thus, many starches are plant derived starches or "plant starch". Typically, starch produced in the USA is derived from corn, potatoes, rice, and wheat.
- useful starches can come from any source e.g., seeweed and other macroalgae, uni-cellular micro-algaes; arrowroot; guar gum; locust bean; tapioca; arracacha; buckwheat; banana; barley; cassava; konjac; kudzu; oca; sago; sorghum; sweet potato; taro; yams; soybeans; and beans e.g., favas, lentils and peas.
- starch also refers to "modified starch” which has been modified by human intervention such that it differs from the raw, unmodified form as extracted from a plant.
- starch can be modified by methods known in the art such as e.g., by inter alia chemical crosslinking and/or by stabilization through the introduction of anionic groups to the starch granule.
- aquaculture refers to the farming of aquatic organisms e.g., fish, mollusks, crustaceans etc, with some sort of intervention in the rearing process to facilitate production, e.g., regular stocking, feeding, protection from predators, etc.
- slurrying refers to combining a solid or semi solid substance with a liquid and mixing to form a more or less homogeneous mixture.
- slurrying the ground starch containing grain refers to mixing the ground starch containing grain e.g., barley, with a liquid e.g., water, to form a more or less homogeneous mixture.
- transfer substrate e.g., a carbohydrate e.g., a starch, sugar, etc, into a
- fermentation e.g., an acid or an alcohol.
- fermentation utilizes a fungus to convert a sugar into an alcohol.
- fermentation utilizes a bacterium to convert a sugar into an acid.
- fermentation is carried out in an anaerobic environment.
- fermentation is carried out in an aerobic environment.
- a fermenting organism refers to any organism which, under appropriate conditions e.g. anaerobic conditions, is capable of fermenting a fermentation substrate e.g. glucose.
- a fermenting organism is a fungus e.g., Saccharomyces cerevisiae, Pichia stipitis, Rhizopus oryzae, Rhizopus microsporus.
- a fermenting organism is a bacterium e.g. Zymomonis mobilis.
- drying at a temperature below that which would denature or damage proteins refers to temperatures which are lower than those which are known to cause proteins to become denatured.
- a "temperature below that which would denature or damage proteins” is a temperature that is below about 100 0 C, e.g., about 2O 0 C, about 25 0 C, about 3O 0 C, about 4O 0 C, about 5O 0 C, about 6O 0 C, about 7O 0 C, about 8O 0 C, or about 9O 0 C.
- a "temperature below that which would denature or damage proteins” is a temperature that is in a range that is between about 40 0 C and about 100 0 C.
- a "temperature below that which would denature or damage proteins” is a temperature that is about 4O 0 C, about 5O 0 C, about 60 0 C, about 7O 0 C, about 8O 0 C, about 9O 0 C, or aboutlOO°C.
- a fermentation product refers to a substance or product that is produced by fermentation.
- a fermentation product is an alcohol e.g., ethanol, methanol, butanol.
- a fermentation product is an antibiotic e.g., penicillin ' .
- a fermentation product is an amino acid.
- a fermentation product is an organic acid e.g., lactic acid, acetic acid, citric acid.
- beta glucan refers to a carbohydrate compound having the formula (CeHiOOs) n , where the subscript "n” denotes the total number of glucose monomer units with the glucose monomer units linked by beta 1,3 linkages.
- the invention provides a process for producing a protein concentrate from a starch containing grain.
- the invention provides a high quality, highly digestible protein concentrate that, in an exemplary embodiment, is suitable for use in the preparation of aquaculture feeds.
- the process is optimized for protein production and utilizes hulless grains.
- the hulless grains are ground, mixed with water to produce a mash, which may then be processed according to either a cooked or no-cook process.
- the mash is enzymatically treated to hydrolyze starch and beta glucans (if present) thereby freeing insoluble proteins and producing fermentable sugars, primarily glucose. Either simultaneously, or subsequently, the enzymatically treated mash is fermented to produce ethanol.
- the liquid fraction, and solid fraction which comprises the protein are separated e.g., by centrifugation.
- the protein containing solid fraction is dried at temperatures not exceeding 100 0 C.
- the liquid fraction is used as a fermentation feedstock for ethanol production.
- the liquid fraction is used for the production of other fermentation products e.g., methanol, citric acid, amino acids, etc.
- the process produces a fermentation feedstock, and also a protein concentrate, wherein the protein comprising the protein concentrate is a high quality, highly digestible protein suitable for use in aquaculture feeds.
- Barley is a short-season, early maturing crop that is grown commercially in both irrigated and in dry land environments. Barley is resistant to drought, flood and frost is thus adapts well to many different types of growing environments. Because of its adaptability and the ability to tolerate a wide range of environmental conditions, barley can be grown in many regions of the world where it may be difficult or impossible to grow other types of cereal grain.
- the process disclosed herein for producing a protein concentrate utilizes barley as a starch containing grain. Any barley variety e.g., hulled or hulless (see e.g., Cereal Chemistry 76: 589-599) can be used. [0043] In one exemplary embodiment, the process utilizes a hulled barley variety. "Hulled” or equivalently “covered” barley varieties produce kernels with a tough inedible outer hull. Typically, the tough outer hull or husk is removed before use in the methods disclosed herein.
- Dehulled barley is prepared by any method known in the art (e.g., using a vertical sheller) and refers to barley kernals that have had their outer hull removed, but which still have the germ and the outer layer of bran.
- hulled barley kernals are first dehulled using standard techniques well known in the art.
- "Dehulled” barley can be further processed to remove the bran layer and thereby provide a further dehulled barley referred to in the art as '"pearled barley".
- “pearled barley” is processed by the methods disclosed herein. However, in this embodiment, the pearling process removes some of the protein that could otherwise be recovered using the methods disclosed herein.
- Hulled barley is typically dehulled
- "'hulled barley” is processed according to the methods disclosed herein without removing the tough outer hull or husk. In this embodiment, a more fibrous protein concentrate is obtained.
- the barley is a hulless variety of barley.
- Hulless barley is typically used without dehulling prior to use.
- the hulless barley variety is Merlin.
- barley processed as disclosed herein is a useful source of plant protein for use in aquaculture feeds. Indeed, as disclosed in e.g., Example 6, barley protein concentrate prepared as disclosed herein is palatable to fish e.g., trout. Furthermore, barley protein concentrate prepared as disclosed herein supports growth rates of trout comparable to fishmeal based feeds see e.g., Example 7.
- starch containing grain is oats.
- oat protein concentrate having at least about 47% protein concentration is prepared according to the process.
- the starch containing grain is wheat.
- wheat protein concentrate containing at least about 31% protein is prepared according to the process.
- the starch containing grain is soy beans.
- Soy protein concentrate prepared according to the methods disclosed herein is typically free of the anti-nutritional oligosaccharides and phytate that comprise soybean meal and soybean concentrates produced by other methods. Without being bound by theory, it is believed that enzymatic action of selected fermentation microbe/microorganism that are used in the process is responsible for the elimination/reduction of phytate and anti-nutritional oligosaccharides.
- Exemplary microbe/microorganism whose use in the process results in a reduction/elimination of phytate include e.g., Rhizopns oryzae and Rhizopus oligosponis.
- soy protein concentrate prepared according to the methods disclosed herein typically comprises between about 54% to about 62% protein.
- soy protein concentrate containing about 57% protein is prepared according to the process.
- methods that employ mechanical fractionation can produce soy protein concentrates containing 70% protein, it has not been heretofor been appreciated that an enzymatic/biological process could produce a soy protein concentrate having a high concentration (typically between about 54% to about 62%) of protein. Since the biological process disclosed herein is less expensive than other methods known in the art, the process disclosed herein provides high protein soy protein concentrate at reduced cost.
- any starch containing grain or oil seed e.g., barley, wheat, corn, triticale, milo (grain sorghum), oats, rye, millet, soybeans, etc, can be used to prepare a protein concentrate according to the process for preparing a protein concentrate disclosed herein.
- any starch containing grain or oil seed e.g., barley, wheat, corn, triticale, milo (grain sorghum), oats, rye, millet, soybeans, etc.
- the disclosed process for producing a protein concentrate incorporates a dehulling step.
- dehulling utilizes standard, commercially available dehulling equipment ⁇ see e.g., R. D. Reichert et al. (2006) Journal of Food Science 49(1): 267 - 272).
- any milling method that removes and separates the hull from the rest of the grain is suitable for use in the methods disclosed herein.
- dehulling is accomplished in a mill using rotating abrasive stones, resistance bars and screen.
- the hull and dust from the milling process are removed by screening and vacuum to dust collectors.
- the degree of dehulling or pearling is a function of the clearance, speed and grit of the abrasive stones.
- a protein concentrate prepared according to the methods disclosed herein is use for the preparation of an aquaculture feed. Since fiber is typically indigestible in fish, it is desirable to dehull the starch containing grain e.g., barley kernels, at least to a degree wherein the hull (husk) is removed and the bran is left attached to the kernel, prior to preparing a protein concentrate for use in aquaculture feeds.
- fiber is typically indigestible in fish
- dehulling removes between at least about 60% to about 100% of the hull from the grain. In another exemplary embodiment, dehulling removes at least about 75% of the hull from the grain. In another exemplary embodiment, dehulling removes at least about 85% of the hull from the grain. In still another exemplary embodiment, dehulling removes at least about 95% of the hull from the grain.
- the disclosed process for producing a protein concentrate utilizes a hulless grain variety, e.g., a hulless barley variety, thereby obviating a dehulling step.
- a hulless grain variety e.g., a hulless barley variety
- particle size of the grain is achieved by use of a hammermill.
- particle size of the grain is achieved by use of a roller mill. Hammermills and roller mils are well known in the art ⁇ see e.g., Kim Koch, Hammennills and Roller mills, Kansas State University, May 2002).
- a standard hammer mill is operated with a screen size to mill grain to wherein at least about 50% or more of the ground grain will pass a 20 mesh US standard screen.
- a coarser ground grain is used.
- a standard hammer mill operated with a screen size to mill grain to wherein at least about 50% or more of the ground grain will pass a 16 mesh US standard screen.
- a course ground grain wherein at least about 50% or more of the ground grain will pass a 10 mesh US standard screen is used.
- a very fine ground flour is used e.g., wherein at least about 50% or more of the ground grain will pass a screen with smaller openings than a 20 mesh US standard screen.
- a grain slurry is typically prepared from the starch containing grain.
- ground starch containing grain is slurried with water to a total solids content of between about 10% to about 40%.
- the protein concentrate is prepared from the slurry using one of several process options disclosed herein below.
- mash is prepared with as high a solids content in the mash as possible without reducing product yield or conversion efficiency.
- mash viscosity is the factor limiting the maximum percent solids in the mash and solids content will vary with grain; e.g., barley typically makes a more viscous mash than corn.
- viscocity of the mash has an upper range of between about 2500 centipoides (cp) to about 5000 cp. The skilled artisan will appreciate the appropriate solids content for their particular application.
- a barley mash is prepared which comprises a solids concentration of about 35%. w/v in water.
- the maximum that can be achieved with effective agitation and heat transfer using the available equipment typically the range for barley mash is between about 10% to about 40% w/v.
- solids content is typically between about 10% to about 50% w/v.
- total solids content can be higher than about 40% in the case of barley, or higher than 50% in the case of other starch containing grains when a fed batch system is used to prepare the mash.
- a fed batch system is employed to provide a barley mash comprising about 60% w/v solids content.
- more starch containing grain e.g., barley can be added as starch is converted to soluble sugars and alcohol.
- enzymatic hydrolysis reduces viscosity, thereby permitting additional grain to be added to the mash.
- the slurry is adjusted to pH 5 to 7.
- the slurry is then heated and held at a temperature that is between about 8O 0 C to about 125 0 C for 5 minutes to 24 hours.
- Any means of heating and holding the slurry known in grain processing may be used e.g., batch tanks heated by steam injection or heat exchangers, jet cookers, hydroheaters feeding into holding tanks, etc.
- a heat tolerant alpha amylase e.g., from Bacillus licheniformis
- Methods for measuring soluble dextrins are well known in the art ⁇ see e.g., L.Serre and C.
- the heat tolerant alpha amylase alpha amylase can be any commercially available, heat tolerant alpha amylase and is typically used as reccommended by the manufacturer.
- the heat tolerant alpha amylase is Termamyl® SC from Novozymes is added in an amount equal to 0.025 to 0.05% of the weight of grain. Fermentation samples can be analyzed to determine the amount and composition of remaining starch to establish a baseline consistent with time or viscosity.
- the slurry is cooled to at least about 70 0 C or less, and pH adjusted to a pH that is between about pH 5 to about pH 7.
- Glucoamylase is added to the cooled cooked mash in an amount sufficient to hydrolyse the soluble dextrins to glucose.
- Glucoamylase can be any commercially available or in house produced, glucoamylase. Commercially available glucoamylase is typically added at a temperature and in an amount in accordance with manufacturer's recommendations.
- the glucoamylase Spirizyme® fuel from Novozymes is added in an amount equal to 0.04 to 0.06% of the weight of grain.
- the starch containing grains e.g., barley comprise beta glucans.
- beta glucosidase enzymes are also added.
- beta glucosidase is added in an amount and under conditions of temperature and pH consistent with manufacturer's recommendations such that it is sufficient to hydrolyze essentially all of the beta glucans present.
- Methods for measuring beta glucans are known in the art ⁇ see e.g., Maximov, V.I. et al. (1975) Prikl Biokhim Mikrobiol. 11(3):455-459, EP0709681, etc).
- the beta glucanase Viscozyme® L from Novozymes is added in an amount between 0.1 and 0.3% of the weight of grain.
- the slurry comprising the glucoamylase and beta glucanase enzymes is typically held at a temperature of between about 35 0 C to about 7O 0 C for a period of up to about 12 hours and cooled to 2O 0 C to 35 0 C, or until a viscosity range of about l OOcp to about lOOOcp is obtained.
- a viscosity of 100-lOOOcp is an indicator that all the starch has been converted to sugar.
- Cooking and enzymatic hydrolysis of starch to glucose can also be monitored by assaying for glucose by any standard assay method such as e.g., high performance liquid chromatography.
- Glucanase and beta glucosidase enzymes can be any commercially available preparations e.g., enzyme preparations from Novozymes or Genencore. Typically, the amount of enzyme used is estimated based on the solids loading and starch/glucan content of the starch containing grain e.g., barley. In general, the enzyme dosage is chosen such that it is sufficient to solubalize essentially all of the starch and glucans. Thus, a person of skill in the art will thus appreciate the appropriate enzyme dosage for their particular application.
- gelatinization temperatures for starch vary depending on the type and source of starch and can be determined by any method known in the art ⁇ see e.g., Li F. -D.; et al. (2004) J. Food Eng. 62(2): 1 13-120). Most grain derived starches typically begin gelatinization around about 65 0 C, where as potato starches typically begin gelatinization at temperatures of around about 51 0 C. In the cooking process, the range of temperatures can vary from 65 0 C to 140 0 C. The preferred temperature range for our process is 90 0 C to 12O 0 C.
- the temperature range is high enough to impede or kill off contaminating microbes, the amylase enzymes still work, but low enough that the protein molecules aren't altered. In the '"no-cook" process, it is not necessary to heat the starch to gelatinization temperatures.
- Cooking systems can be batch tanks or continuous flow using "jet cookers".
- a commercial scale process employing barley is cooked with a jet cooker of the type routinely employed in dry mill ethanol production.
- the jet cooker is operated to achieve 8O 0 C to 12O 0 C and to feed a holding tank or vessels to hold 8O 0 C to 100 0 C for at least 1 hour.
- Process development employed a batch tank fitted with steam coils for heating and agitation that cooked the mash at 9O 0 C.
- a temperature and acid tolerant alpha amylase is used to solubalize and/or liquefy gelatinized starch.
- Temperature and acid tolerant alpha amylase enzymes typically tolerate up to H O 0 C and typically have pH optima in the range of about 4 to about 6.
- Some exemplary temperature and acid tolerant alpha amylase enzymes suitable for use with cooked grain mash include, but are not limited to Genencor SPEZYME FRED, Novozymes Liquozyme®.
- a barley mash is pH adjusted to about pH 5 and the mash is heated to a temperature that is an a range that is between about 9O 0 C to about 105 0 C, and alpha amylase is added at a ratio of enzyme to starch that matches the manufacturers recommendation for dose rate.
- Any alpha amylase enzyme may be used in the process, including enzyme prepared by culture of a selected microorganism in a separate but parallel process on site or elsewhere ⁇ see e.g., W/O 1989/012679; US Patent 4,536,477; Aehle, Wofgang ed Enzymes in Industry: Production and Applications. Wilet-VCH, 2007) or alpha amylase purchased from commercial suppliers.
- a glucoamylase with activity up to 7O 0 C is used.
- Genecor Distillase or Novozymes Spiritzyme are a commercially available glucoamylases suitable for use in the process.
- a cooked barley mash is cooled to about 65 0 C, pH adjusted and enzyme added at a dose rate consistent with the manufacturer's recommendation. The mash is held for a period of between about a few minutes to about 24 hours at a temperature of between about 5O 0 C to about 7O 0 C then cooled to fermentation temperature of 20 0 C to 35 0 C.
- Any glucoamylase may be used in the process including glucoamylase produced by a selected microorganism in a separate parallel process at the site of protein production or elsewhere, using methods known in the art (see e.g., U.S. Patent
- the procees utilizes barley and employs a beta glucanases to hydrolyze the beta glucans in barley to glucose.
- Beta glucan hydrolysis reduces viscosity, solubalizes carbohydrate thereby increasing protein concentration and ethanol yield.
- Beta glucanases are added at the temperature an pH recommended by manufacturers.
- Exemplary beta glucanases include, but are not limited to Genencor Optimash and Novozymes Viscozyme.
- any beta glucanase may be used in the process including beta glucanase produced by a selected microorganism in a separate parallel process at the site of protein production or elsewhere as is known in the art.
- beta glucanase is added after cooked mash is cooled to the appropriate temperature.
- beta glucanase is added to the mash prior to cooking to reduce mash viscosity.
- fermentation temperature is about 35 0 C.
- fermentation temperature is in a range that is between about 15 0 C to about 4O 0 C.
- pH is about 4.0. In other exemplary embodiments pH varies within a range that is between about 3.3 to about 7.
- the fermentation organism is a yeast that produces ethanol from glucose e.g., Saccharomyces cerevisiae (see e.g., Frelot, D., et al. (1982) Biotechnology Letters 4(ii):705-708 ⁇
- the fermentation organism is bacteria that produces ethanol or other fermentation products from glucose e.g., Zymomonas.
- yeast with rapid fermentation and tolerance up to 12%w/w ethanol in the fermentation beer is used.
- yeast are prepared in an inoculum culture approximately 1% of the mash volume.
- Inoculum culture is grown in a nutrient broth for 24 hours to a cell density of about 100 million per ml. When added to the saccharified mash, the initial yeast cell density is typically about one million per ml. However, a wide range of yeast strains and inoculum procedures may be used in the process provided that the selected yeast and inoculum procedure results in an acceptable conversion of glucose to ethanol. In one exemplary embodiment, fermentation is inoculated directly with commercially available dried yeast preparations. Fermentation is maintained at 15 0 C to 4O 0 C or preferably 35 0 C until maximum conversion of glucose and other fermentable sugars to ethanol is complete.
- Fermentation completeness can be monitored by methods know to the art e.g., by sampling and assay of ethanol concentration (see e.g., Hyun-Beom Seo et al. (2009) J. Ind. Microbiol. Biotechnol. 36:285-292) or by assay for residual starch and soluble sugars (see e.g., Vidal, Bernardo C. et al. (2009) Cereal Chemistry 86(2): 133-135; Saglio, P.H and Pradet, A.(1980) Plant Physiol. (1980) 66:516-519) in the fermented mash. Fermentation time will vary with initial grain solids loading and available starch content.
- barley at 35% solids, to a final fermentation ethanol concentration of 8 to 12%w/w fermentation time is 48 to 60 hours. Range of fermentation time typically varies from between about 8 to about 72 hours.
- the organism added to the cooked saccharified mash is a fungus e.g., Rhizopus that converts sugars to cell mass which increases protein production or protein concentration.
- the invention provides a process for hydrolysis of grain starch and fermentation of sugars is a no cook process.
- a no-cook process temperature of the mash is kept temperatures below the gelatinization point for the starch.
- No-cook processes are known in the art (see e.g., U.S. Patent 7,037,704; U.S. Patent Application Publication 20040234649; U.S. Patent Application Publication 20050233030; U.S. Patent Application Publication 20040219649).
- barley mash is prepared comprising about 10% to about 35% solids (w/v).
- the mash is adjusted to a pH between about 3.5 and about pH 4.0 followed by the addition of yeast culture and an enzyme preparation that hydrolyzes ungelatinized raw starch.
- an enzyme preparation comprising alpha amylase and beta glucanase is suitable for this process.
- commercially available enzyme preparations may also be used in a no cook process at temperatures below the gelatinization point for the starch.
- Mash with enzyme and yeast is incubated, preferably with agitation, at a temperature that is a member selected from a temperature that is between about 3O 0 C to 7O 0 C for about 48 hours to about 72 hours in a simultaneous starch hydrolysis and fermentation.
- Soluble protein is recovered e.g., from distillation bottoms, by any known processes e.g., evaporation, and added to the solids.
- Solids from the separation are dried in air at temperatures of less than about 100 0 C in the solids. In some exemplary embodiments, Solids from the separation are dried in air at temperatures in a range that is between about 4O 0 C to about 100 0 C. However any temperature that will not denatuture or damage the protein is suitable.
- the dried solids are ground to a powder using a hammer mill or other suitable equipment to form the grain protein concentrate.
- the resulting protein concentrate is a barley protein concentrate comprising at least about 53% protein. In another exemplary embodiment, the resulting protein concentrate is a barley protein concentrate wherein the protein concentration is selected from protein concentrations in a range of between about 30% protein to about 70% protein.
- the protein concentrate has greater than 80% protein digestibility, phosphate availability of about 70%, is palatable to trout and thus, is used as one of the protein ingredients in trout feeds.
- grain protein concentrates are prepared according to the processes disclosed herein and the protein concentrates comprise protein in a concentration selected from protein concentrations in a range of between about 36% protein to about 75% protein and are suitable for use in aquaculture feeds.
- the solid and liquid fractions are separated before fermentation by mechanical means such as by centrifuge or by filtration.
- the damp solids fraction is collected, reslurried in water to wash our residual sugars and reseparated by centrifuge or filtration.
- the solids are recovered and dried.
- the solids fraction is the protein concentrate and typically comprises between about 30% protein and about 70% protein.
- the liquid fraction is fermented by addition of a fermentation organism as disclosed herein above and then distilled.
- the solids and liquid fraction are separated before fermentation by mechanical means such as by centrifuge or by filtration.
- the moist solids are inoculated with a fungal culture.
- the innoculated fungi utilize the residual sugars and thus, remove the sugars from the protein concentrate.
- the solids are dried and ground to prepare the protein concentrate.
- the liquid stream is fermented to ethanol or other product. Because the fungus more efficiently removes soluble sugars, this embodiment does not utilize a wash step therefore, the liquid stream to fermentation is not diluted and can be processed more efficiently.
- Suitable fungi for innoculation of the moist solids include, but are not limited to any fungus that it is a fungus that utilizes sugars and is non toxic and non pathogenic and safe to use in animal feeds. Additionally, in an exemplary embodiment, the fungus is palatable to the fish or animals. In one exemplary embodiment, the fungus is Rhizopus oryzae. In another exemplary embodiment, the fungus is and Rhizopus microsporia. Typical protein concentrations using this approach are about 60%. [0085] In another exemplary embodiment, soluble protein in distillation bottoms is recovered by culturing a yeast or fungus in the distillation bottoms.
- Sugar or hydrolyzed grain mash is added in an amount that is between about 0.5 to 1% to provide a carbon source.
- the microorganism utilizes the soluble protein and any added sugar, converting the nutrients to insoluble cell mass which is recovered by filtration or centrifugation and add to the solids.
- Any yeast fungus can be used provided that it is a fungus that is non toxic and non pathogenic and safe to use in animal feeds; is palatable to the fish or animals and which preferentially utilizes sugars.
- Rhizopus oryzae or Rhizopus microsporia are two organisms suitable for this embodiment.
- yeast or other fermentation organism is added to the slurry prior to solid liquids separation.
- the fermentation proceeds until all of the soluble sugars are converted to ethanol or other fermentation product. Typically in about 24 to about 72 hours.
- solids and liquids fractions are separated by centrifuge or filtration or other suitable means. The solids are recovered and dried. In the drying step any residual ethanol or other volatile fermentation product evaporates.
- the liquid fraction is sent to distillation or other fermentation product recovery step. This differs from a a conventional ethanol process in that solids are separated prior to distillation. Distillation may denature or alter protein making the protein less digestible.
- soluble sugars in the slurry are converted to additional protein rather than to ethanol by addition of an appropriate microbe.
- Soluble sugars are converted to cell mass by addition of a suitable yeast or fungus grown under conditions of agitation, aeration, temperature and pH designed to produce the maximum cell mass. Cell mass is recovered together with the grain solids resulting in an increase in protein concentrate recovered from the process.
- Appropriate microbes include, but are not limited to any fungus or yeast that is non toxic and non pathogenic and safe to use in animal feeds; is palatable to the fish or animals and which preferentially utilizes sugars.
- Exemplary appropriate microbes include e.g., Rhizopus oryzae. Rhizopus microsporus.
- Rhizopus is added to increase protein concentration of the final protein concentrate.
- Rhizopus is added in any one of three ways to increase protein concentration.
- the Rhizopus is added after yeast fermentation, but before distillation to utilize soluble proteins and increase recoverable protein.
- the Rhizopus is added instead of a another fungus e.g., a yeast e.g., Saccharomyces c ⁇ reviceae.
- the Rhizopus utilizes soluble sugars and soluble protein to produce cell mass instead of ethanol. increasing total protein recovery.
- the ground starch containing grain is slurried and the starch and glucans are solublized with enzymes.
- Yeast are then added to the liquid fraction to ferment the soluble sugars to ethanol.
- Rhizopus is added to the moist solids fraction from the centrifuge prior to drying.
- the moist solids contain sugars which are converted to cell mass by the Rhizopus.
- the starch containing grain used for this embodiment is soybeans.
- fermentation with the Rhizopus reduces or eliminates phytate and other anti-nutritional compounds typically associated with soybeans, and so provides a protein concentrate with reduced or eliminated phytate and reduced or eliminated anti-nutritional factors.
- drying steps are conducted at temperatures below the point where proteins are denatured, burned or otherwise adversely altered.
- air drying at about 5O 0 C is used.
- air drying at or below 5O 0 C is used.
- the protein concentrate is a fine brown to off-white powder comprising between about 30% to about 70% or more protein.
- the protein concentrate comprises about 35% protein.
- the protein concentrate comprises about 40% protein.
- the protein concentrate comprises about 45% protein.
- the protein concentrate comprises about 50% protein.
- the protein concentrate comprises about 55% protein.
- the protein concentrate comprises about 60% protein.
- the protein concentrate comprises about 65% protein.
- fish feeds are typically processed into water- stable, particulate forms (granules, pellets) for efficient consumption by the fish and to minimize fouling of the water.
- Most manufactured fish feed is processed by compression pelleting or extrusion; other manufactured forms include moist (or semimoist), microencapsulated, and micropulverized feeds. These processes are well known in the art.
- the protein concentrate produced by methods disclosed herein is suitable for use as an ingredient in aquaculture feed and in other animal feeds where a plant-derived, high protein source is needed or desired.
- Ethanol is produced from the liquid fermentation stream by methods known in the art (see e.g., Gyamerah, M. and Glover, J. (1996) J. of Chem. Tech. and Biotech. 66(2): 145- 152; Minier M. and Coma, G ( 1981) Biotechnology Letters 3(8):405-408; P. Christen et al (1990) Biotechnology and Bioengineering 36(2):116-123).
- the liquid stream and the wash water are combined in an appropriate tank.
- a suitable fermentation organism is added and the liquid stream fermented.
- Exemplary suitable organisms include, but are not limited to yeasts which ferment the liquid stream to ethanol.
- Ethanol concentration in the fermented liquid typically depends on the solids content of the original slurry and the carbohydrate content of the barley but is typically in a range that is between about 3% to 15%.
- the fermented liquid stream is distilled by known methods to separate the ethanol from the water (see e.g., Kister, Henry Z. (1992). Distillation Design (1st Edition ed.). McGraw-Hill).
- Hulless barley, Merlin variety was ground in a hammer mill using a fine screen. Ground barley was mixed with water to make a mash with 20% solids content in a 300 liter batch tank equipped with agitation and heat exchanger. The pH was approximately 5.5. Mash heating was initiated and when the mash reached 5O 0 C, alpha amylase (Genecor SPEZYME FRED) was added in the mount of 3.3ml per kg barley. Heating continued until the mash was 9O 0 C. Mash was held at 9O 0 C for two hours. Mash was then cooled to 65 0 C.
- Genecor SPEZYME FRED alpha amylase
- Glucoamylase (Genencor Distillase 500L) at the rate 1.6 ml per kg of grain and Beta glucanase (Genencor Optimash BG) was added at the rate of 1.66 ml per kg of grain.
- Mash cooled slowly for 16 hours to 35 0 C at which time a yeast culture was added.
- Initial cell density was about one million yeast per ml of mash. Fermentation was maintained at 35 0 C with agitation for approximately 52 hours. Fermentation beer was then centrifuged using a Sharpies centrifuge operated at 70,000 rpm. Liquid stream was sampled and ethanol concentration determined by gas chromatography to be 5% w/w. Solids were recovered from the centrifuge, dried and ground.
- Protein concentration of the dry powder was determined as total N using a Leco nitrogen analyzer with protein concentration equal to %N X 6.25. Protein concentration from 7 production batches of barley protein concentrate averaged 54% Protein concentrates prepared as in this example were used to formulate feeds for trout feeding trials as described in Examples 5 and 6.
- Example 2
- Oats were ground in a hammer mill using a fine screen as disclosed in Example 1. Ground oats were mixed with water to make a mash with 20% solids content in a 300 liter batch tank equipped with agitation and heat exchanger. The pH was approximately 5.5. Mash heating was initiated and when the mash reached 5O 0 C, alpha amylase (Genecor SPEZYME FRED) was added at the 3.3ml per kg barley barley. Heating continued until the mash was 107 0 C. Mash was held a 107 0 C for seven minutes. Mash was then cooled to 65 0 C.
- Genecor SPEZYME FRED alpha amylase
- Glucoamylase (Genencor Distillase 500L) at the rate 1.6 ml per kg of grain and Beta glucanase (Genencor Optimash BG) was added at the rate of 1.66 ml per kg of grain.
- Mash cooled overnight to 35 0 C at which time a yeast culture was added.
- Initial cell density was about one million yeast per ml of mash. Fermentation was maintained at 35 0 C with agitation for approximately 52 hours. Fermentation beer was then centrifuged at 70,000 rpm. Liquid stream was sampled and ethanol concentration determined by gas chromatography to be 5% w/w. Solids were recovered from the centrifuge, dried and ground. Protein concentration of the dry powder was determined as total Nitrogen (N) using a Leco nitrogen analyzer with protein concentration equal to %N X 6.25.
- a standard hulled barley variety (Baronesse) was dehulled and treated by the process disclosed in Example 1. Except that the mash was cooked by pumping through a hydroheater at 105 0 C then into a 200 gallon tank where the mash was cooled to 90 0 C alpha amylase added and held for about 2 hours. Mash was further cooled to 65°C when glucoamylase and beta glucanase were added and held for about 2 hours. Mash was further cooled to 35 0 C when yeast were added. Fermentation and protein recovery were as described in example 1. Beginning protein content of the barley prior to dehulling was 15.7%, final protein content of the barley protein concentrate was 53.1%.
- the following Example illustrates a no-cook process for preparing a protein concentrate from barley
- the exemplary process utilizes Merlin barley.
- a barley mash is prepared at 30 to 35% solids and is adjusted to a pH between 3.5 and 4.0 followed by the addition of yeast culture and an enzyme preparation that hydrolyzes ungelatinized raw starch.
- the enzyme preparation comprises a culture of a selected strain of Aspergillus grown in solid substrate culture as disclosed in co-pending U.S. Patent Application Patent Application No. 12/264,875, filed November 4, 2008 which is incorporated herein by reference in its entirety. Some suitable enzyme mixtures are also commercially available.
- Mash with enzyme and yeast is incubated preferably with agitation at 3O 0 C to 35 0 C for 48 to 72 hours in a simultaneous starch hydrolysis and fermentation.
- Each diet was fed to triplicate tanks of 30 rainbow trout (Oncorhynchus mykiss) held in 75 liter tanks supplied with 14 0 C water for 3 weeks, after a 2 week conditioning period. Fish were fed by hand to apparent satiation and feed intake was monitored. After 3 weeks, feed consumption was averaged and expressed a grams feed per gram of fish weight per day. There was a significant difference in feed consumption among trout fed any of the three diets (Table 2).
- ADC Apparent Digestibility Coefficients
- Rainbow trout Oncorhynchus mykiss ( ⁇ 250 g/fish) were held in300-L fiberglass tanks and supplied with 6 1/m of 15 0 C water throughout the experiment. Lighting was maintained on a 14:10 h diurnal cycle.
- the reference diet was composed of corn gluten meal (37.42%), wheat gluten meal (7.61%), soybean meal (20.62%), wheat flour (24.62%), lysine-HCL (1.47%). taurine (0.50%), fish oil (13.43%), stay-C (0.30%), choline-CL (0.50%), vitamin premix (0.80%), Trace mineral premix (0.10%), yttrium oxide (0.01%).
- Each test ingredient was mixed with the reference diet at a 30/70 ratio, w/w, before processing.
- the diets containing barley protein concentrates were produced by cold extrusion due to limited quantities of material available and all other diets were produced by cooking extrusion.
- Diets were fed to triplicate groups of fish, and were replicated over time. Diets were randomly assigned to a tank of fish and the fish were fed to apparent satiation twice daily. The fish were fed their respective diets for seven days prior to fecal collection. Fecal samples were obtained in one collection by manual stripping 16-18 h post-feeding. Manual stripping of fish was accomplished by netting and anesthetizing all fish in the tank, followed by gently drying and then applying pressure to the lower abdominal region to express fecal matter into a plastic weighing pan. Care was taken to exclude urinary excretions from the collection. Thereafter, fish were removed, replaced with new fish, and each diet was randomly assigned to a tank of fish for the second and third replicates of diet.
- ADCN d , et 100-100 ⁇ % Yt in diet X % nutrient in feces ⁇ / ⁇ % Yt in feces X % nutrient in diet ⁇
- ADCN ingr ed,em ⁇ (a+b) ADCN, -(a) ADCN r ⁇ b "1 where,
- ADCN m g r edie n t apparent digestibility coefficient of the nutrient in the test ingredient
- ADCN t apparent digestibility coefficients of the nutrient in the test diets
- the ADC " s for protein and amino acids for the fermented BPC were very high (Table 3) and equivalent or better than standard fish meal.
- the ADC for protein was significantly increased by the concentration of both the washed and fermented BPCs.
- FIG. 3 shows the effect of substituting fish meal or soy protein concentrate with barley protein concentrate.
- feeding a diet with 1/3 and 2/3 replacement of fish meal with barley protein concentrate increased growth rate compared to feeding the 100% fish meal diet (means with different letters are different P ⁇ 0.05). Feeding diets to trout with complete replacement of fish meal with barley protein concentrate resulted in equivalent growth.
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EP09732865.2A EP2276352B1 (en) | 2008-04-15 | 2009-04-15 | Protein concentrate from starch containing grains: composition, method of making, and uses thereof |
CA2721617A CA2721617C (en) | 2008-04-15 | 2009-04-15 | Protein concentrate from starch containing grains: composition, method of making, and uses thereof |
MX2010011325A MX2010011325A (en) | 2008-04-15 | 2009-04-15 | Protein concentrate from starch containing grains: composition, method of making, and uses thereof. |
PL09732865T PL2276352T3 (en) | 2008-04-15 | 2009-04-15 | Protein concentrate from starch containing grains: composition, method of making, and uses thereof |
JP2011505174A JP5746614B2 (en) | 2008-04-15 | 2009-04-15 | Protein concentrate obtained from starch-containing grains, its composition, production method and use |
MX2013009287A MX361478B (en) | 2008-04-15 | 2009-04-15 | Protein concentrate from starch containing grains: composition, method of making, and uses thereof. |
ES09732865T ES2781760T3 (en) | 2008-04-15 | 2009-04-15 | Protein concentrate from grains containing starch: composition, manufacturing method and uses of it |
DK09732865.2T DK2276352T3 (en) | 2008-04-15 | 2009-04-15 | PROTEIN CONCENTRATE OF STARCH GRAIN: COMPOSITION, PROCEDURE FOR PREPARATION AND APPLICATIONS THEREOF |
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Also Published As
Publication number | Publication date |
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EP2276352A2 (en) | 2011-01-26 |
AU2009236220A1 (en) | 2009-10-22 |
US9644228B2 (en) | 2017-05-09 |
EP2276352A4 (en) | 2012-04-25 |
ES2781760T3 (en) | 2020-09-07 |
US20090259018A1 (en) | 2009-10-15 |
JP5746614B2 (en) | 2015-07-08 |
PL2276352T3 (en) | 2020-07-13 |
AU2009236220B2 (en) | 2014-10-23 |
MX2010011325A (en) | 2011-02-23 |
EP2276352B1 (en) | 2020-01-01 |
US8481677B2 (en) | 2013-07-09 |
HUE049048T2 (en) | 2020-08-28 |
WO2009129320A3 (en) | 2009-12-23 |
US20130274443A1 (en) | 2013-10-17 |
JP2015128429A (en) | 2015-07-16 |
CA2721617C (en) | 2016-06-21 |
MX361478B (en) | 2018-12-06 |
DK2276352T3 (en) | 2020-04-06 |
JP2011516095A (en) | 2011-05-26 |
CA2721617A1 (en) | 2009-10-22 |
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