COMPOSITION INCLUDING A PROTEIN OF NON-ANIMAL ORIGIN, ANIMAL FEED INCLUDING SUCH COMPOSITION AND MET

专利摘要:
composition, animal feed, animal feed diet, method of producing a protein concentrate of non-animal origin, and, biologically pure strain culture. the present invention describes a biological process for producing high quality protein concentrate (hqpc) by converting plant-derived celluloses into bioavailable protein via aerobic incubation, including the use of such hqpc so produced as a nutrient, including use as a substitute for bran. fish in aquaculture diets.
公开号:BR112014013279B1
申请号:R112014013279-8
申请日:2012-12-02
公开日:2021-08-10
发明作者:William R. Gibbons；Michael L. Brown
申请人:Prairie Aqua Tech；
IPC主号:

专利说明:

HISTORY OF THE INVENTION
[001] This work was done with government support from the National Science Foundation under contract DBI-1005068. The Government has certain rights in this invention. REFERENCE TO RELATED PATENT APPLICATION
[002] This patent application claims benefit under 35 USC §119(e) to US Provisional Patent Application No. 61/566,487, filed December 2, 2011, and US Provisional Patent Application No. 61/566,557, filed December 2, 2011, each of which is incorporated by reference herein in its entirety. FIELD OF THE INVENTION
[003] The invention relates, in general, to incubation processes, and specifically microbial aerobic incubation processes to produce high quality protein concentrates, including products made from them and the use of such products in the formulation of nutritional rations . HISTORY INFORMATION
[004] In 2008, approximately 28% of the world's wild marine fish were overexploited and 52% were fully exploited, even as the demand for per capita consumption of fish and seafood products increased with the increase in human population. With declining wild fish populations, in an effort to meet this increased demand, commercial aquaculture production has increased dramatically. However, one of the main components of aquaculture diet formulations, fishmeal protein, is also derived from wild fish. It is estimated that at least 6.7 mmt of fishmeal will be needed to support commercial aquaculture production by 2012. This is clearly an unsustainable trend.
[005] Lower cost and more sustainable plant-derived protein sources have been used to partially replace fishmeal in aquaculture diets. Defatted soybean meal (SBM, 42-48% protein) has typically been used to replace up to 20% of total protein in growth diets for various species, while soy protein concentrate (SPC, 65% protein) has been successfully tested at higher levels of total protein replacement, largely regulated by the trophic state of the species. These soy products provide a high protein content and relatively good amino acid profile, but are still deficient in some critical amino acids (eg, taurine) required by carnivorous marine fish. SPC can be used at higher levels than soy meal, mainly because the solvent extraction process used to produce SPC removes anti-nutritional factors (eg oligosaccharides) and thus increases protein bioavailability. In addition, a thermal step has been used to inactivate heat-labile antigenic factors. The main limitations of the current solvent extraction process are its cost, the lack of use for the oligosaccharides extracted in the process, and quality issues that often limit the inclusion to 50% of the total protein in the diet. In addition, processing soy material into soy meal or soy protein concentrates can be environmentally problematic (eg, chemical waste disposal issues associated with hexane extraction).
[006] Corn co-products, including soluble distillers dried grains (DDGS), were also evaluated in aquaculture diets at fishmeal replacement levels of up to 20%. DDGS has less protein (28-32%) and more fiber than soy products, but is generally traded at ~50% of the value of defatted soy flour. Some ethanol plants have incorporated a dry fractionation process to remove some of the fiber and oil prior to the conversion process, resulting in a dry DDGS fraction of up to 42% protein. Although this product has been used to replace between 20 and 40% of fishmeal in aquaculture feeds, there is still a need for a higher protein and more digestible DDGS aquaculture feed product. Such a product would be especially attractive if the protein component has higher levels of critical amino acids such as taurine, lysine, methionine and cysteine.
[007] Therefore, a source of plant-derived protein that is cost effective and “green” and that is of high enough quality to completely or substantially replace more of the fish meal in an aquaculture diet is needed. SUMMARY OF THE INVENTION
[008] The present disclosure relates to an organic microbial system for converting plant material into a highly digestible and concentrated protein source that also contains a microbial gum binder (exopolysaccharide), including such a concentrated source that is suitable for use as a food for animals used for human consumption.
[009] In embodiments, a composition containing a protein concentrate of non-animal origin is disclosed, wherein the composition contains at least 55% protein content and no detectable stachyose or raffinose on a dry matter basis. In one aspect, the composition contains Aureobasidium pullulans strain deposited No. NRRL 50792, No. NRRL 50793, No. NRRL 50794, No. NRRL 50795, or a combination thereof.
[010] In one aspect, the non-animal-based protein concentrate is isolated from cereal grains and oleaginous plant material including, but not limited to, soybeans, peanuts, rapeseed, canola, sesame seeds, barley, cotton seeds, palm seeds , grape seeds, olives, safflowers, sunflowers, copra, corn, coconut, flaxseed, hazelnuts, wheat, rice, potatoes, cassava, legumes, camelina seeds, mustard seeds, germ, corn gluten bran, distillery by-products /brewery, portions and combinations thereof.
[011] In another aspect, the protein content of the composition is in the range of about 56% to about 90% dry matter produced by a process including the extrusion of plant material above room temperature to form a puree; the addition of one or more cellulose deconstruction enzymes to release sugars in the puree; inoculating the enzyme-treated puree with at least one microbe, which microbe converts released sugars into proteins and exopolysaccharides; precipitation of the resulting proteins, microbes and exopolysaccharides with ethanol or a flocculant; recovery of precipitated material through hydrodynamic force; and drying said precipitated material.
[012] In a related aspect, the at least one microbe includes, among others, Aureobasidium pullulans, Sclerotium glucanicum, Sphingomonas paucimobilis, Ralstonia eutropha, Rhodospirillum rubrum, Kluyveromyces spp, Pichia spp, Trichoderma reesei, Pleurosei, Pleurosei combinations of the same.
[013] In one aspect, the plant material is soybean in the form of defatted soybean flakes or soybean meal. In another aspect, the plant material is oil seeds or their defatted bran. In another aspect, the plant material is soluble distillers dried grains (DDGS).
[014] In one aspect, the protein concentrate includes at least 0.1 g of hydroxylysine/100g of concentrate.
[015] In one embodiment, an animal feed comprising a non-animal protein concentrate is disclosed, where the composition includes at least 1.25g lipid/100g composition, where the composition contains no detectable stachyose or raffinose and at least 55 % protein content in dry matter, and where the composition includes at least 35% of said animal feed by weight.
[016] In a related aspect, the composition is a complete substitute for animal fish meal in a fish feed. In a further related aspect, fish feed is formulated for fish including, but not limited to, Siberian sturgeon, Sterlet sturgeon, star sturgeon, white sturgeon, Pirarucu, Japanese eel, American eel, short fin eel, long fin eel, eel European, Chanos chanos, Milkfish, Sunfish, Green Sunfish, White Crappie, Black Crappie, Asp, Catla, Goldfish, Common Pimpão, Mud Carp, Mrigal Carp, Limo Carp, Common Carp, Silver Carp, Loggerhead Carp , Labeo calbasu, Labeo rohita, Hoven carp, Megalobrama amblycephala, black carp, golden carp, bone-mouth barb, Parabramis pekinensis, Barbonymus gonionotus, java, carp, tench, dojo, Prochilodus magdalenae, bream, tambingaqui, pirapit , pacu, catfish, channel catfish, catfish, blue catfish, Silurus glanis, pangafish (Swai, Tra, Basa), hullfish, Mudfish, Filipino catfish, Hong Kong catfish, North African catfish, catfish bighead, vundú, South American catfish, tamoatá, pike, Ayu, salmon, whitefish, pink salmon, Chum salmon, Coho salmon, Masu salmon, rainbow trout, red salmon, Chinook salmon, Atlantic salmon, trout, Arctic salmon, brook trout, lake trout, Atlantic cod, kingfish, Scomberoides lysan, sea bass, barramundi/big perch, Nile perch, Murray cod, golden perch, striped bass, Morone chrysops, European sea bass, Hong Kong grouper, areola grouper, greasy grouper , Plectropomus maculatus, silver perch, white perch, jade perch, largemouth bass, Micropterus dolomieu, European perch, zander (pike perch), yellow perch, sauger, walleye, anchovy, bull's eye, buri, Trachinotus blochii, Trachinotus carolinus , Trachinotus goodei, Trachurus japonicus, Rachycentron canadum, Lutjanus argentimaculatus, guaiuba, poplar, Diplodus sargus, Evynnis japonica, seabream, snapper, Rhabdosargus sarba, Sparus aurata, Sciaenops ocellatus, Cellanus uruichomas, Aequichudens hthalmum, Etroplus suratensis, Pelmatolapia mariae, blue tilapia, Oreochromis macrochir, Oreochromis mossambicus, Nile tilapia, tilapia, Oreochromis urolepis, Sarotherodon melanotheron, Tilapia rentelli, Tilapia parsley, Lilapia zillili, Lilapia zillili, Lilapia zillili, Lilapia zillili, Lillili , Chelon planiceps, Mugil cephalus, Mugil curema, Mugil liza, Dormitator latifrons, Oxyeleotris marmorata, Siganus canaliculatus, Siganus lineatus, Siganus rivulatus, Thunnus maccoyii, Thunnus thynnus, climber, Trichopoddoram chandeliers, chandeliers, pectorna punctata, Channa striata, Turbot, Paralichthys olivaceus (Japanese language), Paralichthys dentatus, Paralichthys lethostigma, Pseudopleuronectes americanus, Hippoglossus hippoglossus, Rhombosolea tapirina, Solea solea, and combinations thereof.
[017] In one aspect, fish feed effects greater performance in one or more aspects of performance including, but not limited to, growth, weight gain, protein efficiency ratio, feed conversion ratio, total consumption, survival, and Fulton's condition factor compared to fish feeds characterized by comprising fishmeal of animal origin or soy protein concentrate.
[018] In another aspect, said fish feed effects the performance aspects at a crude protein content that is less than or equal to the protein content of fish feeds comprising animal fish meal or soy protein concentrate .
[019] In one aspect, animal feed is supplemented with lysine, methionine, lipids, biotin, choline, niacin, ascorbic acid, inositol, pantothenic acid, folic acid, pyridoxine, riboflavin, thiamine, vitamin A, vitamin B12, vitamin D , vitamin E, vitamin K, calcium, phosphorus, potassium, sodium, magnesium, manganese, aluminum, iodine, cobalt, zinc, iron, selenium or a combination thereof.
[020] In another embodiment, a method of producing a non-animal protein concentrate is disclosed, including extruding plant material above room temperature to form a puree and transferring the puree to a bioreactor; the addition of one or more cellulose deconstruction enzymes to release sugars in the puree in the biological reactor; inoculating the enzyme-treated puree with at least one microbe, which microbe converts released sugars into proteins and exopolysaccharides; precipitation of the resulting proteins, microbes, and exopolysaccharides with ethanol, a flocculant, or a combination thereof; recovery of precipitated material through hydrodynamic force; and drying the precipitated material.
[021] In a related aspect, extrusion is carried out between about 50° to about 170°C, at a compression ratio of about 3:1, and at a screw speed sufficient to provide a shear effect against grooved channels on both sides of an extrusion drum.
[022] In another related aspect, the method includes mixing the extruded materials with water to achieve a solids loading rate of at least 5% into the bioreactor; and optionally, autoclaving and cooling the extruded materials, where the one or more cellulose deconstruction enzymes are selected from the group consisting of endo-xylanase and beta-xylosidase, glycoside hydrolase, β-glucosidases, hemicellulase activities, and combinations thereof.
[023] In a related aspect, the method includes reducing the temperature of the enzyme-treated puree to between about 30°C to about 37°C; inoculation of the cooled puree with 2% (v/v) of a 24-hour culture of at least one microbe, where the at least one microbe includes, among others, Aureobasidium pullulans, Sclerotium glucanicum, Sphingomonas paucimobilis, Ralstonia eutropha, Rhodospirillum rubrum , Kluyveromyces spp, Pichia spp, Trichoderma reesei, Pleurotus ostreatus, Rhizopus spp, and combinations thereof; optionally, aeration of the inoculated puree at about 0.05 L/L/min; and incubating until sugar utilization ceases or after about 96 to 120 hours of incubation in the presence of the at least one microbe.
[024] In one aspect, the method includes adding about 0.6 L of ethanol/L of the puree; centrifuging the ethanol treated puree; the recovery of ethanol; optionally, the recovery of suspended fine particles, the recovery of centrifuged solids; and drying the solids recovered from the centrifuge. In another aspect, the supernatant can be dried, the dry solids recovered, and then mixed with solids from the centrifuge.
[025] In one embodiment, a biologically pure culture of Aureobasidium pullulans strain selected from the group consisting of No. NRRL 50792, No. NRRL 50793, No. NRRL 50794, and No. NRRL 50795 is disclosed. BRIEF DESCRIPTION OF THE DRAWINGS
[026] Figure 1 shows a flowchart for the HQSPC conversion process.
[027] Figure 2 shows a flowchart for the process of converting HQSPC to aquatic feeds.
[028] Figure 3 shows bench scale extended incubation assays for evaluating the composition and yield of HQSPC.
[029] Figure 4 shows a flowchart for the conversion process from HP-DDGS to aquatic feeds.
[030] Figure 5 shows the effect of moisture content and extrusion speed on glucose recovery after extrusion of HP-DDGS at 100°C. DETAILED DESCRIPTION OF THE INVENTION
[031] Before the present composition, methods and methodologies are described, it should be understood that this invention is not limited to specific compositions, methods and experimental conditions described, since such compositions, methods and conditions may vary. It is also to be understood that the terminology used in this document is for the purposes of describing specific embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.
[032] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references, unless the context clearly indicates otherwise. Thus, for example, references to "a nucleic acid" include one or more nucleic acids, and/or compositions of the type described herein that will become apparent to those skilled in the art upon reading this disclosure, and so forth.
[033] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as commonly understood by a person skilled in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the invention, as it will be understood that modifications and variations are encompassed within the spirit and scope of the present disclosure.
[034] As used in this document, "about", "approximately", "substantially" and "significantly" will be understood by a person skilled in the art and will vary to some degree depending on the context in which they are used. If there are uses of the term that are not clear to experts in the field, given the context in which they are used, "about" and "approximately" will mean more or less <10% of the specific term and "substantially" and "significantly" will mean more or less >10% of the specific term.
[035] As used herein, the term "animal" means any organism belonging to the kingdom Animalia and includes, without limitation, humans, birds (eg, poultry), mammals (eg, cattle, swine, goats, sheep, cats , dogs, mice and horses), as well as aquaculture organisms such as fish (eg trout, salmon, perch), molluscs (eg shellfish) and crustaceans (eg lobster and shrimp).
[036] The use of the term “fish” includes all vertebrate fish, which may be bony or cartilaginous fish.
[037] As used in this document, "non-animal protein" means that the protein concentrate comprises at least 0.81 g of crude fiber/100g of the composition (dry matter), which crude fiber is mainly cellulose and lignin material obtained as a residue in the chemical analysis of plant substances.
[038] As used in this document, "incubation process" means the provision of ideal conditions for growth and development of bacteria or cells, where such bacteria or cells use biosynthetic pathways to metabolize various raw materials. In embodiments, the incubation process can be carried out, for example, aerobic conditions. In other embodiments, the incubation process can include fermentation.
[039] As used in this document, the term “incubation products” means any residual substances directly resulting from an incubation process/reaction. In some cases, an incubation product contains microorganisms so that it has a higher nutritional content compared to an incubation product that is deficient in such microorganisms. Incubation products may contain suitable constituent(s) of an incubation broth. For example, incubation products can include dissolved and/or suspended constituents of an incubation broth. Suspended constituents may include undissolved soluble constituents (eg where the solution is supersaturated with one or more components) and/or insoluble materials present in the incubation broth. Incubation products can include substantially all of the dry solids present at the end of an incubation (e.g., by spray drying an incubation broth and the biomass produced by the incubation) or can include a portion thereof. Incubation products can include raw incubation material where a microorganism can be fractionated and/or partially purified to increase the nutrient content of the material.
[040] As used herein, a "conversion culture" means a culture of microorganisms that are contained in a medium that comprises sufficient material for the growth of the microorganisms, for example, water and nutrients. The term “nutrient” means any substance with nutritional value. It can be part of an animal feed or food supplement for an animal. Exemplary nutrients include, among others, proteins, peptides, fats, fatty acids, lipids, water- and fat-soluble vitamins, essential amino acids, carbohydrates, sterols, enzymes and trace elements, such as phosphorus, iron, copper, zinc, manganese, magnesium, cobalt, iodine, selenium, molybdenum, nickel, fluorine, vanadium, tin and silicon.
[041] Conversion is the process of cultivating microorganisms in a conversion culture under suitable conditions to convert protein/carbohydrate/polysaccharide materials, eg soy material, into a high quality protein concentrate. Proper conversion means using 90% or more of specified carbohydrates to produce microbial cell mass and/or exopolysaccharides. In embodiments, the conversion can be aerobic or anaerobic.
[042] As used herein, a "flocculant" or "scavenging agent" is a chemical that promotes colloids to come out of suspension through aggregation, and includes, among others, a multivalent ion and polymer. In embodiments, such flocculant/scavenging agent may include bioflocculants such as exopolysaccharides.
[043] A large number of plant protein sources can be used in connection with the present disclosure as raw materials for conversion. The main reason for using plant proteins in the animal feed industry is to replace more expensive protein sources such as animal protein sources. Another important factor is the danger of disease transmission by feeding animal proteins to animals of the same species or related species. Examples for vegetable protein sources include, but are not limited to, protein from the Fabaceae plant family as exemplified by soybeans and peanuts, from the Brassiciaceae plant family as exemplified by canola, cottonseed, from the Asteraceae plant family including, but not limited to, sunflower , and the Arecaceae plant family, including copra. These protein sources, also commonly defined as oilseed proteins, can be served in their entirety, but they are most commonly served as a by-product after the oils have been removed. Other vegetable protein sources include vegetable protein sources from the Poaceae family, also known as Gramineae, such as cereals and grains, especially corn, wheat and rice or other staple crops such as potatoes, cassava and legumes (peas and beans), some milling by-products , including corn gluten germ or bran, or distillery/brewery by-products. In embodiments, raw materials for proteins include, among others, plant materials from soybeans, peanuts, rapeseed, barley, canola, sesame seeds, cotton seeds, palm seeds, barley, grape seeds, olives, safflowers, sunflowers, copra, corn, coconut, flaxseed, hazelnuts, wheat, rice, potatoes, cassava, pulses, camelina seeds, mustard seeds, germ, corn gluten bran, distillery/brewery by-products, and combinations thereof.
[044] In the fish farming industry, the main fish meal substitutes of vegetable origin allegedly used include, among others, soy meal (SBM), corn gluten meal, rapeseed/canola meal (Brassica sp.), lupine (Lupinus sp. as proteins in shelled white lupine (Lupinus albus), sweet (L. angustifolius) and yellow (L. luteus) seed bran, sunflower seed bran (Helianthus annuus), crystalline amino acids; as well as pea bran (Pisum sativum), cottonseed (Gossypium sp.), peanut bran and bagasse (peanut; Arachis hypogaea), soy protein concentrate, corn gluten bran (Zea mays) and wheat gluten (Triticum aestivum), Potato Protein Concentrate (Solanum tuberosum L.), as well as other plant foods such as Moringa leaves (Moringa oleifera Lam.), all in various concentrations and combinations.
[045] Protein sources can be in the form of untreated plant materials and treated and/or extracted plant proteins. As an example, heat-treated soy products have greater protein digestibility.
[046] A protein material includes any type of protein or peptide. In embodiments, protein material or the like can be used, such as whole soybeans. Whole soybeans can be standard marketed soybeans; soybeans that have been genetically modified (GM) in some way; or non-GM identity preserved soybeans. Exemplary GM soybeans include, for example, soybeans designed to produce carbohydrates other than stachyose and raffinose. Exemplary non-GM soybeans include, for example, Schillinger varieties that are bred by in-line crossbreeding for low oil, low carbohydrate, and low trypsin inhibition.
[047] Other types of soy material include soy protein meal, soy protein concentrate, soy meal and soy protein isolate, or mixtures thereof. Traditional processing of whole soy into other forms of soy protein, such as soy protein flours, soy protein concentrate, soy meal and soy protein isolates, includes breaking clean raw whole soy into several pieces , typically 6 (six) to 8 (eight), to produce soy chips and husks, which are then removed. The soy chips are then conditioned at about 60°C and flocculated to about 0.25mm thick. The resulting flakes are then extracted with an inert solvent, such as a hydrocarbon solvent, typically hexane, in one of several types of countercurrent extraction systems to remove the soybean oil. For soy protein flours, soy protein concentrates, and soy protein isolates, it is important that the flakes are desolventized in a way that minimizes the amount of cooking or browning of the soy protein to preserve a high soy protein content. soluble in water. This is typically accomplished using steam desolventizers or flash steam desolventizers. The flakes resulting from this process are generally called “edible defatted flakes” or “white soy (bean) flakes”.
[048] White soy flakes, which are the raw material for soy protein flour, soy protein concentrate, and soy protein isolate, have a protein content of approximately 50%. The white soy flakes are then milled, usually in an open mesh milling system, by a hammer mill, classifier mill, roller mill or impact pin mill into grains first, and with additional milling, into soy flours with desired particle sizes. Screening is typically used to size the product into uniform particle size ranges, and can be performed with agitator sieves or cylindrical centrifugal sieves. Other oil seeds can be processed in a similar way.
[049] In embodiments, soluble and distillery dry grains (DDGS) can be used. DDGS are currently manufactured by the corn ethanol industry. Traditional DDGS comes from dry milling plants, where the whole corn grain is ground and processed. The DDGS in these factories typically contains 28-23% protein.
[050] Protein sources can be in the form of untreated plant materials and treated and/or extracted plant proteins. As an example, heat treated soy products have high protein digestibility. Still, the upper inclusion level for inclusion of full-fat or defatted soy meal in carnivorous fish diets is at a 20 to 30% inclusion level, even if heat-labile antinutrients are eliminated. In fish, soy protein has shown that feeding fish with protein concentration inclusion levels greater than 30% causes intestinal damage and, in general, reduces growth performance in different fish species. In fact, most fish farmers are reluctant to use more than 10% vegetable protein in the total diet due to these effects.
[051] The present invention solves this problem and allows vegetable protein inclusion levels of up to 40 or even 50%, depending, among other factors, on the species of animal being fed, the origin of the vegetable protein source, the ratio of different vegetable protein sources, the concentration and quantity of proteins, the origin, the molecular structure and the concentration of glucan and/or mannan. In embodiments, vegetable protein inclusion levels are up to 40%, preferably up to 20 or 30%. Typically, the vegetable protein present in the diet is between 5 and 40%, preferably between 10 or 15 and 30%. These percentages define the percentage amount of a total vegetable protein source in the feed or animal feed, this includes fat, ash, etc. In embodiments, pure protein levels are up to 50%, typically up to 45%, in embodiments, 5 to 95%.
[052] The ratio of vegetable protein to other protein in the total feed or diet can be 5:95 to 95:5, 15:85 to 50:50, or 25:75 to 45:55. MICRO-ORGANISMS
[053] Microorganisms must be able to convert carbohydrates and other nutrients into a high quality protein concentrate in a conversion culture. In embodiments, the microorganism is a yeast-like fungus. An example of a yeast-like fungus is Aurobasidium pullulans. Other exemplary microorganisms include yeasts such as Kluyveromyces and Pichia spp, lactic acid bacteria, Trichoderma reesei, Pleurotus ostreatus, Rhizopus spp, and many types of lignocellulose degrading microbes. In general, exemplary microbes include microbes that can metabolize stachyose, raffinose, xylose, and other sugars. However, it is within the skill of a person skilled in the art to choose, without undue experimentation, other appropriate microorganisms based on the disclosed methods.
[054] In embodiments, microbial organisms that can be used in the present process include, among others, Aureobasidium pullulans, Sclerotium glucanicum, Sphingomonas paucimobilis, Ralstonia eutropha, Rhodospirillum rubrum, Kluyveromyces and Pichia spp, Trichoderma reesei, Rhizoppus, Pleurotus and combinations thereof. In embodiments, the microbe is Aureobasidium pullulans.
[055] In embodiments, A. pullulans is adapted to various environments/stressors encountered during conversion. In achievements, a strain of A. pullulans denoted by deposit no. NRRL 50793, which was deposited in the Agricultural Research Culture Collection (NRRL), Peoria, Ill., under the terms of the Budapest Treaty on November 30, 2012 , exhibits lower gum production and is adapted to DDGS. In achievements, a strain of A. pullulans denoted by deposit no. NRRL 50792, which was deposited in the Agricultural Research Culture Collection (NRRL), Peoria, Ill., under the terms of the Budapest Treaty on November 30, 2 012, is adapted for high levels of antibiotic tetracycline (eg tetracycline about 75 µg/ml to tetracycline about 200 µg/ml). In achievements, a strain of A. pullulans denoted by NRRL Deposit No. 50794, which was deposited in the Agricultural Research Culture Collection (NRRL), Peoria, Ill., under the terms of the Budapest Treaty on November 30, 2012 , is adapted for high levels of the antibiotic LACTROL® (eg virginiamycin about 2 μg/ml to virginiamycin about 6 μg/ml). In achievements, a strain of A. pullulans denoted by deposit no. NRRL 50795, which was deposited in the Agricultural Research Culture Collection (NRRL), Peoria, Ill., under the terms of the Budapest Treaty on November 30, 2012 , is acclimated to condensed corn solubles. CONVERSION CULTURE
[056] In exemplary embodiments, after pretreatment, the protein material (such as extruded white soy flakes) can be mixed with water at a solids loading rate of at least 5%, with pH adjusted to 4, 5 to 5.5. Then, the appropriate dosages of hydrolytic enzymes can be added and the slurry incubated with agitation at 150 to 250 rpm at 50°C for 3 to 24 h. After cooling to 35°C, an inoculum of A. pullulans can be added and the culture can be incubated for an additional 72 to 120 h, or until the carbohydrates are consumed. During incubation, sterile air can be injected into the reactor at a rate of 0.5 to 1 L/L/h. In embodiments, the conversion culture undergoes conversion by incubation with the soy material for less than about 96 hours. In embodiments, the conversion culture will be incubated for between about 96 hours and about 120 hours. In embodiments, the conversion culture can be incubated for more than about 120 hours. The conversion culture can be incubated at about 35°C.
[057] In embodiments, the pH of the converting culture, while in conversion, can be about 4.5 to about 5.5. In embodiments, the pH of the conversion culture can be less than 4.5 (for example, at pH 3). In embodiments, the conversion culture can be actively aerated, as disclosed in Deshpande et al., Aureobasidiumpullulans in applied microbiology: A status report, Enzyme and Microbial Technology (1992), 14(7):514.
[058] High quality protein concentrate (HQPC), as well as pullulan and siderophores, can be recovered from the conversion culture after the conversion process by optionally precipitation in alcohol and centrifugation. An exemplary alcohol is ethanol, although the person skilled in the art understands that other alcohols should work. In embodiments, salts can also be used to precipitate. Exemplary salts can be potassium, sodium and magnesium chloride salts. In embodiments, a polymer or multivalent ions can be used alone or in combination with alcohol.
[059] In embodiments, the recovery of solids from final protein concentrations can be modulated by varying incubation times. For example, about 75% protein can be achieved with a 14-day incubation, where solids recovery is about 16 to 20%. In embodiments, incubation for 2 to 2.5 days increases solids recovery to about 60 to 64%, and protein level to 58 to 60% in the HQPC. In embodiments, incubation for 4 to 5 days can maximize both protein content (eg, greater than about 70%, among others) and solids recovery (eg, greater than about 60%, among others). These numbers can be higher or lower depending on the raw material. In embodiments, protein concentrates (i.e., HQSPC or HP-DDGS) can have a specific lipid:protein ratio, for example, about 0.010:1 to about 0.03:1, about 0.020:1 to about 0.025:1 or about 0.021:1 to about 0.023:1.
[060] In embodiments, raw materials can be extruded into a single-screw extruder (eg Brabender Plasti-corder Model PL2000, Hackensack, NJ) with a barrel length to screw diameter of 1:20 and a compression ratio 3:1 although other geometries and ratios may be used. The raw materials can be adjusted to about 10% to about 15% humidity, to about 15%, or to about 25% humidity. The temperature of the feed, barrel and exit sections of the extruder can be maintained between about 40°C to about 50°C or about 50°C to about 100°C, about 100°C to about 150°C, about 150°C to about 170°C, and the screw speed can be set to about 50 rpm to about 75 rpm or about 75 rpm to about 100 rpm or about 100 rpm to about from 200 rpm to about 250 rpm. In embodiments, the screw speed is sufficient to provide a shear effect against rough grooves on both sides of a drum. In embodiments, screw speed is selected to maximize sugar release.
[061] In embodiments, extruded raw material (eg, vegetable proteins or DDGS) can be mixed with water to achieve a solids loading rate of at least 5% into a reactor (eg, a New Brunswick Bioflo 3 de bioreactor 5 L; 3-4 L working volume). The slurry can be autoclaved, cooled, and then saccharified by subjecting it to enzymatic hydrolysis using a cocktail of individual enzymes, among others, endo-xylanase and beta-xylosidase, glucoside hydrolase, β-glucosidases, hemicellulase activities. In one aspect, the enzyme cocktail includes enzymes from Novozyme®. Dosages may include 6% CellicCtek® (per gm glucan), 0.3% CellicHtek® (per gm total solids), and 0.15% Novozyme 960® (per gm total solids). The saccharification can be conducted for about 12 h to about 24 h at 40° to about 50°C and about 150 rpm to about 200 rpm to solubilize the fibers and oligosaccharides into simple sugars. The temperature can then be reduced to between about 30°C to about 37°C, in embodiments, to about 35°C, and the slurry can be inoculated with 2% (v/v) of a culture. 24 h of the microbe. The slurry can be aerated at 0.5 L/L/min and incubation can be continued until sugar utilization ceases or from about 96h to about 120h. In fed batch process conversions, more extruded raw material can be added during the saccharification and/or microbial conversion stage.
[062] In embodiments, the raw material and/or the extrudate may be treated with one or more antibiotics (for example, but not limited to, tetracycline, penicillin, erythromycin, tylosin, virginiamycin, and combinations thereof) prior to inoculation with the conversion microbe to avoid, for example, contamination by unwanted bacterial strains.
[063] During incubation, samples can be removed at intervals of 6 to 12 h. Samples for HPLC analysis can be boiled, centrifuged, filtered (eg, through 0.22 µm filters), placed in autosampler vials, and frozen until analysis. In embodiments, samples can be evaluated for carbohydrates and organic solvents using a Waters HPLC system, although other HPLC systems may be used. Samples can be subjected to plate counts or hemocytometers to assess microbial populations. Samples can also be evaluated for cellulose, hemicellulose, and pectin levels using national renewable energy laboratory procedures. DIETARY FORMULATIONS
[064] In exemplary embodiments, high quality protein concentrate recovered from the conversion culture that has undergone conversion is used in dietary formulations. In embodiments, recovered high quality protein concentrate (HQPC) will be the primary protein source in the dietary formulation. Percentages of protein sources in dietary formulations are not intended to be limiting and may include 24 to 80% protein. In embodiments, the high quality protein concentrate (HQPC) will be greater than about 50%, greater than about 60%, or greater than about 70% of the protein source of the total dietary formulation. The recovered HQPC can replace protein sources such as fish meal, soybean meal, wheat and corn flours and concentrates and glutens, and animal by-product such as blood, poultry and feather meal. Dietary formulations using recovered HQPC may also include supplements such as mineral and vitamin premixes to meet the remaining nutrient requirements as appropriate.
[065] In certain embodiments, the performance of HQPC, such as high quality soy protein concentrate (HQSPC) or high quality DDGS (HP-DDGS), can be measured by comparing growth, feed conversion, efficiency of protein, and animal survival in a high quality protein concentrate dietary formulation with animals fed control diet formulations such as fish meal. In embodiments, the test formulations contain consistent protein, lipid and energy levels. For example, when the animal is a fish, visceral characteristics (fat deposition) and organs (liver and spleen), carcass weight percentage, and immediate fillet analysis, as well as intestinal histology (enteritis) can be measured to assess the answer to diet.
[066] As is understood, individual dietary formulations containing the recovered HQPC can be optimized for different types of animals. In embodiments, the animals are fish cultivated in commercial aquaculture. Methods for optimizing dietary formulations are well known and easily verifiable by the skilled person without undue experimentation.
[067] Complete growth diets can be formulated using HQPC in accordance with known nutritional requirements for various animal species. In embodiments, the formulation can be used for yellow perch (eg 42% protein, 8% lipids). In embodiments, the formulation can be used for rainbow trout (eg 45% protein, 16% lipid). In embodiments, the formulation can be used for one or more of the aforementioned animals.
[068] Premixes of minerals and basal vitamins for plant diets can be used to ensure micronutrient requirements are met. Any supplements (as deemed necessary by analysis) can be evaluated against an identical formulation without supplementation; thus, the feeding trial can be done in a factorial design to take into account the effects of supplementation. In embodiments, feeding trials may include a fishmeal based control diet and reference diets based on ESPC and LSPC [traditional SPC (TSPC) is produced from washing white soy flakes with solvent to remove soluble carbohydrates; Textured SPC (ESPC) is produced by extruding TSPC under humidity and high temperature; and low antigen SPC (LSPC) is produced from TSPC by changing the solvent wash and temperature during processing. corder Model PL2000). FOOD TESTS
[069] In embodiments, a replication of four experimental units per treatment (ie, each mixture of experimental and control diet) can be used (eg, about 60 to 120 days each). The tests can be performed in 110-L circular tanks (20 fish/tank) connected in parallel to a closed-loop recirculation system driven by a centrifugal pump and consisting of a solid pit, and bioreactor, filters (100 bag µm, carbon and ultraviolet). Heat pumps can be used as needed to maintain optimal temperatures for species-specific growth. Water quality (eg dissolved oxygen, pH, temperature, ammonia and nitrite) can be monitored in all systems.
[070] In embodiments, experimental diets can be administered according to the size of the fish and divided into two or five daily feeds. Growth performance can be determined by total mass measurements taken in one to four weeks (depending on fish size and trial duration); rations can be adjusted according to earnings to allow for feeding to satiety and to reduce waste flow. Consumption can be assessed fortnightly from collections of unconsumed feed from individual tanks. Unconsumed feed can be dried at a constant temperature, cooled, weighed to estimate feed conversion efficiency. Protein and energy digestibilities can be determined from faecal material manually taken during the midpoint of each experiment or by necropsy of the lower intestinal tract at the conclusion of a feeding trial. Survival, weight gain, growth rate, health indices, feed conversion, protein and energy digestibilities, and protein efficiency can be compared between treatment groups. Immediate analysis of necropsied fish can be performed to compare fillet composition among dietary treatments. Amino acid and fatty acid analysis can be performed as needed for fillet constituents, in accordance with the purpose of the feeding test. Dietary treatments feeding trial responses can be compared to a control diet response (eg, fish meal) to see if the performance of HQPC diets meets or exceeds the control responses.
[071] Statistical analyzes of diets and feeding trial responses can be completed with an a priori α = 0.05. Analysis of performance parameters across treatments can be performed with appropriate analysis of variance or covariance (Proc Mixed) and multiple post hoc comparisons as required. The analysis of fish performance and tissue responses can be evaluated by non-linear models.
[072] In embodiments, the present disclosure proposes to convert fiber and other carbohydrates in flakes/soybean meal or DDGS into additional protein using, for example, a GRAS state microbe. A microbial exopolysaccharide (i.e., gum) can also be produced which can facilitate the formation of extruded feed pellets, eliminating the need for binders. This microbial gum can also provide immunostimulating activity to activate defense mechanisms that protect fish from common pathogens resulting from stressors. Immunoprophylactic substances, such as β-glucans, bacterial products, and plant constituents, are increasingly used in commercial feeds to reduce economic losses due to infectious diseases and minimize the use of antibiotics. The microbes of the present disclosure also produce extracellular peptidases, which should increase corn protein digestibility and absorption during metabolism, providing greater feed efficiency and yield. As revealed in this document, this microbial incubation process provides a valuable and sustainable aquaculture feed that is less expensive per unit of protein than SPC or fishmeal.
[073] As revealed, the present microbes can metabolize individual carbohydrates into soy flakes/meal or DDGS, producing both cell mass (protein) and a microbial gum. Several strains of these microbes also enhance fiber deconstruction. The microbes of the present invention can also convert soy and corn proteins into more digestible peptides and amino acids. In realizations, the following actions can be taken: 1) determine the efficiency of using certain microbes of the present disclosure to convert pretreated soy protein, oil seed proteins, DDGS and the like, producing a high quality protein concentrate ( HQPC) with a protein concentration of at least 45%, and 2) evaluate the effectiveness of HQPC in replacing fish meal. In embodiments, optimization of soybean, oilseed, and DDGS pretreatment and conversion conditions can be performed to improve microbe performance and robustness, test the resulting growth feeds for a range of commercially important fish, validate process costs and energy requirements, and complete steps for scaling up and commercialization. In embodiments, the HQPC of the present disclosure may be able to replace at least 50% of fish meal, while providing higher growth rates and conversion efficiencies. Production costs must be lower than commercial soy protein concentrate (SPC) and substantially lower than fish meal (including harvest).
[074] Figures 1 and 2 show the approach of the present disclosure in the pretreatment of vegetable-based products, converting sugars into cell mass (protein) and gum, recovering HQSPC and generating aquaculture feeds, and testing aquaculture feeds in trials of feeding on fish.
[075] After pretreatment by extrusion, cellulose deconstruction enzymes can be evaluated to generate sugars, which the microbes of the present disclosure can convert into protein and gum. In embodiments, sequential omission of these enzymes and evaluation of co-culture with cellulolytic microbes can be used. Ethanol can be evaluated to precipitate the gum and improve the centrifugal recovery of the HQPC. After drying, HQPC can be incorporated into practical dietary formulations. In embodiments, test growth diets can be formulated (with premixes of minerals and vitamins) and comparisons with a fishmeal control and commercial SPC diets (SPC is distinctly different from soybean meal as it contains residues of oligopolysaccharides and antigenic substances glycinin and b-conclinin) in feeding trials with a commercially important fish, eg yellow perch or rainbow trout, can be performed. Performance (eg growth, feed conversion, protein efficiency), visceral characteristics, and intestinal histology can be examined to assess fish responses.
[076] In other embodiments, optimizing the HQPC production process by determining optimal pretreatment and conversion conditions while minimizing process inputs, improving microbe performance and robustness, testing growth feeds results for a range of commercially important fish, validation of process costs and energy requirements, and completion of initial steps for scale-up and commercialization can be accomplished.
[077] In recent years, a handful of facilities have installed a dry mill capacity that removes corn husk and germ prior to the ethanol production process. This dry fractionation process produces a DDGS with up to 42% protein (hereinafter called DDGS dryfrac). In embodiments, conventional DDGS and dryfrac under predetermined conditions to rapidly generate a sufficient amount of high protein DDGS (HP-DDGS) for use in bass feeding trials can be compared. In realizations, careful monitoring of the performance of this conversion (through chemical composition changes) is carried out and the parameters with the greatest impact on the quality of HP-DDGS are identified. In some embodiments, low oil DDGS can be used as a substrate for conversion, where such low oil DDGS has a higher protein level than conventional DDGS. In a related aspect, low oil DDGS increases the growth rates of A. pullulans compared to conventional DDGS.
[078] Several groups are evaluating the partial replacement of fish meal by plant-derived proteins such as soybean meal and DDGS. However, low protein content, inadequate amino acid balance, and the presence of anti-nutritional factors have limited replacement levels to 20 to 40%. Preliminary growth trials indicate that no current DDGS or SPC based diets provide similar performance to fishmeal control diets. Several deficiencies have been identified among commercially produced DDGS and SPCs, mainly in protein and amino acid composition, which produce variability in growth performance and fish composition. However, the HP-DDGS and HQSPC diets disclosed in this document containing nutritional supplements (formulated to meet or exceed all requirements) have provided growth results that are similar to or exceed fishmeal controls. Thus, the processes disclosed in this document and the products developed therefrom provide a higher quality HQSPC or HP-DDGS (relative to nutritional requirements) and support growth performance equivalent to or better than diets containing fish meal.
[079] Fish that can feed on the fish feed composition of the present disclosure include, among others, Siberian sturgeon, Sterlet sturgeon, star sturgeon, white sturgeon, Pirarucu, Japanese eel, American eel, short-finned eel, finned eel Long, European Eel, Chanos chanos, Milkfish, Sunfish, Green Sunfish, White Crappie, Black Crappie, Asp, Catla, Golden, Common Goldfish, Mud Carp, Mrigal Carp, Slime Carp, Common Carp, Silver Carp , loggerhead carp, Labeo calbasu, Labeo rohita, Hoven carp, Megalobrama amblycephala, black carp, golden carp, bone-mouth barb, Parabramis pekinensis, Barbonymus gonionotus, java, carp, tench, dojo, Prochilodus magdalenae, bream, tambaqui, pirapitinga, pacu, catfish, channel catfish, catfish, blue catfish, Silurus glanis, panga fish (Swai, Tra, Basa), hull fish, Mudfish, Filipino catfish, Hong Kong catfish, northern catfish African, bighead catfish, vundú, South American catfish, tamoatá, pike, Ayu, salmon, whitefish, pink salmon, Chum salmon, Coho salmon, Masu salmon, rainbow trout, red salmon, Chinook salmon, Atlantic salmon, trout, Arctic salmon, brook trout, brown trout lake, Atlantic cod, kingfish, Scomberoides lysan, sea bass, barramundi/big perch, Nile perch, Murray cod, golden perch, striped bass, Morone chrysops, European sea bass, Hong Kong grouper, grouper, fatty grouper, Plectropomus maculatus, silver perch, white perch, jade perch, largemouth bass, Micropterus dolomieu, European perch, zander (pike perch), yellow perch, sauger, walleye, anchovy, bull's eye, buri, Trachinotus blochii, Trachinotus carolinus, Trachinotus goodei, Trachurus japonicus, Rachycentron canadum, Lutjanus argentimaculatus, guaiuba, hornbeam, Diplodus sargus, Evynnis japonica, sea bream, snapper, Rhabdosargus sarba, Sparus aurata, Sciacaudense cdenequis odenichomas, mangrove soma urophthalmum, Etroplus suratensis, Pelmatolapia mariae, blue tilapia, Oreochromis macrochir, Oreochromis mossambicus, Nile tilapia, tilapia, Oreochromis urolepis, Sarotherodon melanotheron, Tilapia parrelli, Tilapia par- ched, Lapia- roma, taylpilli saliens, Chelon planiceps, Mugil cephalus, Mugil curema, Mugilliza, Dormitator latifrons, Oxyeleotris marmorata, Siganus canaliculatus, Siganus lineatus, Siganus rivulatus, Thunnusmaccoyii, Thunnus thynnus, Climber, Trichopodramus goopodynates, pectoralis pectoralis , Channa striata, Turbot, Paralichthys olivaceus (Japanese language), Paralichthys dentatus, Paralichthys lethostigma, Pseudopleuronectes americanus, Hippoglossus hippoglossus, Rhombosolea tapirina, Solea solea, and combinations thereof.
[080] It will be appreciated by the person skilled in the art that the fish feed composition of the present disclosure can be used as a convenient vehicle for pharmaceutically active substances, such as, for example, antimicrobial agents and immunologically active substances, including vaccines against bacterial or viral infections, and any combination thereof.
[081] The fish feed composition according to the present disclosure may be provided as a liquid, pourable emulsion, or in the form of a paste, or in dry form, for example, as a granulate or pellets, or powder, or as flakes. When the fish feed composition is provided as an emulsion, a lipid-in-water emulsion, it may be in a relatively concentrated form. Such a concentrated emulsion form can also be called a pre-emulsion as it can be diluted in one or more steps in an aqueous medium to provide the final enrichment medium for organisms.
[082] In embodiments, the raw material containing cellulose for the microbial processes as disclosed is corn. Corn has about two-thirds of starch, which is converted during a fermentation and distillation process into ethanol and carbon dioxide. The remaining nutrients or fermentation products can result in condensed distillery solubles or distillery grains, such as DDGS, which can be used in feed products. In general, the process involves an initial preparation step of dry milling or grinding the corn, the processed corn is then subjected to hydrolysis and enzymes are added to break down the main starch component in a saccharification step. The following fermentation step is allowed to proceed after the addition of a microorganism (eg yeast) provided according to an embodiment of the disclosure to produce gaseous products such as carbon dioxide. Fermentation is conducted to produce ethanol that can be distilled from the fermentation broth. The remainder of the fermentation medium can then be dried to produce fermentation products including DDGS. This step generally includes a solid/liquid separation process by centrifugation where a solid phase component can be collected. Other methods including filtration and spray drying techniques can be employed to effect such separation. The liquid phase components can then undergo an evaporation step that can concentrate soluble co-products, such as sugars, glycerol and amino acids, into a material called condensed corn syrup or solubles (CCS). The CCS can then be recombined with the solid phase component to be dried as incubation products (DDGS). It should be understood that the subject compositions can be applied to new or existing ethanol plants based on dry milling to provide an integrated ethanol production process that also generates higher value fermentation products.
[083] In embodiments, the incubation products produced in accordance with the present disclosure have a greater commercial value than conventional fermentation products. For example, incubation products can include improved dry solids with increased amino acid and micronutrient content. A “golden colored” product can thus be provided, which generally indicates greater amino acid digestibility compared to darker colored HQSP. For example, a light colored HQSO can be produced with a higher concentration of lysine in accordance with embodiments herein compared to relatively darker colored products with generally lower nutritional value. Product color can be an important factor or indicator in assessing the quality and nutrient digestibility of fermentation or HQSP products. Color is used as an indicator of exposure to excessive heat during drying, causing caramelization and Maillard reactions of free amino groups and sugars, reducing the quality of some amino acids.
[084] The basic steps in an ethanol manufacturing process by grinding or dry grinding can be described as follows: grinding or grinding corn or other grain product, saccharification, fermentation and distillation. For example, selected whole corn kernels can be ground or crushed with typically hammer mills or roller mills. Particle size can influence cooking hydration and subsequent enzymatic conversion. The ground or ground corn can then be mixed with water to make a puree that is cooked and cooled. It may be useful to include enzymes during the initial steps of this conversion to reduce the viscosity of the gelatinized starch. The mixture can then be transferred to saccharification reactors, held at selected temperatures, such as 140°F, where the starch is converted by the addition of saccharification enzymes to fermentable sugars, such as glucose or maltose. The converted puree can be cooled to desired temperatures, such as 84° F, and fed to fermentation reactors, where fermentable sugars are converted to carbon dioxide by using selected strains of microbes provided according to the revelation that results in more products. of nutritional fermentation compared to more traditional ingredients such as Saccharomyces yeasts. The resulting product can be flash evaporated to separate carbon dioxide and the resulting liquid can be fed to a recovery system consisting of distillation columns and an extraction column. The ethanol stream can be directed to a molecular sieve where the remaining water is removed using adsorption technology. Purified ethanol, denatured with a small amount of gasoline, can produce fuel-grade ethanol. Another product can be produced by further purifying distilled ethanol to remove impurities, resulting in about 99.95% ethanol for non-combustible uses.
[085] All vinasse can be removed from the bottom of the distillation unit and centrifuged to produce wet distillery grains (DWG) and fine vinasse (liquids). DWGs can leave the centrifuge at 55 to 65% humidity, and can be sold as cattle feed or dried as improved fermentation products provided in accordance with the disclosure. These products include an improved final product which may be referred to in this document as dry distillery grains (DDG). Using an evaporator, the thin stillage (liquid) can be concentrated to form distillery solubles, which can be added back, and combined with a dry and distillery grain process stream. This combined product according to embodiments of the disclosure can be marketed as an improved fermentation product having higher amino acid and micronutrient content. It should be understood that various concepts of the disclosure can be applied to other fermentation processes known in the field than those illustrated herein.
[086] Another aspect of the invention is directed to complete fish bran compositions with a better concentration of nutrients that include microorganisms characterized by a higher concentration of nutrients such as, among others, fats, fatty acids, lipids such as phospholipid, vitamins, amino acids essential, peptides, proteins, carbohydrates, sterols, enzymes, and trace elements such as iron, copper, zinc, manganese, cobalt, iodine, selenium, molybdenum, nickel, fluorine, vanadium, tin, silicon, and combinations thereof.
[087] In an incubation process of the present disclosure, a carbon source can be hydrolyzed to its component sugars by microorganisms to produce alcohol and other gaseous products. Gaseous products include carbon dioxide and alcohol includes ethanol. The incubation products obtained after the incubation processes are typically of greater commercial value. In embodiments, the incubation products contain microorganisms that have higher nutrient content than products deficient in the microorganisms. Microorganisms can be present in an incubation system, in the incubation broth and/or in the incubation biomass. The hatching broth and/or biomass can be dried (eg spray dried) to produce hatchery products with a higher nutritional content.
[088] For example, used and dry solids recovered after the incubation process are improved according to the disclosure. These hatchery products are generally non-toxic, biodegradable, readily available, inexpensive, and nutrient-rich. The choice of microorganism and incubation conditions are important in producing a low-toxicity or non-toxic hatching product for use as a food or nutritional supplement. Although glucose is the main sugar produced by the hydrolysis of starch in grains, it is not the only sugar produced in carbohydrates in general. Unlike SPC or DDG produced from the traditional dry milling ethanol production process, which contain a large amount of carbohydrates other than starch (eg as much as 35% cellulose and arabinoxylans measured as neutral detergent fiber, in dry weight), the nutrient-enriched incubation products in question produced by enzymatic hydrolysis of carbohydrates other than starch are more palatable and digestible to non-ruminants.
[089] The nutrient-enriched incubation product of this disclosure may have a nutrient content of at least about 1% to about 95% by weight. Nutrient content is preferably in the range of at least about 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, and 70%-80% by weight. The available nutrient content may depend on the animal it is fed and the context of the rest of the diet, and the stage in the animal's life cycle. For example, beef cattle need less histidine than lactating cows. The selection of the proper nutrient content for feeding animals is well known to those skilled in the art.
[090] Incubation products can be prepared as spray dried biomass product. Optionally, the biomass can be separated by known methods, such as centrifugation, filtration, separation, decantation, a combination of decantation separation, ultrafiltration or microfiltration. Biomass incubation products can be further treated to facilitate rumen diversion. In embodiments, the biomass product can be separated from the incubation medium, spray dried, and optionally treated to modulate ruminal drift, and added to feed as a nutritional source. In addition to producing nutritionally enriched hatchery products in an incubation process containing microorganisms, nutritionally enriched hatchery products can also be produced in transgenic plant systems. Methods for producing transgenic plant systems are known in the art. Alternatively, when the microorganism host excretes the nutritional contents, the nutritionally enriched broth can be separated from the biomass produced by the incubation and the clarified broth can be used as an animal feed ingredient, for example, in liquid form or in dry form by atomization.
[091] The incubation products obtained after the incubation process using microorganisms can be used as a feed for animals or as a food supplement for humans. The incubation product includes at least one ingredient that has an improved nutritional content that is derived from a non-animal source (for example, a bacteria, yeast and/or plant). In particular, the incubation products are rich in at least one or more of fats, fatty acids, lipids such as phospholipids, vitamins, essential amino acids, peptides, proteins, carbohydrates, sterols, enzymes, and trace elements such as iron, copper, zinc, manganese , cobalt, iodine, selenium, molybdenum, nickel, fluorine, vanadium, tin and silicon. In embodiments, the peptides contain at least one essential amino acid. In other embodiments, essential amino acids are encapsulated within an individual-modified microorganism used in an incubation reaction. In embodiments, essential amino acids are contained in heterologous polypeptides expressed by the microorganism. When desired, heterologous polypeptides are expressed and stored in the inclusion bodies in a suitable microorganism (eg, fungi).
[092] In embodiments, the hatchery products have a high nutritional content. As a result, a greater percentage of hatchery products can be used in a complete animal feed. In embodiments, the feed composition comprises at least about 15% incubation product by weight. In a complete feeding, or diet, this material will be administered with other materials. Depending on the nutritional content of the other materials, and/or the nutritional requirements of the animal to which the feed is provided, modified hatchery products can range from 15% of the feed to 100% of the feed. In embodiments, the incubation products in question can provide mixing at a lower percentage due to the high nutrient content. In other embodiments, the incubation products in question can provide very high fraction feed, for example, above 75%. In suitable embodiments, the feed composition comprises at least about 20%, less about 25%, less about 30%, less about 35%, less about 40%, less about 45%, less about 50 %, minus about 60%, minus about 70%, or minus about 75% of the incubation products in question. Commonly, the feed composition comprises at least about 20% of the incubation product by weight. More commonly, the feed composition comprises at least about 15-25%, 25-20%, 20-25%, 30%-40%, 40%-50%, 50%-60%, or 60%-70 % by weight of the incubation product. When desired, the incubation products in question can be used as a single source of food.
[093] Complete fish meal compositions can have improved amino acid content relative to one or more essential amino acids for a variety of purposes, for example, for weight gain and for improving overall animal health. Complete fish meal compositions may have a higher amino acid content due to the presence of free amino acids and/or the presence of proteins or peptides including an essential amino acid, in the incubation products. Essential amino acids can include arginine, cysteine, histidine, isoleucine, lysine, methionine, phenylalanine, threonine, taurine, tryptophan, and/or valine, which can be present in the complete animal feed as a free amino acid or as part of a protein or peptide which is rich in the selected amino acid. At least one peptide or protein rich in essential amino acids can have at least 1% essential amino acid residues per total amino acid residues in the peptide or protein, at least 5% essential amino acid residues per residue of total amino acids in the peptide or protein, or at least 10% of essential amino acid residues per total amino acid residues in the protein. By administering a nutrient-balanced diet to animals, maximum use of the nutritional content is made, requiring less feed to achieve comparable growth rates, milk production, or a reduction in nutrients present in excreta, reducing waste bioburden.
[094] A complete fish bran composition with an improved essential amino acid content may have an essential amino acid content (including free essential amino acid and essential amino acid present in a protein or a peptide) of at least 2.0% by weight with respect to the weight of crude protein and total amino acid content and, more suitably, at least 5.0% by weight with respect to the weight of crude protein and total amino acid content. The complete fish meal composition includes other nutrients derived from microorganisms including, but not limited to, fats, fatty acids, lipids such as phospholipids, vitamins, carbohydrates, sterols, enzymes and trace elements.
[095] The complete fish bran composition can include composition in complete feed form, composition in concentrate form, composition in blend form, and composition in base form. If the composition is in the form of a complete feed, the percent nutrient level, where nutrients are taken from the microorganism in an incubation product, which may be about 10 to about 25 percent, more suitably about 14 at about 24 percent; whereas, if the composition is in the form of a concentrate, the nutrient level can be from about 30 to about 50 percent, more suitably about 32 to about 48 percent. If the composition is in the form of a mixture, the level of nutrients in the composition can be from about 20 to about 30 percent, more suitably about 24 to about 26 percent; and if the composition is in the form of a masterbatch, the level of nutrients in the composition can be from about 55 to about 65 percent. Unless otherwise stated in this document, percentages are stated on a percentage by weight basis. If the HQPC is high in a single nutrient, eg Lys, it will be used as a supplement at a low rate; if it is balanced in amino acids and vitamins, eg vitamin A and E, it will be a more complete diet and will be given at a higher rate and supplemented with a low-protein, low-nutrient feedstock such as corn husks.
[096] The fish meal composition can include a peptide or a fraction of crude protein present in an incubation product having an essential amino acid content of at least about 2%. In embodiments, a peptide or crude protein fraction may have an essential amino acid content of at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, and in achievements, at least about 50%. In embodiments, the peptide can be 100% essential amino acids. Commonly, the fish meal composition can include a peptide or a fraction of crude protein present in an incubation product having an essential amino acid content of up to about 10%. Most commonly, the fish meal composition can include a peptide or crude protein fraction present in an incubation product having an essential amino acid content of about 2-10%, 3.0-8.0%, or 4.0-6.0%.
[097] The fish meal composition can include a peptide or a fraction of crude protein present in an incubation product having a lysine content of at least about 2%. In embodiments, the peptide or crude protein fraction can have a lysine content of at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, and in achievements, at least about 50%. Typically, the fish meal composition can include the peptide or crude protein fraction having a lysine content of up to about 10%. When desired, the fish meal composition can include the peptide or crude protein fraction having a lysine content of about 2-10%, 3.0-8.0%, or 4.0-6.0%.
[098] The fish meal composition can include nutrients in the hatch product from about 1 g/kg dry solids to 900 g/kg dry solids. In embodiments, nutrients in a fish meal composition can be present in at least about 2 g/kg dry solids, 5 g/kg dry solids, 10 g/kg dry solids, 50 g/kg solids dry, 100 g/kg dry solids, 200 g/kg dry solids, and about 300 g/kg dry solids. In embodiments, nutrients can be present in at least about 400 g/kg dry solids, at least about 500 g/kg dry solids, at least about 600 g/kg dry solids, at least about 700 g/kg dry solids, at least about 800 g/kg dry solids, and/or at least about 900 g/kg dry solids.
[099] The fish meal composition can include an essential amino acid or a peptide containing at least one amino acid present in an incubation product having a content of about 1 g/kg dry solids to 900 g/kg dry solids. In embodiments, the essential amino acid or a peptide containing at least one essential amino acid in a fish meal composition can be present in at least about 2 g/kg dry solids, 5 g/kg dry solids, 10 g/kg dry solids, 50 g/kg dry solids, 100 g/kg dry solids, 200 g/kg dry solids, and about 300 g/kg dry solids. In embodiments, the essential amino acid or a peptide containing at least one essential amino acid can be present in at least about 400 g/kg dry solids, at least about 500 g/kg dry solids, at least about 600 g/ kg dry solids, at least about 700 g/kg dry solids, at least about 800 g/kg dry solids, and/or at least about 900 g/kg dry solids.
[0100] The complete fish bran composition may contain a nutrient-enriched incubation product in the form of a biomass formed during incubation and at least one additional nutrient component. In another example, the fish meal composition contains a nutrient-enriched hatchery product that is dissolved and suspended from a hatchery formed during incubation and at least one additional nutrient component. In a further embodiment, the fish bran composition has a crude protein fraction that includes a protein rich in essential amino acids. The fish bran composition can be formulated to provide a better balance of essential amino acids.
[0101] For compositions comprising DDGS, the complete composition form may contain one or more ingredients, such as wheat bran ("wheat mids"), corn, soybean bran, corn gluten bran, distillery grains or distillery grains with solubles, salt, macrominerals, trace elements and vitamins. Other potential ingredients may commonly include, but are not limited to, sunflower bran, malt sprouts and soybean hulls. The composition in blended form can contain wheat bran, corn gluten bran, distillery grains or distillery grains with solubles, salt, macrominerals, trace elements and vitamins. Alternative ingredients would commonly include, among others, corn, soybean meal, sunflower meal, cottonseed meal, malt sprouts, and soybean husk. The composition in base form can contain wheat bran, corn gluten bran, and distillers grains or distillers grains with solubles. Alternative ingredients would commonly include, among others, soybean meal, sunflower meal, malt rootlets, macrominerals, trace elements and vitamins.
[0102] Highly unsaturated fatty acids (HUFAs) in microorganisms, when exposed to oxidizing conditions can be converted to less desirable unsaturated fatty acids or to saturated fatty acids. However, the saturation of omega-3 HUFAs can be reduced or prevented by introducing synthetic antioxidants or natural antioxidants, such as beta-carotene, vitamin E, and vitamin C, into the diet. Synthetic antioxidants, such as BHT, BHA, TBHQ or ethoxyquin, or natural antioxidants such as tocopherols, can be incorporated into food or food products by adding them to the products or they can be incorporated by in situ production in a suitable organism. The amount of antioxidants incorporated in this manner depends, for example, on further use requirements such as product formulation, packaging methods, and desired shelf-life.
[0103] The hatchery products or the complete fish meal containing the hatchery products of the present disclosure can also be used as a nutritional supplement for human consumption if the process starts with human grade input materials, and food quality standards human beings are observed throughout the process. The incubation product or the complete feed as disclosed in this document has a high nutritional content. Nutrients such as protein and fiber are associated with healthy diets. Recipes can be developed to use the incubation product or the complete feeding of the develop in foods such as cereals, biscuits, pies, cookies, cakes, pizza edges, summer sausage, meatballs, shakes, and in any form of edible food. Another choice might be to develop the incubation product or the complete development feed into snacks or a food bar, similar to a granola bar that could be easily consumed, convenient to distribute. A bar food can include protein, fiber, germ, vitamins, minerals, grain, as well as nutraceutical compounds such as glucosamine, HUFAs, or cofactors such as Vitamin Q-10.
[0104] The fish meal comprising the hatching products in question can be additionally supplemented with flavours. The choice of a specific flavor will depend on the animal to which the food is provided. Both natural and artificial flavors and aromas can be used in the manufacture of more acceptable and palatable foods. These supplements can mix well with ingredients and can be made available as a liquid or dry product. Suitable flavors, attractants, and aromas to be supplemented in animal feeds include, among others, fish pheromones, fenugreek, banana, cherry, rosemary, cumin, carrot, peppermint, oregano, vanilla, anise, in addition to rum, acer, caramel, citrus oils, ethyl butyrate, menthol, apple, cinnamon, any natural or artificial combinations thereof. Flavors and aromas can be exchanged between different animals. Likewise, a variety of fruit flavors, artificial or natural, can be added to food supplements comprising the hatching products in question for human consumption.
[0105] The shelf life of the incubation product or the complete feed of the present disclosure may typically be longer than the shelf life of an incubation product that is deficient in the microorganism. Shelf life may depend on factors such as the moisture content of the product, how much air can flow through the feed mass, environmental conditions and the use of preservatives. A preservative can be added to the complete feed to extend shelf life to weeks and months. Other methods to increase shelf life include management similar to silage management, such as mixing with other feeds and packaging, plastic covering or bagging. Cold conditions, preservatives and the exclusion of air from the feed mass prolong the shelf life of wet co-products. Complete feed can be stored in bins or silo bags. Drying the wet incubation product or the complete feed can also increase the shelf life of the product and improve consistency and quality.
[0106] The complete supply of the present disclosure can be stored for long periods of time. Shelf life can be extended by ensiling, adding preservatives such as organic acids, or mixing with other feeds such as soy husks. Goods bins or mass storage sheds can be used to store complete feeds.
[0107] As used in this document,“ambient temperature” is about 25°C under standard pressure.
[0108] The following examples are illustrative and are not intended to limit the scope of the matter disclosed.EXAMPLESEXAMPLE 1. HIGH QUALITY SOYBEAN PROTEIN CONCENTRATE (HQSPC)
[0109] Figure 1 shows the general approach to pre-treat white flakes, convert sugars into cell mass (protein) and gum, recover HQSPC and generate aquaculture feeds (FIGURE 2), and test the resulting aquaculture feeds in fish feeding trials . The white flakes were first subjected to extrusion pretreatment (Brabender Plasti-corder Model PL2000 single-screw extruder, Hackensack, NJ) at 15% moisture content, 50°C, and 75rpm to disturb the structure and allow greater intrusion of enzymes hydrolytics during posterior saccharification. These conditions provided a shear effect against rough channels on both sides of the drum, and it was also previously observed that this resulted in 50 to 70% greater sugar release after enzymatic hydrolysis. The extruded white flakes were then crushed through a 3 mm hammer mill sieve, mixed with water to achieve a 10% solids loading rate and pH adjusted to 5. After heating to pasteurize or sterilize the puree, the puree was cooled to about 50°C and cellulose and oligosaccharide deconstruction enzymes (15 ml total/kg white flakes) were added to hydrolyze the polymers to simple sugars (hydrolysis 4 to 24h). Specific dosages included were 6% Cellic Ctek (per gm glucan), 0.3% Cellic Htek (per gm total solids), 0.015% NOVOZYME 960 (per gm solids). The resulting puree was then cooled to 30°C, pH adjusted to 3 to 5, inoculated with A. pullulans (1% v/v), and incubated for 4 to 5 days at 50 to 200 rpm of mixing and an aeration rate of 0.5 L/L/min to convert sugars to protein and gum. During incubation, samples were periodically removed and analyzed for sugars, cell counts and gum production. After incubation, the pH was raised to 6.5, and ethanol (0.6 L/L of broth) was added to precipitate the gum. Protein, pullulan and microbial mass (HQSPC) were recovered by centrifugation and dried, while the supernatant was distilled to recover ethanol, and the residual liquid was chemically tested for further recycling at the beginning of the process. The HQSPC was then tested in feeding trials with yellow perch, a fish of importance in the regional market. Growth diets were formulated with HQSPC compared to fishmeal and a competing herbal ingredient. Performance (eg, growth, feed conversion, protein efficiency), visceral characteristics, and intestinal histology were examined to assess fish responses. FOR THE PRODUCTION OF HQSPC
[0110] The system contained a 675 L bioreactor, a variable speed progressive cavity pump, a continuous flow centrifuge, and a 1 x 4 meter drying table. Inoculum for use in the 675L bioreactor was prepared in two 5L New Brunswick Bioflo 3 bioreactors. For each assay, 8-10L amounts of inoculum were prepared by growing A. pullulan as described for 2-3 days. This material was used to inoculate larger amounts of extruded and saccharified white flakes prepared in the 675L bioreactor. After incubation, ethanol was added, the puree was centrifuged to recover the wet solids which were then dried and used in fish feeding assays. When monitoring the performance of the conversion process, yield and composition of the HQSPC, several parameters were observed that significantly affected solids recovery. In the larger scale trials, the parameters shown in Table 1 were varied. Table 1. Pre-Pilot scale trial variables and key performance parameters.


[0111] From the yields of HQSPC and protein levels, the following was observed: 1) an incubation pH of 3 to 3.5 and a temperature of 30 to 32°C, with “high aeration maximized the growth of A. pullulans and minimized pullulan production, 2) an incubation time of 4 to 5 days was ideal for protein content and solids recovery, 3) longer incubation times increased protein content but substantially reduced solids recovery, 4 ) shorter incubation times maintained high solids recovery but limited protein content, and 5) due to lack of stachyose and raffinose in the final product, extrusion and/or reduced enzymatic saccharification (omitted) may be possible.
[0112] Preliminary bench scale trials in 5L bioreactors were conducted to optimize the process conditions. A solids loading rate of 10% of extruded white flakes was used and saccharified for 24h, followed by inoculation with A. pullulans and incubated at pH 5, 0.5 L/L/min aeration, 200 rpm agitation for 10 days . The extended incubation time was tested to establish an optimal harvest window to maximize both solids recovery percentage and protein content in solids. Samples (100 ml) were removed daily and, on alternate days, were subjected to the following: Precipitation of all solids with ethanol, centrifugation and drying of solids, measurement of residual solids in the resulting supernatant. Centrifugation of the broth first to recover solids, drying the solids, precipitating pullulan from the resulting supernatant and drying.
[0113] The first ethanol precipitation method recovered about 97% of solids (solids, cells and soy gum) using a laboratory centrifuge (10,000g), with about 3% solids remaining in the fluid phase. The centrifugation method first recovered about 81.7% solids (soy solids and cells), with ethanol precipitation of the supernatant recovering about 14.8% solids (exopolysaccharide), and about 3.5% remaining solids in the fluid.
[0114] Through these bench-scale assays, the levels of protein, pullulan, and total solids that could be recovered each day were measured. It was expected that as the incubation proceeded, protein and pullulan levels would increase, but that the total solids recovered would decrease as some nutrients were catabolized to water and CO2. The average protein levels of the solids from the three replications are shown in Figure 3. Protein levels reached 70% on days 3-5, while the total solids recovered began to fall on days 5-6. Thus, it appears that an incubation time of 4-5 days may be ideal.
[0115] Several differences among commercially available SPCs were previously identified, mainly in protein and amino acid composition and antinutritional properties, which produced variability in growth performance and fish composition. These experiments justified the need to develop SPC products that would support equivalent or better growth performance than diets containing fish meal. A feeding trial was conducted using yellow perch to provide the evaluation of two HQSPC soy products (fermentation trials 5 and 6) in comparison to a commercial SPC and a Hailberry fish meal control.
[0116] Feed Preparations: Seven diets were formulated as follows: Diet 1 = fishmeal controlDiet 2 = commercial SPCDiet 3 = commercial SPC (supplemented with lysine + methionine) Diet 4 = HQSPC assay 5Diet 5 = HQSPC 5 assay (supplemented with lysine + methionine) Diet 6 = HQSPC 6 assay Diet 7 = HQSPC 7 assay (supplemented with lysine + methionine)
[0117] Approximately 12 kg of each diet were prepared, including 2 kg containing 1 g/100g of chromium oxide for digestibility determinations. Test diets were formulated to contain equivalent amounts of SPC with an appropriate protein:lipid target of 42:10. Soy protein concentrate (SPC, for example, from Netzcon Ltd. Rehovot, Israel) with a minimum protein content of 69% is made by aqueous alcohol extraction of degreased unroasted white flakes. SPC is distinctly different from soybean meal in that it contains oligopolysaccharide residues and antigenic substances glycinin and b-conclinin.
[0118] Large particle ingredients were ground with a laboratory scale hammer mill from Fitzpatrick (Elhurst, IL) with 0.51mm screen prior to dry mixing. The dry ingredients were blended for 20 min using a VI-10 blender with an intensifier bar (Vanguard Pharmaceutical Machinery, Inc., Spring, TX). The dry blended raw materials were then transferred to a Hobart HL200 blender (Troy, OH) where oils and water were added and blended for approximately 5 min. The feeds were then screw pressed using a Hobart 4146 crusher with a 3/16” mold and dried under cooled and forced air conditions. After drying, the feeds were ground into pellets using a food processor, sieved to achieve a consistent pellet size, and placed in frozen storage at -20°C. Chemical analyzes of primary protein sources can be seen in Table 2 .Table 2. Composition of primary protein sources (g/100 g, dry matter basis (dmb)) incorporated in experimental yellow perch diets.


[0119] All ingredients are expressed in dry matter with the exception of moisture (as is).
[0120] Analyzes were performed for crude protein (AOAC 2006, method 990.03), crude fat (AOAC 2006, method 9903), crude fiber (AOAC 2006, method 978.10), moisture (AOAC 2006, method 934.01) chromium oxide (AOAC 2006, method 990.08), ash (AOAC 2006, method 942.05), and amino acids (AOAC 2006, method 982.30 E(a,b,c)). PROPERTIES OF PELLETS
[0121] Samples from each diet were analyzed in triplicate for moisture (%), water activity (aw), unit density (kg/m3), pellet durability index (%), water stability (min), and color ( L, a, b); compressive strength (g), and diameter (mm) were determined with n=10 replications. Moisture (%) was obtained using the standard method 2.2.2.5 (NFTA, 2001). The water activity (aw) of 2g pellet samples was measured with a Lab Touch aw analyzer (Nocasina, Lachen SZ, Switzerland). Three color variables were analyzed with a spectrophotocolorimeter (LabScan XE, HunterLab, Reston, VA) such as Hunter L (brightness/darkness), Hunter a (green redness) and Hunter b (yellowish/blue). Unit density (UD) was estimated by weighing 100 ml of pellets and dividing the mass (kg) by 0.0001 m3. The Pellet Durability Index (PDI) was determined according to the standard method S269.4 (ASAE 2003). The PDI was calculated as: PDI (%) = (Ma/Mb) x 100, where Ma was the mass (g) after tipping and Mb was the mass (g) before tipping. Pellet stability (min) was determined by the static (Wstatic) method (Ferouz et al., Cereal Chem (2011)88:179-188) to mimic the leaching of pellets into tanks until they are consumed. Stability was calculated as weight loss by leaching/dry weight of the initial sample. Pellet diameter was measured using a conventional caliper. The pellets were tested for compressive strength using a TA.XT Plus texture analyzer (Scarsdale, NY). FEEDING TEST
[0122] Yellow perch (2.95g ± 0.05 standard error) were randomly stocked in 21 fish/tank in 28 circular tanks (110 liters) connected in parallel to a closed loop recirculating aquaculture system (RAS). RAS water flow and quality was maintained with a centrifugal pump consisting of double solids sup tanks, bioreactor, bead filter, UV filter, and heat pump. The system water was municipal, which is dechlorinated and stored in a 15,200 L tank. Four replications of each treatment were randomly applied in tanks. Water flow was maintained at ~1.5 L/min/tank. The temperature was maintained at 22°C ± 1°. Temperature and dissolved oxygen were measured with a YSI Pro Plus (Yellow Springs Instrument Company, Yellow Springs, OH). Ammonia-nitrogen, nitrite-nitrogen, nitrate-nitrogen, alkalinity (as CaCO3), and free chlorine were tested using a Hach DR 3900 spectrophotometer (Hach Company, Loveland, CO).
[0123] Fish were fed to satiety by hand twice a day, and feeding rates were modified according to pond weights, observed growth rates, and feed consumption ratings. Consumption (%) was estimated from a known number of pellets administered and the count of pellets not consumed 30 min after feeding. Unconsumed feed collections with subsequent dry weights were also used to estimate consumption. Weekly tank consumption estimates were multiplied by weekly reactions to obtain a weekly consumption (g). The palatability of treatments was determined by the amount of food consumed or rejected. Tank mass (+0.01g) was measured once every two weeks to adjust feed rates and calculate performance indices. Individual lengths (mm) and weights (+ 0.01g) were also measured every two weeks in four randomly selected fish from each treatment.
[0124] The feed conversion ratio (FCR) was calculated as:

[0125] The protein conversion ratio was calculated as:

[0126] The Fulton type condition factor (K) was calculated as:

[0127] The specific growth rate (SGR) was calculated as:

[0128] Statistical analyzes of diets and feeding trial responses were performed with analysis of variance (ANOVA, a priori α = 0.05). Significant F tests were followed by a Turkey post hoc test for separate means of treatment. PELLET AND FOOD RESULTS
[0129] Feed formulations were based on HQSPC nutrient analyzes from Trial 3 (Table 1), while all soy protein concentrates were included equally at 45% (100% fish meal replacement) in trial diets. The analyzes of Trials 5 and 6 were completed after the start date of the scheduled feeding trial (Table 2), generally resulting in similar but not isonitrogenous diets. Diets were formulated to contain 42% protein and 10% lipid, with energy-to-protein (E:P) ratios of 7.91 to 7.94 (kcal/g). Recent analyzes showed that crude protein (dmb) was 44.9% (Diet 1), 43.2% (Diets 2 and 3), 36.8% (Diets 4 and 5), 37.5% (Diets 6 and 7). Crude lipid was approximately 10% for all diets. Amino acid analyzes of feeds revealed no potential deficiencies among unsupplemented diets compared to yellow perch requirements (Table 3). Table 3. Experimental design, dietary formulations, and compositions for perch feeding trials.


Special Select, Omega Protein, Houston, TX; b Consumers Supply Distribution, Sioux City, IA; c Bob's Red Mill Natural Foods, Milwaukie, OR; d USB Corporation, Cleveland, OH; and Virginia Prime Gold, Omega Protein, Houston TX; f Thomas Laboratories, Tolleson, AZ; g ARS 702 Premix, Nelson and Sons, Murray, UT; h SS #3 Trace Mix, Nelson and Sons, Murray, UT; i US Nutrition, Bohemia, NY; j Pure Bulk, Roseburg, OR; k DSM Nutritional Products, Parsippany, NJ; l Diamond V Mills, Cedar Rapids, IA; in Fisher Scientific, Pittsburg, PA.
[0130] Pellet feeds exhibited significant differences in measurements between treatments, except for diameters (Table 4).Table 4. Physical properties of feed extrudates.

The values given are means (± standard error) associated with the means of treatment. Values not significantly different (P > 0.05) have the same letter within a given line. MC (% db) = moisture content; aw (-) = water activity; BD (kg/m3) = unit density; CS (g) = compressive strength; PDI (%) = pellet durability index; WSIstill (%) = water solubility index in standing water; L--) = Hunter brightness; a (-) = Hunter yellowish; b (-) = Hunter's redness; Dia. (mm) = diameter.
[0131] The moisture content (MC) ranged from about 8.49% (Diet 1) to about 14.07% (Diet 4). MC contributes to an effect on other characteristics such as PDI, compressive strength, and color. No apparent correlation between MC and other variables was identified.
[0132] Water activity, a measure of unbound water in pellets, was high (about 0.58 to about 0.74), with Diet 1 (fish meal) significantly lower (about 0.58 ) and Diet 7 was significantly higher (about 0.74) than all other treatments. Values above 0.6 indicate low storage stability and may allow microbial growth to proliferate. Feeds were stored in a freezer at -20°C.
[0133] Unit density (BD), a measure of feed weight per unit volume, ranged from about 634.87 to about 695.9. Diet 1 (fish meal) had a lower DB, and although not limited by theory, this is likely due to the lower inclusion of fish oil and fish meal. Soy protein concentrate diets had higher BDs because they contained more oil to alter lipid requirements.
[0134] Compressive force (CS) was calculated as a peak fracture force of the stress-strain curve from a perpendicular axial direction. CS ranged significantly from about 24.36 to about 67.03. Diet 1 (fish bran) exhibited the lowest compressive strength (about 24.36). While not limited by theory, this is likely attributable to the heterogeneity of the pellets, which is a result of the pelletizing process (eg screw-pressed rather than extruded). Extrusion cooks the feed with a combination of moisture, pressure, temperature, and mechanical shear. This process gelatinizes starches, which can substantially increase CS.
[0135] The Pellet Durability Index (PDI) was very high in all diets (about 98.05% to about 99.48%), with no significant difference between treatments. These high PDI values can be the result of high MC as well as addition of carboxymethylcellulose (CMC) binder.
[0136] The Water Solubility Index (WSI) was low across all diets (about 9.65 to about 14.43), with Diet 4 exhibiting the highest value, which was significantly different than the lowest , Diet 7, at about 9.65. Due to the nature of screw-pressed feeds relative to extruded feeds, the WSI was expected to be low. Extruded feeds are more stable in water due to gelatinization of starches, reducing water penetration.
[0137] The Hunter color parameters (L, a, b) revealed similarities with the growth performance. Hunter a (redness) was highest in Diet 1 (5.15) and lowest in Diet 2 (2.80), Hunter b (yellowish) was highest in Diet 4 (22.81) and Diet 1 (22.76) and lowest in Diet 2 (18.07). Hunter L (brightness) was highest on Diet 3 (68.48) and lowest on Diet 1 (47.91). Lighter colored feeds were shown to contain higher concentrations and greater availability of lysine compared to darker feeds. FISH PERFORMANCE
[0138] Diet 6 provided the most comparable results to Diet 1 (fish meal) in growth performance. Growth performance was significantly different between treatments in all categories (Table 5). Table 5: Performance Aspects.
Values of mean weight gain (WG, %), specific growth rate (SGR), survival (S, %), total intake (TC, g), feed conversion ratio (FCR), protein efficiency ratio ( PER), and Fulton-type condition factor (K) for experimental diets fed perch. The values given are means (± standard error) associated with the means of treatment. Values not significantly different (P > 0.005) have the same letter within a given column.
[0139] The supplemented diets (Diets 3 and 5) were slightly better than their non-supplemented counterparts, except for Diets 6 and 7. Diet 6 outperformed Diet 7 in all respects. While not bound by theory, this could be the result of timing issues associated with the bioavailability of crystalline amino acid supplements.
[0140] Weight Gain (WG) was highest for Diet 1 (fish meal at 102.1%), followed by Diets 6 and 7 (84.4% and 61.8%, respectively). Diets 2 and 3 (SPCcommercial) exhibited the lowest WG (12.62% and 23.69%, respectively). The Specific Growth Rate (SGR) paralleled the WG results. The SGR was highest on Diet 1 (1.66) and lowest on Diet 2 (0.55).
[0141] The Protein Efficiency Ratio (PER) was highest in Diet 6 (1.87), followed by Diet 1 (1.76) and Diet 4 (1.70). Diets 2 and 3 had the lowest PERs of 0.98 and 1.17, respectively. PER was better in unsupplemented Diets 4 and 6 than in supplemented diets 5 and 7.
[0142] The feed conversion ratio (FCR) followed a similar pattern, with Diet 1 and Diet 6 being the lowest (1.39 and 1.59, respectively). These FCR values indicate very high nutritional food quality. Diets 2 and 3 had the highest FCR (2.91 and 2.24, respectively).
[0143] Total intake (dmb) was highest with Diet 6 (451.5g) and Diet 1 (450.20g) and lowest with Diet 2 (140.91) and Diet 3 (181.60) . All diets were administered until satiety. Diet consumption was considered indicative of palatability, which was also the reason for the significantly lower survival for fish on Diet 2 (82.29%). Survival was 100% for Diet 1 and 98.81% for Diet 6.
[0144] The Fulton Condition Factor (K) was highest for Diet 5 (1.14) and lowest for Diet 2 (1.01). All HQSPC diets met or exceeded the fishmeal control for this specific performance parameter. Commercial SPC diets were smaller than all diets. OTHER TESTS
[0145] Final assay analyzes may include final growth, FCR, PER, consumption, and examination for nutritional deficiencies via necropsy. Plasma assays can be completed for lysine and methionine using standard methods. Individual fish can be euthanized by cervical dislocation to quantify muscle ratio, hepatosomatic index, viscerosomatic index, fillet composition, and large intestine histology (enteritis inflammation scores) . The protein and energy availability of the test diets can be estimated using a chromium oxide (CrO3) marker within feed and faecal matter. Fecal matter can be collected via necropsy from the lower intestinal tract.
[0146] The apparent digestibility coefficients (ADC) for the nutrients in the test diets can be calculated using the following formula:

[0147] where Dref = % nutrient (kJ/g gross energy) of the reference diet puree (as is) and Dingr = % nutrient (kJ/g gross energy) of the test ingredient (as is). SUMMARY
[0148] Donut-pressed compound feeds for yellow perch were exemplified using diets of HQSPC, a control fish meal, and a commercial soy protein concentrate (SPC). Diets based on HQSPC had a performance comparable to fish meal and outperformed commercial SPC. Growth and conversion performance exceeded expectations as the crude protein content in HQSPC diets was approximately 7% less than fishmeal control and 6% less than commercial SPC. Thus, HQSPC diets can serve as a complete replacement for fish meal.
[0149] In addition to the yellow perch results above, a 90-day feeding trial was conducted using a strain of domesticated rainbow trout (Shasta). Table 6 summarizes protein targets by fish species and size. These targets or dietary protein levels (%) can be used in formulating experimental diets for these commercially important bony fish. Table 6. Protein targets for various bone fish by weight range.

[0150] Trout were fed a control fish meal diet or a diet replacing 70% fish meal (Diet 2 - 35% inclusion) with a HQSPC product (Run 6) (see Table 7). Table 7. Test Diet Formulations Used in a Rainbow Trout Feeding Trial

[0151] The 35% inclusion level was used to exceed the 30% inclusion recommended for other soy protein concentrates such as Selecta SPC60. This trial using isocaloric and isonitrogenous diets demonstrated equivalent performance between the control and test diets provided.
[0152] Table 8 summarizes the rainbow trout performance characteristics observed during the 90-day feeding trial. Diet 2, containing HQSPC, showed no decrease in weight gain or feed conversion/efficiency and no mortalities (100% survival). These observations replicate the results observed in the yellow perch assays. Table 8. Performance assays for rainbow trout using HQSPC.
The values given are means (± standard error) associated with the means of treatment. SUMMARY
[0153] Compost pressed porroca feeds for rainbow trout were exemplified using a diet of HQSPC and a control fish meal. In this trial, diets based on HQSPC had a performance comparable to fish meal. Again, these data show that HQSPC diets exceeded results using SPC (eg, HQSPC can be used at a concentration greater than 30% inclusion level), and can serve as an effective substitute for fish meal. 2. PRODUCTION OF HP-DDGS USING MICROBIAL CONVERSION
[0154] The effects of extrusion on the improvement of DDGS scarification using a single-screw extruder (Brabender Plasti-corder Extruder Model PL2000, Hackensack, NJ) with barrel length to screw diameter of 1:20 and a compression ratio of 3:1 were investigated (Figures 4 and 5). It was determined that 25% DDGS moisture content, temperature 100°C to 160°C, and screw speed of 200 rpm resulted in a sugar recovery of 36% corn fiber (Figure 5). The performance of several Novozyme lignocellulose deconstruction enzymes was evaluated separately, and it was found that 6% Cellic Ctek2 (per gm glucan) and 0.3% Cellic Htek2 (per gm total solids) resulted in sugar recoveries of up to 70%. These pretreatment and saccharification conditions can be used to generate HP-DDGS. Then options such as co-culture with cellulase producers to reduce the need for addition of enzymes can be carried out, as well as the use of fed-batch process bioreactors to reduce processing costs.
[0155] The evaluation of the microbe's growth and gum production in the carbohydrates found in soybean meal was carried out and it was found that the protein content can be increased from 42% to at least 60% using the approach disclosed in this document. This showed that the microbe (eg A. pullulans) can efficiently convert a wide range of difficult-to-metabolize oligosaccharides into cell mass (ie protein) and a microbial gum. This soybean meal effort was initiated on the basis of previous studies that evaluated the production of a range of microbial gums (exopolysaccharides) from corn processing by-products such as whole stillage, thin stillage, and condensed corn solubles. Through this work, a variety of microbial strains have been accumulated that efficiently grow into several corn processing by-products and produce high levels of cell mass and gums from available sugars. Key operating parameters and least cost gum recovery methods were identified and developed. Based on this body of work, and in the knowledge that microbe strains produce a wide range of hydrolytic enzymes, robust processes have been identified which allow the efficient conversion of DDGS to HP-DDGS (eg A. pullulans strain of NRRL No. 50793).
[0156] For pretreatment, conventional, dryfrac and/or low oil DDGS are extruded into a single-screw extruder (Brabender Plasti-corder Model PL2000 Extruder, Hackensack, NJ) with a barrel length to screw diameter of 1:20 and a compression ratio of 3:1. DDGS samples (adjusted to 25% humidity), the temperature of the feed, drum, and outer sections of the extruder is maintained at 100°C to 160°C, and the screw speed is set at 200 rpm, providing a shear effect against rough channels on both sides of the drum. These selected levels of temperature, screw speed and humidity were based on previously defined optimized conditions which resulted in 36% sugar release from the corn fiber due to perturbation of the DDGS matrix.
[0157] Extruded conventional and dryfrac DDGS are mixed with water to achieve a solids loading rate of at least 5% in a 5 L New Brunswick Bioflo 3 bioreactor (working volume 3-4 L) at a pH of 5 .8. After autoclaving and cooling, the slurry is saccharified using a Novozyme enzyme cocktail for which preliminary data has been previously collected. Dosages to be used in initial trials include 6% Cellic Ctek2 (per gm glucan) and 0.3% Cellic Htek2 (per gm total solids). Saccharification is conducted for 24h at 50°C and 150 rpm to solubilize fibers and oligosaccharides into simple sugars. The temperature is then reduced to 35°C, the pH is adjusted to 4.0 (to optimize cell growth), and the slurry is inoculated with 2% (v/v) of a 24-h microbe culture. The slurry is aerated at 0.5 L/L/min and incubation is continued until sugar utilization ceases (96-120h anticipated). The following parameters are then evaluated: 1) replacement of cellulase enzymes with cellulase-producing microbes that would be co-cultured with the microbe; 2) maximization of the initial solids loading rate; e3) addition of more extruded substrate during saccharification and/or the microbial conversion steps (i.e. fed batch process operation) to minimize net enzyme dosage, maximize protein and gum concentrations, and minimize recovery costs of product.
[0158] During incubation, samples are removed at intervals of 6 to 12 h. Samples for HPLC analysis are boiled (to inactivate enzymes), centrifuged, filtered through 0.22 µm filters, placed in auto-display vials, and frozen until the moment of analysis. These samples are evaluated for carbohydrates and organic solvents using a Waters HPLC system. Samples are subjected to microbial counts to assess microbial populations. Samples are also evaluated for cellulose and hemicellulose levels using national renewable energy laboratory procedures.
[0159] The converted slurry is then subjected to ethanol precipitation and centrifugation to separate the protein, microbial gum and microbial biomass (HP-DDGS) from the remaining culture fluid. Although not bound by theory, the presence of a precipitant gum improves the centrifugation efficiency in recovering suspended solids. The composition of HP-DDGS is then determined and used in fish feeding trials. Ethanol is recovered from the liquid stream via distillation, and the residual liquid is chemically analyzed to assess possible uses (eg incorporation into HP-DDGS or biogas production).
[0160] The HP-DDGS thus produced contains a microbial gum to serve as a binding agent and potentially as an immunostimulant. Other hydrolytic enzymes excreted by the microbe should release peptides, amino acids, and any remaining lipids, thus increasing feed absorption, growth performance, and nutrient utilization in fish feeding trials. EVALUATION OF THE PERFORMANCE OF HP-DDGS AS A SUBSTITUTE OF FISH MEAL IN PASS FEEDS
[0161] Conventional HP-DDGS and dryfrac of the above are analyzed for nutritional competences in view of the requirements of the target species, especially focusing on yellow perch. The samples are subjected to chemical analysis: immediate analysis, Van Soest fibers, insoluble carbohydrates, amino acids, fatty acids and minerals. This ensures that nutritional benchmarks are met. Any anti-nutritional properties (eg phytic acid content) of HP-DDGS are compared to current DDGS and provide a basis for further modification of the process.
[0162] Practical complete diets are formulated using conventional HP-DDGS and dryfrac according to known nutrient requirements for yellow loss (eg 42% protein, 8% lipid). Baseline mineral and vitamin premixes for plant diets are used to ensure micronutrient requirements are met. Any supplements (as deemed necessary by analysis) are evaluated against an identical formulation without supplementation; thus, the feeding trial is carried out in a factorial design to take into account the effects of supplementation. All feeding trials include a control diet based on fishmeal and diets containing graded levels of HP-DDGS. Pellets for feeding tests are produced using a laboratory scale single-screw extruder (Brabender Plasti-corder Extruder Model PL2000).
[0163] Replications of four experimental units per treatment are used in all feeding trials (60 to 120 days each). The tests are carried out in 110-L circular tanks (20 fish/tank) connected in parallel to a closed-loop recirculation system consisting of a solid pit, bioreactor and filters (100 μm, carbon and ultraviolet bag) powered by a Centrifugal pump. Heat pumps are used as needed to maintain optimal temperatures for species-specific growth. Water quality (eg dissolved oxygen, pH, temperature, ammonia and nitrite) is monitored in all systems.
[0164] Experimental diets are administered according to the size of the fish, divided into two to five daily feeds. Growth performance is determined by total mass measurements taken in one to four weeks (depending on fish size and trial duration); rations are adjusted according to earnings to allow feeding until satiety and to reduce the waste stream. Consumption is assessed fortnightly from collections of unconsumed feed from individual tanks. Unconsumed feed is dried at a constant temperature, cooled, weighed to estimate conversion efficiency. Protein, energy, and phosphorus digestibility are determined from faecal material manually removed during the midpoint of each experiment or by necropsy of the lower intestinal tract at the conclusion of a feeding trial. Survival, weight gain, growth rate, health indices, feed conversion, protein and energy digestibilities, protein efficiency, and phosphorus utilization are compared across treatment groups. Immediate analysis of necropsied fish is performed to compare fillet composition among dietary treatments. Amino acid and fatty acid analysis is performed as needed for fillet constituents, in accordance with the purpose of the feeding test.
[0165] Statistical analyzes of diets and feeding trial responses are completed with an a priori α = 0.05. Analysis of performance parameters across treatments is done with appropriate analysis of variance or covariance (Proc Mixed) and multiple post hoc comparisons as required. The analysis of fish performance and tissue responses is evaluated by non-linear models. DETERMINATION OF PRELIMINARY MASS BALANCE, ENERGY REQUIREMENTS, AND COSTS
[0166] Inputs and outputs of the conventional HP-DDGS and dryfrac conversion process are monitored and used to establish a process mass balance. Likewise, the energy requirements for the process are measured and/or estimated to calculate total energy usage. Together, these inputs are used to assess preliminary costs, which are compared with the value of conventional HP-DDGS and dryfrac.
[0167] All references cited in this document are incorporated by reference in their entirety.
[0168] From the above discussion, a person skilled in the art can determine the essential characteristics of the invention, and without departing from the spirit and scope of the invention, can carry out various changes and modifications of the realizations to adapt them to different uses and conditions . Thus, various modifications of the embodiments, in addition to those shown and described in this document, will become apparent to those skilled in the art from the above description. Such modifications are also intended to fall within the scope of the appended claims.

权利要求:
Claims (12)
[0001]
1. COMPOSITION COMPRISING A PROTEIN OF NON-ANIMAL ORIGIN, characterized in that said composition contains a protein content, and non-detectable stachyose or raffinose based on dry matter, in which the protein concentrate of non-animal origin is isolated from plant material of the group that consists of soybeans, peanuts, rapeseed, canola, sesame seeds, cotton seeds, palm seeds, barley, grape seeds, olives, safflower, sunflowers, copra, corn, coconut, flaxseed, hazelnuts, wheat, rice, potatoes, cassava, legumes, camelina seeds, mustard seeds, germ, corn gluten bran, distillery/brewery by-products, and combinations thereof, in which the protein content of said composition is in the range of 56% to 90% based in dry matter produced by a process including: a) pre-treatment of plant material by solvent extraction, extrusion, addition of one or more cellulose deconstruction enzymes or a combination thereof to form a puree; b) aino culation of the puree with one or more microbes, which microbe converts released sugars into proteins and exopolysaccharides; c) precipitation of proteins, microbes and resulting exopolysaccharides with ethanol or a flocculant; d) recovery of the precipitated material by hydrodynamic force; and e) drying said precipitated material.
[0002]
2. COMPOSITION, according to claim 1, characterized by comprising one or more microbe(s) selected from the group consisting of Aureobasidium pullulans, Sclerotium glucanicum, Sphingomonas paucimobilis, Ralstonia eutropha, Rhodospirillum rubrum, Kluyveromyces spp, Pichia spp, Trichoderma reesei, Pleurotus ostreatus, Rhizopus spp, and combinations thereof.
[0003]
3. COMPOSITION according to claim 1, characterized in that the plant material is soy in the form of soy flakes or soy bran.
[0004]
4. COMPOSITION according to claim 1, characterized in that the plant material is oilseed.
[0005]
5. COMPOSITION according to claim 1, characterized in that the plant material is dried grains from distillers with solubles (DDGS).
[0006]
6. COMPOSITION according to claim 1, characterized in that the protein concentrate comprises 0.1 g of hydroxylysine/100g of concentrate or more and 1.26 g of lipid/100g of concentrate or more.
[0007]
7. ANIMAL FEED COMPRISING SUCH COMPOSITION, as defined in any one of claims 1 to 6, characterized in that said composition comprises from 35% to 45% of said animal feed by weight.
[0008]
8. ANIMAL FEED, according to claim 7, characterized in that said composition is a complete replacement for fish meal of animal origin in a fish feed, and wherein said animal feed is formulated for fish selected from the group consisting of Siberian Sturgeon, Sterlet Sturgeon, Star Sturgeon, White Sturgeon, Pirarucu, Japanese Eel, American Eel, Shortfin Eel, Longfin Eel, European Eel, Chanos Chanos, Milkfish, Sunfish, Green Sunfish, White Crappie, black crappie, Asp, Catla, golden, common beetle, mud carp, mrigal carp, slime carp, common carp, silver carp, loggerhead carp, Labeo calbasu, Labeo rohita, Hoven carp, Megalobrama amblycephala, black carp, golden carp , bone-mouth barbel, Parabramis pekinensis, Barbonyus gonionotus, java, carp, tench, dojo, Prochilodus magdalenae, bream, tambaqui, pirapitinga, pacu, catfish, channel catfish, catfish, blue catfish, Silurus glanis , foot Panga ixe (Swai, Tra, Basa), shellfish, Mudfish, Filipino catfish, Hong Kong catfish, North African catfish, loggerhead catfish, vundú, South American catfish, tamoatá, pike, Ayu, salmon, whitefish, pink salmon, Chum salmon, Coho salmon, Masu salmon, rainbow trout, red salmon, Chinook salmon, Atlantic salmon, trout, Arctic salmon, brook trout, lake trout, Atlantic cod, kingfish, Scomberoides lysan, sea bass, barramundi/big perch, Nile perch, Murray cod, gold perch, striped bass, Morone chrysops, European bass, Hong Kong grouper, areola grouper, greasy grouper, Plectropomus maculatus, silver perch, white perch , jade perch, largemouth bass, Micropterus dolomieu, European perch, zander (pike perch), yellow perch, sauger, walleye, anchovy, bull's eye, buri, Trachinotus blochii, Trachinotus carolinus, Trachinotus goodei, Trachurusjaponicus, Rachycentron canadum Lutjanus argentimaculatus, guaiuba, poplar, Diplodus sargus, Evynnis japon ica, sea bream, snapper, Rhabdosargus sarba, Sparus aurata, Sciaenops ocellatus, Aequidens rivulatus, Paraneetroplus maculicauda, Cichlasoma managuense, Cichlasoma urophthalmum, Etroplus suratensis, Pelmatolapia mariae, Ore. , Sarotherodon melanotheron, Tilapia rendalli, Tilapia zilli, mullet, Liza macrolepis, Liza parsia, Liza ramada, Liza saliens, Chelon planiceps, Mugil cephalus, Mugil curema, Mugil liza, Dormitator latifrons, Siga canalatus marmoculata, Sigatus marmoratanus Siganus rivulatus, Thunnus maccoyii, Thunnus thynnus, climber, Trichopodus pectoralis, kisser, Osphronemus goramy, Channidae, Channa micropeltes, Channa punctata, Channa striata, Turbot, Paralichthys olivaceus (Japanese tongues lettusses, Paralichsu , Rhombosolea tapirina, Solea solea, and combinations of me smos.
[0009]
9. METHOD OF PRODUCING A PROTEIN CONCENTRATE OF NON-ANIMAL ORIGIN OF SUCH COMPOSITION, as defined in any one of claims 1 to 6, characterized in that it comprises: a. extruding plant material above room temperature to form a puree and transferring the puree to a biological reactor; b. the addition of one or more cellulose deconstruction enzymes to release sugars in the puree in the biological reactor; c. inoculation of the enzyme-treated puree with one or more microbes, which microbe converts released sugars into proteins and exopolysaccharides; d. the precipitation of the resulting proteins, microbes and exopolysaccharides with ethanol, a flocculant, or a combination thereof; e. recovery of precipitated material through hydrodynamic force; and drying said precipitated material.
[0010]
10. METHOD, according to claim 9, characterized in that it further comprises: mixing the extruded materials with water to achieve a solids loading rate of 5% or more in the biological reactor; Optionally, autoclaving and cooling of extruded materials, in which the one or more cellulose deconstruction enzymes are selected from the group consisting of endo-xylanase and beta-xylosidase, glycoside hydrolase, β-glucosidases, hemicellulase activities, and combinations thereof.
[0011]
11. METHOD, according to claim 9, characterized in that it further comprises: the reduction of the temperature of the enzyme-treated puree to between 30°C to 37°C; the inoculation of the cooled puree with 2% (v/v) of a culture 24-hour period of one or more microbes, wherein said one or more microbes is(are) selected from the group consisting of Aureobasidium pullulans, Sclerotium glucanicum, Sphingomonas paucimobilis, Ralstonia eutropha, Rhodospirillum rubrum, Kluyveromyces spp, Pichia spp , Trichoderma reesei, Pleurotus ostreatus, Rhizopus spp, and combinations thereof; optionally, aeration of the inoculated puree at 0.05 L/L/min; and incubation until: i. the use of sugar cease or ii. after 96 to 120 hours of incubation with said one or more microbes.
[0012]
12. METHOD, according to claim 9, characterized in that it further comprises: the addition of 0.6 L of ethanol/L of puree; centrifuging the puree treated with ethanol; and optionally; recovering ethanol from the supernatant; recovering fine particles suspended in said supernatant; drying recovered fine particles; and combining the recovered fine particles with the dry precipitated material.

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EP2785855A4|2015-08-19|

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KR20210000725A|2021-01-05|

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US20210251256A1|2021-08-19|

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BR112014013279A2|2017-06-13|

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JP6243848B2|2017-12-06|

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WO2013082574A2|2013-06-06|

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HK1202897A1|2015-10-09|

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KR20140099528A|2014-08-12|

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US20200329733A1|2020-10-22|

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IL232941A|2020-10-29|

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KR20190077104A|2019-07-02|

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CN107821795B|2021-12-21|

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CA2857667A1|2013-06-06|

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US20160249638A1|2016-09-01|

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IN2014CN04931A|2015-09-18|

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JP2017060493A|2017-03-30|

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CL2014001429A1|2015-02-27|

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EP2785855B1|2018-09-26|

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法律状态:
2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: C12P 21/04 (2006.01), C12N 1/16 (2006.01) |

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2019-08-06| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-12-15| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-04-20| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-06-01| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-07-13| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-08-10| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 02/12/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161566557P| true| 2011-12-02|2011-12-02|

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US201161566487P| true| 2011-12-02|2011-12-02|

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US61/566,557|2011-12-02|

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US61/566,487|2011-12-02|

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PCT/US2012/067508|WO2013082574A2|2011-12-02|2012-12-02|Microbial-based process for high-quality protein concentrate|

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