Microorganism-based process for protein concentrate of improved quality

Abstract

To provide a method for generating a plant-derived protein source which is highly cost effective and sustainable, and is sufficiently high-quality for completely or substantially substituting more of animal protein in an animal food, a product and a use thereof.SOLUTION: Provided is a bio-based process for generating a high-quality protein concentrate (HQPC) by converting plant-derived cellulose and carbohydrate into biologically available protein via aerobic incubation, which includes use of such an HQPC, which is generated in such a manner, as a nutrient including use as a fish meal alternative in aquaculture food.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention generally relates to an incubation process, particularly a microorganism-based incubation process for producing high-quality protein concentrates, including products made therefrom and the use of such products in the formulation of nutritional feeds.

Background Art

[0002] Despite the increasing demand for per capita seafood consumption with the growth of the population, in 2008, approximately 28% of the world's wild marine fish resources were overfished and only 52% were fully utilized. As the wild fish resources decline, commercial aquaculture production has increased dramatically to meet this growing demand. However, fish meal protein, one of the main components of aquaculture diets, also comes from wild capture fisheries. It is estimated that at least 6.7 mmt of fish meal will be required by 2012 to support commercial aquaculture production. This is clearly unsustainable based on current trends.

[0003] Low-cost and more sustainable plant-derived protein sources are being used to replace some of the fishmeal in aquaculture feeds. Defatted soybean meal (SBM, 42 - 48% protein) is commonly used to replace up to 20% of the total protein in the grow-out diets of some species, while soy protein concentrate (SPC, 65% protein) has been successful in trials at higher total protein replacement levels, although this is highly dependent on the nutritional status of the species. These soybean products provide high protein and a relatively good amino acid profile, but still lack some important amino acids (e.g., taurine) required by carnivorous marine fish. SPC can be used at higher levels than soybean meal, mainly because the solvent extraction process used to produce SPC removes antinutritional factors (e.g., oligosaccharides), thereby increasing the biological availability of the protein. In addition, a heating step is used to inactivate heat-labile antigenic factors. The main limitations of the current solvent extraction process are its cost, the fact that the oligosaccharides removed in the process are not utilized, and quality issues where the total protein included in the diet is often limited to 50% at most. Furthermore, processing soybean materials into soybean meal or soy protein concentrate can also be environmentally problematic (e.g., issues with the disposal of chemical waste associated with hexane extraction).

[0004] Producers of animal-based proteins are facing various sustainability risks. By diversifying into sustainable proteins, these businesses can spread these risks and gain access to a rapidly growing market.

[0005] At the same time, food technology innovation is accelerating, creating opportunities for protein production that could disrupt the current industry. For intensive animal-based protein producers, not engaging with this innovation is a risk. Therefore, diversification into alternative (i.e., non-animal) protein production is key both for risk management of resource-constrained supply chains and for seizing opportunities for market growth.

[0006] Meat alternatives and broader protein alternatives that can function as substitutes for conventional animal foods have attracted significant financial investment.

[0007] Therefore, there is a need for plant-derived protein sources that are cost-effective, sustainable, and of high enough quality to fully or substantially replace more of the animal protein in animal diets, including their use in meat alternatives generally from such plant-derived sources (e.g., for human consumption).

SUMMARY OF THE INVENTION

[0008] The present disclosure relates to an organic microorganism-based system for converting plant materials into highly digestible concentrated protein sources that also contain microbial gum (exopolysaccharide) binders, including such concentrated sources suitable for use as animal feed and human food for consumption.

[0009] In a plurality of embodiments, a composition comprising a non-animal protein concentrate, wherein the concentrate contains a fermented plant product containing A. pullulans with a low pullulan yield at a protein content of at least about 65% to about 75% (dry matter basis), and the protein concentrate exhibits one or more of the properties selected from a degree of hydrolysis (DH) of at least about 2%, an ash content of up to 4%, or a potassium and magnesium content of less than about 0.1 ppm, is disclosed.

[0010] In one aspect, A. pullulans produces less than about 3.0 g / L of pullulan when grown in a medium containing 0.35 - 0.5 g / L of yeast extract.

[0011] In another aspect, the non-animal protein concentrate is isolated from plant materials including soybeans, sorghum, peanuts, legumes, rapeseed, triticale, barley, rye, lupinus, broad beans, canola, peas, sesame, cottonseed, palm kernel, barley, grape seeds, olives, safflower, sunflower, copra, corn, coconuts, flaxseeds, hazelnuts, wheat, rice, potatoes, cassava, leguminous plants, camelina seeds, nasturtium seeds, mustard seeds, wheat germ meal, corn gluten meal, corn gluten feed, distillery / brewing by-products, and combinations thereof.

[0012] In a related aspect, the plant material is derived from soybeans in the form of soy flakes or soy meal.

[0013] In one aspect, a feed or foodstuff comprising the above composition is disclosed.

[0014] In a related aspect, the composition is combined with one or more meat substitutes. In a further related aspect, the meat substitutes include frozen tofu that has been thawed and sliced, onchom, tempeh, tofu, tofurkey, artificial turkey, paneer, glamorgan, breadfruit seeds, sapal, eggplant, jackfruit, falafel, cancer imitation, and combinations thereof. In a further related aspect, the concentrate improves one or more of the sensory properties of one or more meat substitutes, including texture, aroma, mouthfeel, crunch, flavor, appearance, or combinations thereof, compared to the same meat substitutes lacking the concentrate. In another related aspect, the foodstuff is for human consumption.

[0015] In one aspect, the feed is formulated for animals including fish, shellfish, crustaceans, breeding animals, livestock, and combinations thereof.

[0016] In another aspect, A. pullans is NRRL-Y-2311-1.

[0017] In one aspect, 4664 cm of the raw NIR spectrum for the final product -1~4836 cm -1 There is a significant downward shift compared to the feedstock. In a related aspect, the downward shift is at least about 10% to about 20%.

[0018] In a plurality of embodiments, a method of treating a plant material, comprising: a) transferring the plant material to a first mixing tank, wherein the plant material is mixed with one or more first solvents to produce a washed mash; b) separating the washed mash into at least one centrifugate and a washed cake; c) transferring the washed cake to one or more second mixing tanks, wherein one or more second solvents are mixed with the washed cake to produce a washed cake suspension; d) transferring the washed cake suspension to one or more fermenters, wherein the transferred washed cake suspension is inoculated with at least one microorganism and the inoculated washed cake suspension is incubated for a sufficient time to produce a fermented mixture; e) heating the fermented mixture for a sufficient time to achieve a degree of hydrolysis (DH) of about 2% to about 80% for the protein therein; f) separating the heated fermented mixture into a fermented centrifugate and a fermented cake; g) transferring the fermented centrifugate to (i) the first mixing tank and / or (ii) one or more second mixing tanks such that the mixing tank contains the plant material or the washed cake, and steps (c)-(f) and h) are repeated at least once for sub-step (i) or (ii); and h) drying the fermented cake, wherein at least one microorganism does not produce sufficient exopolysaccharide to produce a viscous fermented cake during drying, and the resulting dried fermented cake has a higher protein content and / or substantially reduced antinutritional factors compared to the transferred plant material. A method is disclosed.

[0019] In one aspect, at least one microorganism produces less than about 3 g / L of pullulan when grown in a medium containing 0.35 to 0.5 g / L of yeast extract.

[0020] In another aspect, the method further includes transferring at least one of the centrifugates of step (b) to one or more of the mixing tanks before inoculation.

[0021] In one aspect, by reusing the centrifugate, a) the amount of fresh solvent added to the first mixing tank and / or one or more second mixing tanks is reduced, and / or b) the protein yield and recovery rate are increased.

[0022] In another aspect, the method does not include the addition of cellulolytic enzymes.

[0023] In one aspect, the method further includes heating the washed cake suspension before transferring it to one or more fermenters. In a related aspect, the washed cake suspension is heated to 100 °C or higher.

[0024] In another aspect, the fermentation centrifugate is transferred to the first mixing tank.

[0025] In a related aspect, the centrifugate and the cake are generated by fluid force, and the method includes a system in which four mixing tanks and four centrifuges are continuous. Before the second fermentation, the fermentation centrifugate of mixing tank 4 is transferred to mixing tank 3, the centrifugate of mixing tank 3 is transferred to mixing tank 2, and the centrifugate of mixing tank 2 is transferred to mixing tank 1.

[0026] In one aspect, the solvent includes water, an acid, an aqueous enzyme mixture, an antifoaming agent, or a combination thereof, and the aqueous enzyme mixture includes phytase.

[0027] In another aspect, the centrifugate from the first mixing tank, the fermented centrifugate, or both, is transferred to at least one evaporator that produces a liquid protein condensate. In a related aspect, the centrifugate is evaporated at a temperature of about 60 °C to 90 °C and / or at about 1 psia to 6 psia.

[0028] In one aspect, the non-animal protein concentrate is isolated from plant materials including soybeans, sorghum, peanuts, legumes, rapeseed, triticale, barley, rye, lupinus, fava beans, canola, peas, sesame, cottonseed, palm kernel, barley, grape seeds, olives, safflower, sunflower, copra, corn, coconuts, flaxseeds, hazelnuts, wheat, rice, potatoes, cassava, leguminous plants, camelina seeds, mustard seeds, germ meal, corn gluten meal, distillery / brewery by-products, and combinations thereof.

[0029] In another aspect, the drying is carried out at a temperature higher than 100 °C, and the dried fermented cake exhibits a water content of less than about 7%.

[0030] In one aspect, at least one microorganism is NRRL Y-2311-1. In another aspect, the microorganism can be identified by targeting the presence of an amplification product from SEQ ID NO: 1 as a template via PCR. In a related aspect, the amplification product can be used to identify the source of the HQPC disclosed herein.

[0031] In one aspect, for the raw NIR spectrum of the final product at 4664 cm -1 ~4836 cm -1 a significant downward shift is observed compared to the feedstock. In a related aspect, the downward shift is at least about 10% to about 20%.

[0032] In another aspect, the treatment does not include adding one or more cellulolytic enzymes.

[0033] In multiple embodiments, compositions are disclosed that include a solid protein concentrate produced by the above-described method.

[0034] In multiple embodiments, feeds or foodstuffs are disclosed that include the above-described composition.

[0035] In a related aspect, the feed is formulated for animals including fish, shellfish, crustaceans, breeding animals, livestock, and combinations thereof. In a further related aspect, the composition is for human consumption.

[0036] In multiple embodiments, a method is disclosed for improving the survival rate of juvenile shrimp, the method comprising feeding the juvenile shrimp the above-described feed, wherein the degree of hydrolysis (DH) of the protein in the feed by shrimp enzymes is at least 7%. In a related aspect, the predicted apparent protein digestibility (PPD) of the feed is at least 90%.

[0037] In multiple embodiments, a feed for juvenile shrimp is disclosed that includes the above-described composition and exhibits a degree of hydrolysis (DH) of at least 7%, a predicted apparent protein digestibility (PPD) of at least 90%, or a combination thereof.

Brief Description of the Drawings

[0038]

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Modes for Carrying Out the Invention

[0039] Before describing the compositions, methods, and methodologies, it should be understood that the present invention is not limited to the specific compositions, methods, and experimental conditions described, as such compositions, methods, and conditions can vary. Also, since the scope of the present invention is limited only to the appended claims, it should be understood that the terms used herein are for the purpose of describing only particular embodiments and are not intended to be limiting.

[0040] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a formulation" includes one or more formulations and / or compositions of the kind described herein that would be apparent to one of ordinary skill in the art upon reading this disclosure.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Modifications and variations are understood to be included within the spirit and scope of the disclosure, so any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0042] As used herein, "about", "approximately", "substantially", and "significantly" are considered to be understood by those of ordinary skill in the art and will vary somewhat depending on the context in which they are used. Considering the context in which these terms are used, where the use of a term is not clear to one of ordinary skill in the art, "about" and "approximately" are considered to mean within plus or minus 10% of the particular term, and "substantially" and "significantly" are considered to mean more than plus or minus 10% of the particular term.

[0043] Internal research to develop a practical diet for RAS operations using the described microbial enhanced protein has shown that the HQPC disclosed herein is a promising solution for the production of environmentally friendly aquaculture feeds. In addition to having a crude protein content of over 70% and a highly available phosphorus content, the HQPC disclosed herein can also be manufactured from non-GM soybeans, making it optimal for the European aquaculture industry. Results from numerous in-house feeding trials have demonstrated that the HQPC disclosed herein can maintain the health, high performance growth, and feed efficiency of fish and shrimp at very high dosage levels reaching up to 70% of the total amount of ingredients in the feed. Feeding trials using rainbow trout, barramundi, and coho salmon have shown that fish fed the disclosed HQPC-based feeds consistently utilized the feed more efficiently than fish fed the control feed. In tests conducted using such species reared in RAS systems, good growth rates were shown and feed conversion rates decreased when feeds containing the disclosed HQPC at levels up to 25% were fed.

[0044] The central problems faced by sustainable RAS relate to two interrelated limitations that operators need to balance to succeed in aquaculture operations. The first limitation relates to the understanding of current water reuse systems that limit the production of fish or shrimp. The second limitation is centered on sustainable fish feeds, and the use of soybean-based feeds in such current water reuse systems makes management more difficult. The resulting situation requires current aquaculture operators to balance the risks and benefits of each limitation in order to meet customer demands while achieving profitability.

[0045] Formulating aquaculture feeds with less fishmeal for RAS systems requires the use of combinations of several ingredients, because most feeds have been shown to have significant limitations with respect to nutrition and functionality. Fermentation of plant ingredients can reduce anti-nutritional factors and improve digestibility. Without wishing to be bound by theory, the large differences observed in the growth performance of fish and shrimp given HQPC in this disclosure are probably due to the removal of anti-nutritional factors and the improvement in the digestibility of the ingredients. The results presented herein show that at least 80% of the dietary fishmeal can be directly replaced with HQPC, as described for shrimp larvae and some juvenile and adult fish species in particular, as well as for commercially available feeds.

[0046] As used herein, the term "animal" means any organism belonging to the animal kingdom, including humans, birds (e.g., poultry), mammals (e.g., humans, cows, pigs, goats, sheep, cats, dogs, mice, and horses), and aquaculture organisms such as fish (e.g., trout, salmon, sea bream), mollusks (e.g., clams), and crustaceans (e.g., crabs, lobsters, prawns, and shrimp), but not limited thereto.

[0047] The use of the term "fish" includes all vertebrate fish, which may be either bony fish or cartilaginous fish.

[0048] As used herein, "non-animal protein" means that the substance contains at least 0.81 g of crude fiber per 100 g of composition (dry matter basis), which crude fiber is mainly cellulose and lignin material obtained as a residue in the chemical analysis of plant substances.

[0049] As used herein, the term "incubation process" means providing appropriate conditions for the growth and development of bacteria or cells, such bacteria or cells using biosynthetic pathways to metabolize various feedstocks. In multiple embodiments, the incubation process may be carried out, for example, under aerobic conditions. In other embodiments, the incubation process may include fermentation.

[0050] As used herein, the term "incubation product" means any residual material that results directly from an incubation process / reaction. In some cases, the incubation product contains microorganisms, whereby the nutrient content in this product is enhanced compared to an incubation product lacking such microorganisms. The incubation product may contain suitable components from the incubation broth. For example, the incubation product may include dissolved and / or suspended components from the incubation broth. The suspended components may include undissolved soluble components (e.g., the solution is supersaturated with one or more components, e.g., proteins) and / or insoluble materials present in the incubation broth. The incubation product may include substantially all of the dry solid material present at the end of incubation (e.g., by spray drying the incubation broth and the biomass produced by incubation), or a portion thereof. The incubation product may include crude material from the incubation, and the microorganism / solid / centrifugate / cake can be fractionated and / or partially purified to increase the nutrient content of the material.

[0051] As used herein, "conversion culture" means a culture of microorganisms contained in a medium containing materials sufficient for the growth of the microorganisms, such as water and nutrients. The term "nutrients" means any substance having nutritional value. This may also be part of animal feed, food for animals or dietary supplements. Exemplary nutrients include, but are not limited to, proteins, peptides, fats, fatty acids, lipids, water-soluble vitamins and fat-soluble vitamins, essential amino acids, carbohydrates, sterols, enzymes and trace minerals such as phosphorus, iron, copper, zinc, manganese, magnesium, cobalt, iodine, selenium, molybdenum, nickel, fluorine, vanadium, tin, silicon, and combinations thereof.

[0052] Conversion is the process of culturing microorganisms in a conversion culture under conditions suitable for converting protein / carbohydrate / polysaccharide materials, such as soybean constituents, into high-quality protein concentrates. Appropriate conversion involves utilizing 90% or more of a specific carbohydrate to produce a microbial cell population and / or extracellular polysaccharides, enzymes and microbial metabolites, a specific decrease in the oligosaccharide concentration, achieving a selected degree of hydrolysis of the protein, a specific % change in the NIR spectrum at 4664 cm -1 ~4836 cm -1 or a combination thereof, but is not limited thereto. In multiple embodiments, the conversion may be aerobic or anaerobic, or a combination thereof.

[0053] As used herein, "flocculant" or "clarifying agent" means a chemical substance that promotes the precipitation of colloids from a suspension by aggregation, including but not limited to polyvalent ions and polymers. In multiple embodiments, such flocculants / clarifying agents may include biological flocculants, such as extracellular polysaccharides (e.g., pullulan).

[0054] As used herein, "formulation" means a material or mixture prepared according to a specific formulation.

[0055] As used herein, "foodstuff" means a substance suitable for consumption as a nutritional composition that humans or animals eat or drink, or that plants absorb to maintain life and growth.

[0056] As used herein, "centrifugate" means a liquid separated from a fluid stream after most of the solids have been removed, and the resulting dry product is referred to as "cake".

[0057] As used herein, "suspension" means a heterogeneous mixture containing solid particles large enough to sediment.

[0058] As used herein, "evaporation" means the process of changing from a liquid to a vapor. The main difference between evaporation and distillation is that evaporation is a process involving a change in the state of a substance, while distillation is a process of separation. Both of these processes can be used for various purposes. Vaporization in evaporation occurs below the boiling point, while vaporization in distillation occurs at the boiling point.

[0059] As used herein, "exopolysaccharide" means a high-molecular-weight polymer containing sugar residues that is produced by a microorganism and released into the surrounding environment, and the high-molecular-weight polymer includes assimilation products and metabolites.

[0060] As used herein, "degree of hydrolysis (DH)" means the proportion of peptide bonds cleaved in a protein hydrolysate. There are several methods for determining DH, and the most commonly used ones are pH-stat, trinitrobenzenesulfonic acid (TNBS), o-phthalaldehyde (OPA), trichloroacetic acid-soluble nitrogen (SN-TCA), and the official titration method.

[0061] As used herein, "trypsin inhibitor unit (TIU)" means the amount of trypsin inhibitor in a sample. For example, in one method, N-benzoyl-DL-arginine p-nitroanilide can be used as a chromogenic substrate for trypsin, and the ability of an aliquot of a soybean meal extract to inhibit the activity of trypsin against this substrate is used to estimate the amount of trypsin inhibitor in the soybean meal sample. The amount of p-nitroaniline formed during a 10-minute incubation is measured spectrophotometrically, and the absorbance values in the presence and absence of the soybean extract are used in calculations that give the number of trypsin inhibitor units (TIU) per gram of the original soybean sample.

[0062] As used herein, "meat substitute" or "meat analogue" means a composition that mimics certain aesthetic qualities (mainly texture, flavor, and appearance) or chemical properties of a particular meat. In multiple embodiments, such substitutes or analogues include dairy-based ones: paneer cheese, gramorgan sausage, paneer; fungus-derived ones: edible mushrooms, mycoprotein, beefsteak fungus (fistulina hepatica), shimeji mushroom (lyophyllum decastes); fruit-based ones: tempeh, breadfruit, coconut burger, green jackfruit pulp, eggplant, jackfruit; legumes: Burmese tofu, falafel, ganmodoki, freeze-dried tofu, onchom (red onchom and black onchom), tempeh burger, plant-based protein artificial meat, tofurkey or artificial turkey, vegetarian bacon, vegetarian hot dog, vegetarian sausage, and veggie burger, but are not limited thereto. In one aspect, the protein concentrate disclosed herein is combined with a meat substitute.

[0063] As used herein, "NIR (near-infrared spectroscopy)" is a non-invasive detection method for measuring protein content.

[0064] As used herein, "hydrodynamic force" means the energy acting on a solid immersed in a fluid and moving relative to the fluid. In a related aspect, such forces may be applied through processes including, but not limited to, centrifugation and filtration.

[0065] As used herein, "cellulolytic enzyme" means an enzyme that acts by hydrolyzing glycosidic bonds of linear glucose β-1,4-linked polymers to produce glucose and other simple or complex sugars.

[0066] As used herein, "antifoaming agent" or "defoaming agent", including its grammatical variations, is a chemical additive that reduces and prevents the formation of foam in industrial process liquids. In a related aspect, such chemicals include, but are not limited to, petroleum-based defoaming agents; powder defoaming agents; aqueous defoaming agents; silicone-based defoaming agents; EO / PO-based defoaming agents and alkyl polyacrylates. In a related aspect, such defoaming agents include aqueous food-grade emulsions designed to control foam in aqueous food canning processes, non-aqueous silicone-free defoaming agents utilizing defoaming polymers and biodegradable oils, and food-grade 100% active food-grade kosher defoaming agents designed to break foam in aqueous environments including food manufacturing, fermentation, agricultural and industrial-grade processes.

[0067] As used herein, "predicted apparent protein digestibility (PPD)" is a measure of the regression calculation between the in vivo apparent protein digestibility (APD) of a feeding ingredient by different digestive enzymes and the in vitro protein digestion (e.g., degree of hydrolysis).

[0068] As used herein, "room temperature" is about 25 °C under standard pressure.

[0069] As used herein, "APD" is a measure of the ratio of the difference between the nitrogen ingested and the nitrogen in the feces to the nitrogen ingested, expressed as a percentage.

[0070] As used herein, "HQPC" means a high-quality protein concentrate from one or more fermented plant materials. Such HQPC can be used as a feed, a supply ingredient, alone or in combination with other feeds or supply ingredients, including as a probiotic or as a component thereof, and as a means of delivering nutritional supplements and / or pharmaceuticals to animals. In multiple embodiments, the protein content of the HQPC may be from about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75% or more (dry matter basis (dmb)).

[0071] As used herein, "solvent" means a substance, usually a liquid, in which other substances dissolve to form a solution. Polar solvents (e.g., water, aqueous solutions) are favorable for ion formation, and nonpolar solvents (e.g., hydrocarbons) are not. Solvents may be predominantly acidic, predominantly basic, amphoteric, or aprotic. Organic compounds used as solvents include, but are not limited to, aromatic compounds and other hydrocarbons, alcohols, esters, ethers, ketones, amines, and nitrated and halogenated hydrocarbons.

[0072] Plant protein sources In connection with the present disclosure, a number of plant protein sources can be used as feedstocks for conversion. The main reason for using plant proteins in the feed industry is to replace more expensive protein sources, such as animal protein sources. Another important factor is the risk of disease transmission by feeding animal protein to animals of the same species.

[0073] Examples of plant protein sources include, but are not limited to, plants of the family Fabaceae, such as soybeans and peanuts; plants of the family Brassiciaceae, such as canola; plants of the family Asteraceae, such as cottonseed, sunflower; plants of the family Arecaceae, such as copra. These protein sources are generally also defined as oilseed proteins and may be given whole, but more commonly are given as a byproduct after the oil has been removed. Other plant protein sources include grains and cereals, particularly plant protein sources of the family Poaceae, also known as Gramineae, such as corn, wheat and rice, or other major crops, such as potatoes, cassava, and legumes (round and oval beans), milling byproducts including germ meal or corn gluten meal, or distillery / brewing byproducts. In multiple embodiments, the protein feedstock includes, but is not limited to, plant materials derived from soybeans, corn, peanuts, legumes, rapeseed, triticale, barley, rye, canola, sesame, cottonseed, palm kernel, grape seed, olive, safflower, sunflower, copra, coconut, flaxseed, hazelnut, wheat, rice, potato, cassava, leguminous plants, camelina seeds, mustard seeds, karashi seeds, wheat germ meal, corn gluten meal, corn gluten feed, distillery / brewing byproducts, and combinations thereof.

[0074] Major plant-derived proteins reported for use in agriculture include soybean meal (SBM), maize gluten meal, rapeseed / canola (Brassica sp.) meal, lupinus (Lupinus sp.), e.g., the proteins in the kernel meal of dehulled white lupin (Lupinus albus), sweet lupin (L. angustifolius) and yellow lupin (L. luteus), sunflower (Helianthus annuus) seed meal, crystalline amino acids; as well as pea (Pisum sativum) meal, cottonseed (Gossypium sp.) meal, Lemnoidae (duckweed or watermeal), peanut (Arachis hypogaea) meal and oil cake, soy protein concentrate (SPC), soy protein isolate (SPI), maize (Zea mays) gluten meal and wheat (Triticum aestivum) gluten, potato (Solanum tuberosum L.) protein concentrate, and other plant-based feedstuffs such as the leaves of Moringa oleifera Lam., are included in various concentrations and combinations, but are not limited to these.

[0075] Protein sources may be in the form of untreated plant material, as well as processed and / or extracted plant proteins. For example, heat-treated soybean products have high protein digestibility.

[0076] The protein material includes all kinds of proteins or peptides. In multiple embodiments, a soybean material such as, for example, whole soybeans may be used. The whole soybeans may be standard commercialized soybeans; soybeans genetically modified (GM) in some manner; or non-GM identity-preserved (IP) soybeans. Exemplary GM soybeans include, for example, soybeans engineered to produce carbohydrates other than stachyose and raffinose. Exemplary non-GM soybeans include, for example, the Syngenta varieties bred for low carbohydrate and low trypsin inhibitor. High protein varieties include, but are not limited to, N2358 (Benson Hill Inc., St. Louis, MO).

[0077] Other types of soybean materials include soy protein powder, soy protein concentrate, soy meal and soy protein isolate, or mixtures thereof. Traditional processing to convert whole soybeans into other forms of soy protein, such as soy protein powder, soy protein concentrate, soy meal, soy protein isolate, includes crushing the cleaned raw whole soybeans into several pieces, typically 6 to 8, to form soy chips and hulls, and then removing the hulls. The soy chips are then pretreated at about 60°C and formed into flakes about 0.25 millimeters 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 powder, soy protein concentrate, and soy protein isolate, it is important to desolventize the flakes in a manner that minimizes the amount of cooking or toasting of the soy protein to keep the content of water-soluble soy protein high. This is typically achieved by using a steam desolventizer or a flash desolventizer. The flakes obtained in this step are generally referred to as "edible defatted flakes" or "white soy (bean) flakes".

[0078] White soybean flakes, the starting material for soy protein flour, soy protein concentrate, and soy protein isolate, have a protein content of approximately 50%. The white soybean flakes are then first ground into coarse particles, usually in an open-loop grinding system, by hammer mills, classifier mills, roller mills, or impact pin mills, and then further ground into soy flour with the desired particle size. Screening is typically used to size the product to a uniform particle size range, which can be achieved with vibrating screens or cylindrical centrifugal separators. Other oilseeds can be processed in a similar manner.

[0079] In addition to glycinin and β-conglycinin, soybeans contain minor but very important 2S albumin storage proteins. Soybeans also contain biologically active or metabolic proteins, such as enzymes, trypsin inhibitors, hemagglutinin, and cysteine ​​proteases similar to papain.

[0080] Soy products have high protein digestibility, but for example, the upper limit for full fat or defatted soybean meal in the diet of carnivorous fish is 20-30% inclusion level, even if heat labile antinutrients are removed. In fish, soy protein has been shown to cause intestinal damage when fed to fish at protein concentration inclusion levels above 30% and generally reduces growth performance in various fish species. In fact, most fish farmers are reluctant to use more than 10% plant protein in the total diet due to these effects.

[0081] The present invention solves this problem and enables plant protein content levels of up to 40%, and even up to 50%, which depends, among other factors, on the animal species being fed, the origin of the plant protein source, the ratio of the various plant protein sources, the protein concentration, as well as the amount, origin, molecular structure and concentration of glucan and / or mannan. In a plurality of embodiments, the plant protein content level is up to 40%, preferably up to 20% or 30%. Typically, the plant protein present in the diet is 5 to 40%, preferably 10 to 15% or 10 to 30%. These percentages define the percentage amount of the total plant protein source in the animal feed or diet, which includes fat, ash, etc. In a plurality of embodiments, the level of pure protein is up to 50%, typically up to 45%, and in a plurality of embodiments is 5 to 95%.

[0082] The ratio of plant protein to other proteins in the entire feed or diet may be 5:95 to 95:5, 15:85 to 50:50, or 25:75 to 45:55.

[0083] In addition to providing food, the HQPCs disclosed herein may be formulated for use with meat substitutes. In a plurality of embodiments, the HQPCs may be combined with fermented soybean-based products. Examples of fermented soybean-based products include: thawed and sliced frozen tofu; oncom, one of the traditional staple foods of West Java (Sunda) cuisine in Indonesia, which comes in two types, red oncom and black oncom (oncom is closely related to tempeh, both being foods fermented using mold); soy protein; soy pulp used in vegetable burgers and croquettes); tempeh, a traditional Indonesian soybean product made from fermented soybeans; textured vegetable protein, a defatted soybean flour product that is a byproduct of soybean oil extraction and is often used as a meat analog or meat extender, with a protein content comparable to that of certain meats); tofu (although not traditionally considered a meat substitute in Asia, it is widely used for that purpose in the Western Hemisphere); and tofurkey, a vegetarian protein loaf or casserole-shaped meat substitute usually made from tofu (soy protein) or wheat gluten (wheat protein), accompanied by a stuffing made from grains or bread, flavored with gravy, herbs, and spices; paneer cheese; gramorgan sausage; mushrooms including, but not limited to, Ganoderma lucidum, Laetiporus spp., and Pleurotus ostreatus; breadfruit; coconut burger (made from sapi, a coconut pulp byproduct of traditional coconut milk extraction); eggplant; jackfruit; falafel; and mock meat, among others.

[0084] In multiple embodiments, the disclosed HQPC composite fermented soybean product may be used alone as a meat substitute or in combination with other meat substitutes or meat analogs to produce various products for human consumption, including various combinations including the above examples. In one aspect, the addition of HQPC is combined with various meat substitutes and / or meat analogs to improve the texture, aroma, mouthfeel, chewiness, crispness, flavor, or appearance of the meat substitute and / or meat analog. Such aesthetic senses may be determined, for example, using Caswell's food quality classification (see, for example, Caswell, J Agr Res Econ (1998) 42:409-474).

[0085] Microorganisms The disclosed microorganisms must be able to convert carbohydrates and other nutrients into high-quality protein concentrates in the processes as disclosed herein. In multiple 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 microorganisms. Generally, exemplary microorganisms include microorganisms capable of metabolizing stachyose, raffinose, starch, glucose, fructose, lactose, sucrose, xylose, and other sugars. However, based on the disclosed methods, selecting other suitable microorganisms without undue experimentation is within the ability of those skilled in the art.

[0086] In multiple embodiments, the microorganism exhibits low extracellular polysaccharide (e.g., pullulan) production. For example, low pullulan yield is considered to be less than about 3.0 g / L when grown in a yeast extract (YE)-containing medium (as a nitrogen source) where yeast extract (YE) is present at 0.35 - 0.5 g / L (see, e.g., page 232, column 2, paragraph 3, and Table 3 of Leathers et al., J Indus Micro (1988) 3:231 - 239, which is incorporated herein by reference). Without wishing to be bound by theory, high extracellular polysaccharide-producing bacteria produce end products that can be difficult to dry (e.g., extended drying times) and / or produce viscous products after drying. The method for determining pullulan content can be used as disclosed in Leathers et al., J Indus Micro (1988) 3:231 - 239, page 232, columns 1, paragraphs 3 through 2, column 1, which is incorporated herein by reference, although those skilled in the art will recognize that alternative methods are available.

[0087] In multiple embodiments, A. pullulans adapts to various environmental / stress factors encountered during conversion. In one aspect, the A. pullulans strain is selected from NRRL deposit numbers Y - 2311 - 1, Y - 6754a, YB - 4026, YB - 4588, Y - 6992, Y - 17000, or Y - 17001, and combinations thereof. In a related aspect, the A. pullulans strain represented by NRRL deposit number Y - 2311 - 1 can be used as disclosed herein.

[0088] In a related aspect, the microorganism can be identified using PCR. In multiple embodiments, the organism can be targeted by directing primers to the following nucleic acid sequence (Genbank accession number: AY495375) encoding alpha - arabinofuranosidase.

[0089] (SEQ ID NO: 1): 1 gatcccgccg gattacggaa aataacagag cgagttcgta tgcgatgatc ttcgctggag 61 atgtgctaca tccacagctc gaacataaat agagaagaca atgccgcctg gctgtccaac 121 atcaactcct ctcatatccg caagcttcct gtcaaccctc ctcacagttc gctcatcact 181 caaacatgcg ttccaggacg aacatcgctc ttggcctagc tgccactggt tccctagtcg 241 ctgccgcgcc ttgcgatatc tatcagaatg gcggtactcc ttgcgtagct gctcacggca 301 caactcgcgc attgtatgat tcctacactg gtcctctcta ccaacttaag agaggctcag 361 atggcactac gaccgatatt tctcctttgt ctgctggtgg tgttgccaat gctgctgctc 421 aggactcttt ctgcaagggt actacctgtc ttatcagtat tatctacgat cagtctgggc 481 gtgcaaacca tctttatcag gcccagaaag gtgctttcag cggaccagat gtcaacggaa 541 acgacaactt ggcaggcgct attggagcac cagtgacttt gaatggcaag aaggcatatg 601 gcgtgttcat ctcgcccggc actgggtaca gaaacgacga agtcagcggc acggccactg 661 gaaacgaacc tgagggcatg tatgctgttc ttgacggcac tcattacaac gatgcttgct 721 gctttgacta cggaaacgcg gaaatcagca acacggatac tggtaacgga catatggagg 781 ccgtctacta tggtaacaac acgatttggg gcagtggctc tggcagcggt ccttggctca 841 tggccgacct tgagaacggt ttgttctctg gccagggtac caagcagaac actgcagacc 901 cttcaatctc caacagattc ttcaccggaa tggtcaaggg agagcctaac cagtgggcgc 961 ttcgcggtag caatgccgcg tccggttcct tgtcgaccta ctacagtggc gctcgtccca 1021 ccgtcggcgg ttacaacccc atgagcctcg agggcgccat cattcttggc atcggtggcg 1081 ataacagcaa tggcgctcag ggcactttct atgagggggt catgacctcg ggctacccgt 1141 ctgatgccac tgaagcctcg gtgcaggcca acattgtggc tgcgaagtac gctaccacat 1201 ctttgaacac agcaccactc actgtcggca acaagatttc gatcaaggtg accacccccg 1261 gctacgacac ccgctatctg gcacacaccg gagccaccgt caacacgcag gttgtctctt 1321 catctagcgc gactagcctc aagcagcagg ccagctggac tgttcgcaca ggcctcggta 1381 acagcggctg ttactctttc gagtcggttg atacacctgg aagcttcatc agacactaca 1441 acttccagct ccagctcaac gcgaatgaca acaccaaggc tttcaaggaa gacgcgactt 1501 tctgctctca gaccggtctt gttaccggca acactttcaa ctcgtggagc taccctgcca 1561 agttcatccg tcactacaac aatgttggat acatcgccag caacggtggt gttcacgact 1621 ttgactctgc tacaggcttc aacaacgatg tcagctttgt ggttggaagc agctttgctt 1681 agatgtaaaa ggtcaggatg aatatgatgg atgtttatga caaaagaagt tatgagtttg 1741 tagttatgga atcttagctg tagcttttga aagcctttgg gatatcagat gtttgtctct 1801 tgttcatgtg ccgttgcaaa gaagaaaaga aggagcagca agcagtgagg ctcttatcgg 1861 gcgatagggc tagatc

[0090] In a related aspect, the following primers can be used.

Table 1

[0091] In one aspect, primer pairs can be used alone or in combination with each other or with other primer pairs. In another aspect, primer pairs can be used to track and identify the origin of products created by the methods described herein. PCR can be performed by standard methods (see, for example, U.S. Patent No. 4,800,159, which is incorporated herein by reference), although alternative PCR methods will be apparent to those skilled in the art.

[0092] Although not intended to be bound by theory, it has been observed that the viscosity of the final product appears to depend on exopolysaccharides that are only present when the novel materials resulting from fermentation, such as plant materials, contain plant substrates that produce high viscosities other than pullulan and substrates that are metabolized by microorganisms that produce fermentation-dependent products (novel viscosity-increasing exopolysaccharides). Therefore, organisms with low exopolysaccharide / pullulan productivity can be alternatively replaced by using organisms that do not produce novel viscosity-increasing exopolysaccharides.

[0093] Production process In an exemplary embodiment, after an optional pretreatment (e.g., to enhance the availability of nutrients to cells, to remove sugars, etc.), the pH can be adjusted to 4.5 - 4.9 at a solids loading rate of at least 5%, and then the plant material can be mixed with water and / or concentrate to form a mash in one or more mixing tanks. In one aspect, the pH is 4.8. The mash can be treated with sulfuric acid and / or a defoaming agent before hydrodynamic forces are applied to separate the suspension into a cake and a centrate. The cake can be further washed with one or more solvents (e.g., water or centrate) and then transferred to a boiling tube - cooler tandem device, where the residence time in the boiling tube can be varied. The temperature in the boiling tube can be varied up to 121.1 °C, and the residence time can be varied from 0 to 2 minutes. In one aspect, the residence time is about 1.5 minutes.

[0094] After cooling to about 30 °C, the cooled mash can be transferred to one or more fermenter vessels, an inoculum of A. pullans can be added to the mash, and the resulting inoculated mash can be incubated for about 7 hours to about 10 hours, about 10 hours to about 14 hours, about 14 hours to about 20 hours, or about 7 hours to about 24 hours, or until a degree of hydrolysis (DH) of 2% to 80% of the protein is achieved. Without wishing to be bound by theory, the DH is substantially due to enzymatic hydrolysis rather than thermal degradation, and also includes the possibility that the DH may be increased by further reuse of the centrifugate from the broth tank. The inoculation volume (e.g., from a 60-hour seed culture, about 1×10 1 ~ about 100×10 9 CFU / ml) may be about 1% of the operating volume of one or more fermenter vessels. In a related aspect, the inoculated cells may have a substantially single-cell morphology, and without wishing to be bound by theory, the use of cells having a substantially filamentous morphology may reduce the availability of oxygen and other nutrients. During incubation, sterile air can be introduced into the reactor at a rate of 0.5 to 1 L / L / h. In a plurality of embodiments, the conversion culture is subjected to conversion by incubating with the plant material for less than about 12 hours. In a plurality of embodiments, the conversion culture is incubated for about 7 hours to about 14 hours. The conversion culture can be incubated at about 24 - 35 °C.

[0095] In a plurality of embodiments, the pH of the conversion culture undergoing fermentation may be about 4.5 to about 5.5. In a plurality of embodiments, the pH of the conversion culture may be about 4.8. In a plurality of embodiments, the conversion culture is actively aerated.

[0096] The fermented plant material is transferred from one or more incubation vessels to a broth tank where it is heated to about 60 °C for about 30 minutes to 2 hours. Hydrodynamic forces are applied to the heated fermented plant material, which results in a fermented cake and a fermented centrate. The fermented cake is then dried at a temperature of about 37.8 °C to about 149 °C until the moisture content is less than about 7%. The fermented centrate is transferred to one or more of the previously used mixing tanks (e.g., to conserve and reuse water, increase the protein content including yield and soluble protein), and may be mixed with the incoming plant material, which also includes transferring the fermented centrate to an evaporator to produce a liquid protein concentrate.

[0097] The fermented centrate intended for the evaporator can be subjected to two or more evaporation stages where evaporation is carried out for a sufficient time and at a temperature to obtain a liquid protein concentrate with a solids percentage of about 10 to about 60%. Or, the liquid protein concentrate can be returned to one or more mixing tanks to ultimately form one or more additional centrates / cakes for further processing. In one aspect, the cake / centrate may be washed and / or precipitated with ethanol.

[0098] In multiple embodiments, the solid recovery of the final protein concentrate can be adjusted by the plant feedstock, incubation conditions, pH, drying time, and temperature. For example, about 70% or more protein can be achieved for the final dried cake with a 14-hour incubation, and a solids content of about 76.59% to about 99.65% is obtained. The protein content in the cake may be about 65% to about 70%, about 70% to about 75%, or about 75% to about 80% (dmb).

[0099] In multiple embodiments, for example, to avoid contamination by unwanted bacterial strains, the feedstock may be treated with one or more antibiotics (such as, but not limited to, tetracycline, penicillin, erythromycin, tylosin, virginiamycin, and combinations thereof) prior to inoculation of the conversion microorganism.

[0100] During incubation, samples may be taken at regular intervals during the processing (for example, to determine amino acids, DH, oligosaccharide concentration, pullulan content, etc.). For example, samples for HPLC analysis are boiled, centrifuged and filtered (for example, through a 0.22 μm filter), placed in vials for the autosampler, and frozen until analysis. In multiple embodiments, a WATERS HPLC system can be used to assay samples for carbohydrates and organic solvents, but other HPLC systems may also be used. Those skilled in the art will recognize that other methods (such as UPLC) may also be used. To evaluate the microbial population, samples may be subjected to counting by plate or hemocytometer. Those skilled in the art will recognize that other methods may also be used (such as fluorescence microscopy, flow cytometry, etc.). Samples may be assayed for levels of cellulose, hemicellulose, lignin, starch, and pectin using the procedures of the National Renewable Energy Laboratory.

[0101] Figures 6 - 9 illustrate production processes 100, 100(a), 100(b), 100(c) that can be used to generate the HQPC disclosed herein. Referring to Figure 6, for a plurality of embodiments, a plant material is first fed to a milling device 101 and then transferred to a first mixing tank 102(a) where it is mixed with one or more first solvents, where the first solvents may contain acids, bases, enzymes, defoamers, and / or centrate from a downstream separation step (e.g., centrate 2 during a continuous cycle). The enzymes include non - cellulolytic enzymes (e.g., phytase, protease, etc.). The resulting mash is separated from one or more first solvents by a hydrodynamic device (e.g., a decanter centrifuge) 103(a) to produce a first centrate and a first cake. The first centrate can be transferred to an evaporator 107 to produce soy solubles 108 (i.e., liquid protein concentrate). The first cake is transferred to a second mixing tank 102(b) where the first cake is washed with one or more second solvents and / or downstream centrate (e.g., centrate 3 in the case of a continuous cycle). The washed first cake is separated from one or more second solvents by a hydrodynamic device 103(b) to produce a second centrate and a washed second cake. The second centrate may be transferred to the first mixing tank 102(a) during a continuous cycle. The washed second cake is transferred to a third mixing tank 102(c) where the washed second cake is washed with one or more third solvents and / or downstream centrate (e.g., centrate 3 in the case of a continuous cycle). The washed second cake is separated from one or more third solvents by a hydrodynamic device 103(c) to produce a third centrate and a washed third cake. The third centrate may be transferred to the second mixing tank 102(b) during a continuous cycle. The washed third cake is transferred to a fourth mixing tank 102(d) where the washed third cake is washed with one or more third solvents and / or condensate from the evaporator 107 to form a suspension, and this suspension is transferred to one or more fermenter vessels 104 for inoculation by a seed train 105.Before incubation, in a boiling tube / cooler tandem device (not shown) that heats and cools the suspension, the residence time and temperature within the boiling tube can be adjusted to vary the properties of the final product (e.g., increase the protein content). In multiple embodiments, the residence time can be from 0 seconds to about 5 seconds, about 5 seconds to about 10 seconds, about 10 seconds to about 15 seconds, about 20 seconds to about 30 seconds, or about 30 seconds to 1 minute. In a related aspect, the suspension is heated in a boiling tube at a temperature of at least about 95°C, about 105°C, about 110°C, about 120°C, about 130°C, or about 140°C and then cooled to about 30°C - 32°C before incubation by the seed train 105.

[0102] Continuing from FIG. 6, after incubation, the resulting fermented suspension is separated into a fourth centrate and a final cake by the application of a hydrodynamic device (e.g., a decanter centrifuge / disk stack centrifuge) 103(d). This suspension can be heated in a broth tank (not shown) at at least about 60°C for about 30 minutes to about 2 hours before applying the hydrodynamic force. The cake obtained from 103(d) and / or the broth tank (not shown) is transferred to a dryer 106. Drying is performed by heating the dryer 106 to about 150°C until the moisture content of the final cake is less than about 7%.

[0103] Referring to FIG. 7, the drawing shows the details of the first step of the production process 100(a) (centrate 1). The plant material is first fed to a milling device 101 and then transferred to a first mixing tank 102(a) where it is mixed with one or more first solvents. The resulting mash is separated from the one or more first solvents by a hydrodynamic device 103(a) to produce a first centrate and a first cake. The first centrate can be transferred to an evaporator 107 to produce soy solubles 108.

[0104] Referring to FIG. 8, the drawing shows the details of the production process of the centrifugate 4100(b). The washed third cake is transferred to the fourth mixing tank 102(d), where the washed third cake is washed with one or more third solvents and / or condensate from the evaporator 107 to form a suspension, and this suspension is transferred to one or more fermenter vessels 104 for inoculation. After incubation, the resulting fermented suspension is separated into a fourth centrifugate and a final cake by the application of the hydrodynamic device 103(d), and the centrifugate 4 is transferred to an upstream mixing tank (e.g., 102(c)).

[0105] Referring to FIG. 9, the production process 100(c) shows details regarding the centrifugate 4 and 1. The centrifugate 4 is subjected to disk stack centrifugation 109 and / or ultrafiltration 110 before being transferred to the mixing tank 3102(c), and this includes that the centrifugate 1 may also be subjected to sludge removal and oil removal, ultrafiltration 110 for protein separation, and / or nanofiltration 111 for sugar separation. In multiple embodiments, such separation methods may be used to remove the microorganisms used in fermentation.

[0106] In multiple embodiments, referring to FIGS. 6 - 9, the production processes 100, 100(a), 100(b), 100(c) may be batch or continuous, include at least one washing cycle (from the mill 101 to the dryer 106), and at least two centrifugates, and one concentrate is further processed to concentrate the soy solubles 108.

[0107] In multiple embodiments, the feedstock before milling and the final product (the final dried cake) are analyzed by NIR spectroscopy. In one aspect, the raw spectrum for the final product at 4664 cm -1 ~4836 cm -1There should be a significant downward shift (i.e., "valley") compared to the feedstock (i.e., substantially "flat") (see FIGS. 10 and 11). In a related aspect, the downward shift is calculated using the following formula and should be at least about 10% change, about 10% - about 15% change, or about 15% - about 20% change, or more. (V2 - V1) / |V1|×100 = rate of change

[0108] For example, as shown in FIGS. 10 and 11, the rate of change of the downward shift was about 16.7% for the SBM sample and the final ME-PRO® (HSPQ product) sample produced therefrom. Without wishing to be bound by theory, such a shift indicates an increase in protein content (see, e.g., Fan et al., PLoS ONE (2016) 11(9):e0163145. doi:10.1371 / journal.pone.0163145). NIR spectral data can be generated using the methods disclosed in Fan et al. ((2016); which is hereby incorporated by reference in its entirety), but one of ordinary skill in the art will recognize that other suitable methods, including any relevant hardware and software, are available.

[0109] Dietary composition In an exemplary embodiment, the HQPC recovered from the converted culture is used in a diet formulation. In some embodiments, the recovered HQPC may be the sole protein source in the diet formulation. The percentage of the protein source in the diet formulation is not intended to be limiting and may include a protein concentrate of 24-80%. In some embodiments, the HQPC may be greater than about 50%, greater than about 60%, or greater than about 70% of the total protein source of the diet formulation. The recovered HQPC can replace / supplement protein sources such as fish meal, soybean meal, wheat and corn flour, and gluten and concentrates, as well as animal by-products such as blood, poultry, meat substitutes, meat analogs, and feather meal. Also, diet formulations using HQPC may optionally include dietary supplements such as mineral and vitamin premixes to meet the remaining nutritional requirements.

[0110] In certain embodiments, the performance of the HQPC can be measured by comparing the growth, feed conversion, protein efficiency, DH, APD, PPD, and survival rate of animals fed a high-quality protein concentrate diet formulation, including the aesthetic sense regarding the feedstock for human consumption, with animals fed a control diet formulation. In some embodiments, the test formulations contain consistent protein, lipid, and energy content. For example, if the animal is a fish, the visceral (fat deposition) and organ (liver and spleen) characteristics, dress-out percentage, as well as proximate analysis of the body, and even intestinal tissue structure (enteritis) can be measured to evaluate the diet response. In some embodiments, for shrimp larvae formulations, DH, PPD, and ADP can be measured in vitro to evaluate the diet response. In some embodiments, for piglets, enteric infections characterized by diarrhea are a major cause of reduced growth performance, morbidity, and mortality in weaned pigs, so the reduction of diarrhea can be measured to evaluate the diet response.

[0111] As is understood, individual diet formulations containing the recovered HQPC can be optimized for different types of animals. In multiple embodiments, the animals include, but are not limited to, fish, crustaceans (e.g., shrimp, crab, prawn, and lobster), companion animals (e.g., dogs, cats, birds), and livestock (e.g., cows, pigs, and chickens). In a related aspect, the animals are fish and crustaceans raised in commercial aquaculture. In another related aspect, the crustaceans include juvenile shrimp. Methods for optimizing diet formulations are well known and can be readily identified by those skilled in the art without undue experimentation.

[0112] Complete aquaculture feeds can be formulated using HQPC according to known nutritional requirements for various animal species. In multiple embodiments, the formulation can be used for yellow perch (e.g., 42% protein, 8% lipid). In multiple embodiments, this formulation can be used for rainbow trout (35% protein, 16% lipid). In multiple embodiments, the formulation can be used for any one of the aforementioned animals.

[0113] To ensure that the micro-nutrient requirements are met, a premix of the essential minerals and vitamins of the plant-based diet may be used. Any dietary supplements (as determined necessary by analysis) can be evaluated by comparing them to the same formulation without the dietary supplement; thus, feeding trials can be conducted in a factorial design taking into account the effect of the dietary supplement. In multiple embodiments, the feeding trials can include a fish meal-based control diet as well as reference diets based on ESPC and LSPC [traditional SPC (TSPC) is produced by solvent washing soybean flakes to remove soluble carbohydrates; texturized SPC (ESPC) is produced by extruding TSPC under high humidity and high temperature; low antigen SPC (LSPC) is produced from TSPC by changing the solvent washing and temperature during processing]. The pellets for the feeding trials can be produced using a single-screw extruder (e.g., BRABENDER PLASTI-CORDER EXTRUDER Model PL2000).

[0114] Feeding trial In multiple embodiments, replicate trials with four experimental units (i.e., a mixture of each experimental diet and the control diet) per treatment can be used (e.g., for about 60 to 120 days each). The tests can be conducted 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 sample and a bioreactor, filters (100 μm bag filter, carbon filter, and UV filter). A heat pump may be used as needed to maintain the temperature optimal for species-specific growth. In all systems, the water quality (e.g., dissolved oxygen, pH, temperature, ammonia, and nitrite) can be monitored.

[0115] In multiple embodiments, the experimental diet is given according to the size of the fish and fed in portions 2 to 5 times a day. Growth performance is determined by total mass measurements taken over a period of 1 to 4 weeks (depending on the size of the fish and the test period); the ration can be adjusted according to the increment to allow for satiation feeding and reduce the waste stream. Consumption is evaluated biweekly from the uneaten feed recovered from individual tanks. The uneaten feed can be dried to a constant temperature, cooled, and weighed to estimate feed conversion efficiency. Protein and energy digestibility can be determined from feces manually removed at the midpoint of each experiment or from feces removed from the lower intestinal tract via dissection at the end of the feeding trial. Survival rate, weight gain, growth rate, health indicators, feed conversion, protein and energy digestibility, and protein efficiency can be compared between treatment groups. Proximate analysis of dissected fish can also be performed to compare body composition between diet treatments. Amino acid and fatty acid analyses can be performed as needed on body constituents depending on the purpose of the feeding trial. The feeding trial response to diet treatment can be compared to the response of a control (e.g., fish meal) diet to confirm whether the performance of the HQPC diet meets or exceeds the control response.

[0116] For shrimp, tests can be conducted in 900-gallon tanks or 190-L semi-square tanks, each equipped with a recirculating drain pipe that draws water from below the surface and a drain pipe for sludge attached to the lowest position at the bottom of the center of the tank. The RAS test system can consist of a Cornell-style dual-drain tank, a solids settling tank, mechanical drum filtration, a moving bed biofilm reactor (MBBR), UV sterilization, cooling, and oxygen injection. Water quality is monitored daily to confirm that all parameters are within acceptable ranges (see, e.g., White et al., Aqua Mar Bio Eco (2020) JAMBE:105).

[0117] Furthermore, for example, it is also possible to analyze the shrimp to determine the direct effects of HQPC, such as providing a significant amount of bioactive factors that can increase the gut microbiota, reduce gut inflammation, and promote metabolic processes that improve the health of the animal, which may particularly depend on the conversion of dry matter in the feed to body weight, where the determination of digestibility plays a particularly important role. In a related aspect, in vitro protein digestion by standardized digestive enzymes recovered from the shrimp can be used, including the use of data from such analyses to determine DH, PPD, and APD, as well as overall survival rates.

[0118] Statistical analysis of diet responses and feeding trial responses can be performed with a pre-set α = 0.05. Analysis of performance parameters between treatments can be done, if necessary, by appropriate analysis of variance or covariance (Proc Mixed) and post hoc multiple comparisons. Analysis of animal performance and tissue responses can be evaluated by non-linear models.

[0119] In multiple embodiments, the present disclosure proposes, for example, using GRAS-status microorganisms to convert fibers and other carbohydrates in plant materials into additional proteins. It is also possible to produce extracellular polysaccharides (e.g., glucans) of the microorganisms, which can facilitate the formation of feed pellets by extrusion and may eliminate the need for binders. This microbial gum can also result in immunostimulatory activity that activates innate defense mechanisms that protect animals from common pathogens arising from stress factors. Immunopreventive substances, such as β-glucans, bacterial products, and plant constituents, are increasingly being used in commercial feeds to reduce economic losses due to infections and minimize the use of antibiotics. The microorganisms of the present disclosure also produce extracellular peptidases, which should enhance the digestibility and absorption of proteins during metabolism and result in higher feed efficiency and yields. As disclosed herein, this microbial incubation process provides a valuable and sustainable plant protein-based feed that is lower in cost per protein unit than SBM, SPC, and animal feeds. In multiple embodiments, the components of the centrate can be further subjected to an evaporation stage that can concentrate soluble by-products, such as sugars, glycerol, proteins, peptides, and amino acids, into a substance called syrup or condensed plant-based solubles (PBS).

[0120] As disclosed, the present microorganism can metabolize individual carbohydrates in plant materials to produce any of cell populations (e.g., pullulan), microbial gums (e.g., pullulan), enzyme production, microbial metabolite production, and probiotics. Also, various strains of these microorganisms enhance fiber degradation. The microorganism of the present invention can also convert the protein of plant substances into more digestible peptides and amino acids (see Figure 12). In a plurality of embodiments, the following actions can be performed: 1) determining the efficiency of using the selected microorganism of the present disclosure to convert plant materials to obtain a high-quality protein concentrate (HQPC) having a protein concentration of at least about 65% to about 70% or more, and 2) evaluating the effectiveness of the HQPC to replace animal protein. In a plurality of embodiments, the performance and robustness of the microorganism can be improved, the aquaculture feed can be tested for a range of commercially important animals, and the optimization of process / conversion conditions can be carried out to verify the process costs and energy requirements for commercialization. In a plurality of embodiments, the HQPC of the present disclosure can replace at least 50% of the animal protein, while providing an increased growth rate and conversion efficiency. For example, the production cost should be lower than that of commercially available soy protein concentrate (SPC) and substantially lower than that of fish meal.

[0121] Figures 1, 2, and 6 - 9 show various approaches of the present disclosure in the treatment of plant-based products, which convert sugars into cell populations (proteins) and gums, recover the HQPC to produce aquaculture feed, and test the resulting aquaculture feed in a feeding trial of fish.

[0122] Although not required for all processes disclosed herein, cellulase / pullulanase can be evaluated with respect to the production of sugars that the microorganisms of the present disclosure can convert into proteins, exopolysaccharides, and gums. In multiple embodiments, sequential exclusion of these enzymes and evaluation of co-culture with cellulolytic microorganisms can be used. Ethanol can be evaluated with respect to improving the recovery of protein solubles that may be suspended in various centrifugates by treating various cakes to wash, sediment gums, and produce HQPC. After drying, HQPC can be incorporated into practical diet formulations. In multiple embodiments, test aquaculture feeds can be formulated (along with a mineral and vitamin premix) and compared to animal protein controls and commercial plant protein concentrate / isolate-based feeds in feeding trials with commercially important animals. Performance (e.g., growth, feed conversion, protein efficiency, survival rate), visceral characteristics, effects on the intestine, and intestinal tissue structure can be examined to evaluate the response.

[0123] In other embodiments, while minimizing the input to the process, simultaneously improving the performance and robustness of the microorganisms, testing the resulting aquaculture feeds on a range of commercially important animals, and determining the optimal conversion conditions that can verify / update the process cost and energy requirements, the optimization of the HQPC production process can be implemented.

[0124] In the past few years, at several facilities, a dry milling function has been implemented to remove the husk and germ of corn prior to the ethanol production process. This dry fractionation process yields DDGS (hereinafter referred to as dry fractionated DDGS) containing up to 42% protein. In some embodiments, low-oil DDGS can be used as a substrate for conversion, and such low-oil DDGS has a higher protein level than conventional DDGS. In a related aspect, low-oil DDGS increases the growth rate of A. pullans compared to conventional DDGS.

[0125] Some groups are evaluating the partial replacement of animal-derived proteins with plant-derived proteins. However, due to the low protein content, insufficient amino acid balance, and the presence of nutrient absorption inhibitors, the replacement level is limited to 20-40%. For example, preliminary growth tests have shown that current DDGS- and SPC-based feeds do not achieve results similar to fish meal control feeds. Some defects have been identified in commercially produced DDGS and SPC, mainly in the composition of proteins and amino acids, which lead to variations in growth performance. However, feeds containing the plant-derived proteins disclosed herein that contain (formulated to meet or exceed all requirements) nutritional supplements have resulted in growth outcomes comparable to or better than those of animal protein-based controls. Therefore, the processes disclosed herein and the products developed therefrom provide higher quality HQPC (compared to nutritional requirements) and assist in achieving growth performance equivalent to or better than that of feeds containing animal proteins, including those containing various SPC / SPI.

[0126] In multiple embodiments, the fish to which the fish feed composition of the present disclosure can be given include the Siberian sturgeon, the stellate sturgeon, the starry sturgeon, the beluga sturgeon, the arapaima, the Japanese eel, the American eel, the shortfin eel, the ribbon eel, the European eel, the milkfish, the mackerel scad, the bluegill sunfish, the green sunfish, the white crappie, the black crappie, the asp, the catla, the goldfish, the crucian carp, the silver carp, the grass carp, the common carp, the koi, the bluntnose black bream, the bighead carp, the silver carp, the black carp, the common carp, the white Amur bream, the Thai barb, the Java barb, the roach, the tench, the pond roach, the bocachico, the dourada, the colossoma, the cachimbla, the pacu, the black bullhead, the channel catfish, the European wels catfish, the Pangasius (swai, tra, basa) catfish, the gizzard shad, the bowfin, the Philippine catfish, the Hong Kong catfish, the North African catfish, the bighead catfish, the sampa, the South American catfish, the achip, the northern pike, the ayu, the banded tilapia, the whitefish, the kaluga, the silver salmon, the masu salmon, the coho salmon, the pink salmon, the chum salmon, the chinook salmon, the rainbow trout, the Atlantic cod, the pollock, the common snoek, the barramundi / Asian seabass, the Nile perch, the Malay cod, the golden perch, the silver seabass, the white bass, the European seabass, the Japanese grunt, the bigeye grunt, the smallspotted grunt, the lined grunt, the silver perch, the white perch, the jade perch, the barramundi cod, the cobia, the European perch, the zander (pike perch), the yellow perch, the sauger, the wall eye, the bluefish, the kampachi, the amberjack, the mullet, the Florida pompano, the palometa pompano, the mackerel scad, the Japanese amberjack, the yellowtail snapper, the dusky seabream, the white seabream, the red sea bream, the madai, the red porgy, the goldenlined seabream, the Japanese sea bass, the red drum, the green tera, the blackbelt cichlid, the jaguar guapote, the Mexican mayara, the pearl spot, the three spot tilapia, the blue tilapia, the longfin tilapia, the Mozambique tilapia,Nile tilapia, tilapia, Wami tilapia, blackchin tilapia, redbreast tilapia, Jil tilapia, golden grey mullet, large-scale mullet, gold-spot mullet, chinglip grey mullet, leaping mullet, teide mullet, flathead grey mullet, white mullet, rubramsh mullet, Pacific fat sleeper, marble goby, Simofuria igo, sesame igo, marble spinefoot, southern bluefin tuna, Pacific bluefin tuna, skipjack tuna, snake skin gourami, kissing gourami, giant gourami, sea bream, Indonesian snakehead, spotted snakehead, striped snakehead, stone flounder, flounder (Japanese flounder), summer flounder, southern flounder, winter flounder, Pacific sailfish, greenback flounder, European sole, and combinations thereof, including but not limited to these.

[0127] In multiple embodiments, crustaceans to which the fish feed composition of the present disclosure can be provided include, but are not limited to, kadal shrimp, endeva shrimp, greaseback shrimp, speckled shrimp, northern brown shrimp, kuruma shrimp, Australian tiger, Shonan shrimp, caramo shrimp, banana shrimp, giant tiger shrimp, southern pink shrimp, Sao Paulo shrimp, redtail shrimp, southern white shrimp, kuma shrimp, northern white shrimp, blue shrimp, southern brown shrimp, banana prawn, Atlantic sea bob, akiami, monsoon kawae shrimp, giant kawae shrimp, striped shrimp, American lobster, European lobster, European crayfish, Danube crayfish, Tanka crayfish, American crayfish, yabby, southern crayfish, marron crayfish, canocoi shrimp, gazami, amimeno kogirigazami, Chinese mitten crab, and combinations thereof.

[0128] In multiple embodiments, livestock to which the feed compositions of the present disclosure can be administered include, but are not limited to, cows, sheep, goats, deer, horses, chickens, pigs, rabbits, ducks, alpacas, emus, turkeys, bison, and camels.

[0129] In multiple embodiments, breeding animals to which the feed compositions of the present disclosure can be administered include, but are not limited to, dogs, cats, parrots, goldfish, turtles, budgerigars, hamsters, laboratory rats, guinea pigs, and laboratory mice.

[0130] It will be understood by those skilled in the art that the feed compositions of the present disclosure can be used as a convenient carrier for pharmaceutical active substances including, but not limited to, antibiotics, chemotherapeutic agents, anti-inflammatory agents, NSAIDs, antibacterial agents, immunologically active substances including vaccines against bacterial or viral infections, and any combinations thereof.

[0131] The feed compositions according to the present disclosure can be provided as a liquid, a pourable emulsion, or in the form of a paste, or in a dry form such as, for example, granules, powders, or flakes. When the feed composition is provided as an emulsion, an oil-in-water emulsion, it may be in a relatively concentrated form. Such a concentrated emulsion form can also be referred to as a pre-emulsion since it can be diluted in one or more steps in an aqueous medium to provide the final fortifying medium for the organism.

[0132] In multiple embodiments, the cellulosic starting material for the disclosed microbial-based process is corn. Corn is about two-thirds starch, which is converted to ethanol and carbon dioxide during the fermentation and distillation processes. The remaining nutrients or fermentation products may result in condensed distillery solubles or distillers grains, such as DDGS, which can be used in feed products. Generally, the process includes an initial preparation step of dry milling or grinding the corn. The processed corn is then subjected to hydrolysis, and enzymes are added in the saccharification process to break down the main components of the starch. The next process of fermentation can be advanced by adding a microorganism (e.g., yeast) provided according to an embodiment of the present disclosure to produce gaseous products such as carbon dioxide. Fermentation is carried out for the production of ethanol that can be distilled from the fermentation broth. Subsequently, the remainder of the fermentation broth can be dried to produce a fermentation product containing DDGS. This process typically includes a solid / liquid separation process by centrifugation by which the solid phase components can be collected. Other methods, including filtration and spray drying methods, can also be used to effect such separation. The liquid phase components can then be further subjected to an evaporation step to concentrate soluble by-products, such as sugars, glycerol, proteins, peptides, amino acids, into a material called syrup or condensed corn solubles (CCS). Subsequently, the CCS can be recombined with the solid phase components and dried as an incubation product (DDGS). It should be understood that by applying this composition to a new or existing ethanol plant based on dry milling, an integrated ethanol production process can be provided that also produces valuable fermentation products.

[0133] In multiple embodiments, the incubation products generated in accordance with the present disclosure have a higher commercial value than conventional fermentation products. For example, the incubation products may include fortified dried solids with improved amino acid and micronutrient content. In this way, a "golden" product can be provided, which generally indicates high digestibility of amino acids compared to dark-colored HQSP. For example, in accordance with embodiments herein, a light-colored HQSP with a high lysine concentration can be produced compared to a relatively dark-colored HQSP that generally has a lower nutritional value. The color of the product can be an important factor or indicator when evaluating the quality and nutrient digestibility of fermentation products or HQSP. Color is used as an indicator showing exposure to excessive heat during drying, which causes caramelization and the Maillard reaction of free amino groups and sugars, reducing the quality of some amino acids.

[0134] Another aspect of the present invention is directed to a finished feed composition with enhanced nutrient concentrations, which contains nutrients such as fats, fatty acids, lipids such as phospholipids, vitamins, essential amino acids, peptides, proteins, carbohydrates, sterols, enzymes, and trace minerals such as iron, copper, zinc, manganese, cobalt, iodine, selenium, molybdenum, nickel, fluorine, vanadium, tin, silicon, and combinations thereof, but is characterized by having enhanced concentrations of these. In one aspect, the process allows animals to utilize more phosphorus in a manner that does not cause substantial contamination of wastewater.

[0135] The incubation products obtained after the incubation process typically have a higher commercial value. In multiple embodiments, the incubation products contain microorganisms with enhanced nutrient content compared to products lacking microorganisms. The microorganisms may be present in the incubation system, incubation broth, and / or incubation biomass. To produce incubation products with enhanced nutrient content, the incubation broth and / or biomass may be dried (e.g., spray-dried).

[0136] For example, used dried solids recovered after an incubation process are fortified in accordance with the present disclosure. These incubation products are generally non-toxic, biodegradable, readily available, inexpensive, and nutrient-rich. The selection of microorganisms and incubation conditions are important for producing low-toxicity or non-toxic incubation products for use as feed or nutritional supplements, and this includes the selection of microorganisms that do not produce metabolites at concentrations that would adversely affect the process. Glucose is the major sugar produced by hydrolyzing cereal-derived starch, but glucose is not the only sugar generally produced in carbohydrates. Unlike SPC or DDG produced from traditional dry mill ethanol production processes that contain large amounts of non-starch carbohydrates (e.g., as much as 35% by dry weight of cellulose and arabinoxylan measured as neutral detergent fiber), the nutrient-enriched incubation products produced by enzymatic hydrolysis of non-starch carbohydrates (through metabolism by microorganisms) are more palatable and digestible for non-ruminant animals.

[0137] The nutrient-enriched incubation products of the present disclosure can have a nutrient content of at least about 1% to about 95% by weight. The 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 can depend on the animal to which it is given, as well as the remainder of the diet and the stage of the animal's life cycle. For example, beef cattle require less histidine than dairy cattle. The selection of a suitable nutrient content for feeding animals is well known to those skilled in the art.

[0138] The incubation product can be prepared as a spray-dried biomass product. Optionally, the biomass can be separated by known methods such as centrifugation, filtration, separation, decantation, a combination of separation and decantation, ultrafiltration or microfiltration. The biomass incubation product may be further processed to facilitate rumen bypass. In a plurality of embodiments, the biomass product can be separated from the incubation medium, spray-dried, optionally processed to adjust rumen bypass, and then added to feed as a nutrient source. In addition to generating an incubation product enriched with nutrients in an incubation process containing microorganisms, an incubation product enriched with nutrients can also be produced in a transgenic plant system. Methods for producing transgenic plant systems are known in the art. Alternatively, when the microbial host excretes nutrients, the nutrient-enriched broth can be separated from the biomass produced by incubation, and the purified broth can be used as a component of animal feed, for example, in liquid form or spray-dried form.

[0139] The incubation product obtained after an incubation process using microorganisms can be used as animal feed or as a nutritional supplement for humans. The incubation product contains at least one component with enhanced nutrient content derived from non-animal sources (e.g., bacteria, yeast, and / or plants). In particular, the incubation product is 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 minerals such as iron, copper, zinc, manganese, cobalt, iodine, selenium, molybdenum, nickel, fluorine, vanadium, tin, and silicon. In a plurality of embodiments, the peptide contains at least one essential amino acid. In other embodiments, the essential amino acid is encapsulated within the modified microorganism used in the incubation reaction. In a plurality of embodiments, the essential amino acid is contained in a heterologous polypeptide expressed by the microorganism. Optionally, the heterologous polypeptide is expressed and stored within the inclusion bodies of a suitable microorganism (e.g., a fungus).

[0140] In multiple embodiments, the incubation product has a high nutrient content. As a result, the proportion of the incubation product used in the finished animal feed may be high. In multiple embodiments, the feed composition comprises at least about 15% by weight of the incubation product. In the finished feed, or diet, this material is provided together with other materials. Depending on the nutrient content of the other materials and / or the nutritional requirements of the animals to which the feed is provided, the modified incubation product may range from 15% to 100% of the feed. In multiple embodiments, this incubation product may result in a lower formulation rate due to its high nutrient content. In other embodiments, this incubation product may result in a very high feeding rate, such as greater than 75%. In a preferred embodiment, the feed composition comprises at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 75% of this incubation product. Generally, the feed composition comprises at least about 20% by weight of the incubation product. More generally, 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. Optionally, this incubation product may be used as the sole source of the feed.

[0141] In multiple embodiments, the diet compositions disclosed herein may have an enhanced amino acid content with respect to one or more essential amino acids for various purposes, for example, for weight gain and overall improvement of the health of the animal. The composition may have an enhanced amino acid content due to the presence of free amino acids in the incubation product and / or the presence of proteins or peptides containing essential amino acids. Essential amino acids can include histidine, lysine, methionine, phenylalanine, threonine, taurine, isoleucine, and / or tryptophan, which may be present in the composition as free amino acids or as part of a selected amino acid-rich protein or peptide. A peptide or protein rich in at least one essential amino acid may have at least 1% essential amino acid residues per total amino acid residues of the peptide or protein, at least 5% essential amino acid residues per total amino acid residues of the peptide or protein, or at least 10% essential amino acid residues per total amino acid residues of the protein. By feeding animals a balanced diet, the nutrient content is maximally utilized and less feed is required to achieve growth at an equivalent rate, milk production, or a reduction in nutrients contained in the excreta, thereby reducing the bio-burden of the waste (e.g., less phosphorus in waste treatment).

[0142] The compositions disclosed herein may have an enhanced content of essential amino acids and can have an essential amino acid content (including free essential amino acids and essential amino acids present in proteins or peptides) of at least 2.0% by weight, more suitably at least 5.0% by weight, based on the weight of crude protein and total amino acid content. The feed compositions disclosed herein include, but are not limited to, other nutrients derived from microorganisms, including fats, fatty acids, lipids such as phospholipids, vitamins, carbohydrates, sterols, enzymes, and trace minerals.

[0143] The feed formulations disclosed herein can include a finished feed form composition, a concentrated form composition, a blender form composition, and a base form composition. When the formulation is in the form of a finished feed, the proportion of the nutrient level from which nutrients are obtained from the microorganisms in the incubation product may be about 10% to about 25%, more preferably about 14% to about 24%; on the other hand, when the formulation is in the form of a concentrate, the nutrient level may be about 30% to about 50%, more appropriately about 32% to about 48%. When the formulation is in the form of a blender, the nutrient level in the composition may be about 20% to about 30%, more preferably about 24% to about 26%; when the formulation is in the form of a base mixture, the nutrient level in the formulation may be about 55% to about 65%. Unless otherwise stated herein, the percentages are described as weight percentages. When HQPC is rich in a single nutrient, e.g., Lys, it is considered to be used as a nutritional supplement at a low ratio; when the balance of amino acids and vitamins, e.g., vitamins A and E, is achieved, it becomes a more finished feed and is given at a higher ratio, supplementing low-protein and low-nutrient feed ingredients such as corn stover.

[0144] The feed formulations disclosed herein can include a peptide or crude protein fraction present in an incubation product having an essential amino acid content of at least about 2%. In a plurality of embodiments, the peptide or crude protein fraction can 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 a plurality of embodiments, at least about 50%. In a plurality of embodiments, the peptide may be 100% essential amino acids. For example, a fish meal formulation can include a peptide or crude protein fraction present in an incubation product having an essential amino acid content of up to about 10%. More generally, a fish meal formulation 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%.

[0145] The formulations disclosed herein can include a peptide or crude protein fraction present in an incubation product having a lysine content of at least about 2%. In a plurality of 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 a plurality of embodiments, at least about 50%. For example, a fish meal formulation can include a peptide or crude protein fraction having a lysine content of up to about 10%. Optionally, the fish meal formulation can include a peptide or crude protein fraction having a lysine content of about 2-10%, 3.0-8.0%, or 4.0-6.0%.

[0146] The formulations disclosed herein can include nutrients in the incubation product at about 1 g / kg dry solids to 900 g / kg dry solids. For example, the nutrients in a fish meal formulation can be present at 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 a plurality of embodiments, the nutrients can be present at 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.

[0147] The formulations disclosed herein can include essential amino acids or peptides containing at least one essential amino acid present in the incubation product, having a content of about 1 g / dry solids kg to 900 g / dry solids kg. For example, the essential amino acids or peptides containing at least one essential amino acid in the fish meal composition can be present at at least about 2 g / dry solids Kg, 5 g / dry solids Kg, 10 g / dry solids Kg, 50 g / dry solids Kg, 100 g / dry solids Kg, 200 g / dry solids Kg, and about 300 g / dry solids Kg. In multiple embodiments, the essential amino acids or peptides containing at least one essential amino acid can be present at at least about 400 g / dry solids Kg, at least about 500 g / dry solids Kg, at least about 600 g / dry solids Kg, at least about 700 g / dry solids Kg, at least about 800 g / dry solids Kg and / or at least about 900 g / dry solids Kg.

[0148] The formulations disclosed herein can contain an incubation product enriched in nutrients in the form of biomass formed during incubation, and at least one additional nutrient component. In another example, the formulation contains an incubation product enriched in nutrients dissolved and suspended from the incubation broth formed during incubation, as well as at least one additional nutrient component. In a further embodiment, the formulation has a crude protein fraction containing a protein rich in at least one essential amino acid. The formulation can be prepared to provide an improved balance of essential amino acids.

[0149] For other formulations, the finished formulation may contain HQPC and one or more ingredients, such as wheat mid (containing bran), corn, soybean meal, corn gluten meal, distillers grains with or without solubles, salt, macrominerals, microminerals, and vitamins, which may be produced with or without fermentation. Other possible ingredients may generally include, but are not limited to, sunflower meal, malt sprouts, and soybean hulls. The blender formulation may contain wheat mid, corn gluten meal, distillers grains with or without solubles, salt, macrominerals, microminerals, and vitamins. Alternative ingredients may generally include, but are not limited to, corn, soybean meal, sunflower meal, cottonseed meal, malt sprouts, and soybean hulls. The base form formulation may contain wheat mid, corn gluten meal, and distillers grains with or without solubles. Alternative ingredients may generally include, but are not limited to, soybean meal, sunflower meal, malt sprouts, macrominerals, microminerals, and vitamins.

[0150] Highly unsaturated fatty acids (HUFA) in microorganisms can be converted to less desirable unsaturated or saturated fatty acids when exposed to oxidative conditions. However, the saturation of omega-3 HUFA can be reduced or prevented by introducing synthetic antioxidants or naturally occurring antioxidants, such as beta-carotene, vitamin E, and vitamin C, into the feed. Synthetic antioxidants, such as BHT, BHA, TBHQ, or ethoxyquin, and natural antioxidants, such as tocopherol, can also be incorporated into food or feed products by adding them to the products, or by in situ production in suitable organisms. The amount of antioxidant incorporated in this manner depends on subsequent usage requirements, such as product formulation, packaging method, and desired shelf life.

[0151] In addition, the incubation products of the present disclosure can also be used as nutritional supplements for human consumption when the process starts with human-grade input materials and human food quality standards are observed throughout the process. The incubation products or formulations disclosed herein are high in nutrient content. Nutrients such as protein and fiber are associated with a healthy diet. Recipes can be developed for using the incubation products or finished feeds of the present disclosure in foods such as cereals, crackers, pies, cookies, cakes, pizza dough, smoked sausages, meatballs, shakes, and all forms of edible food. Another option could be to develop the incubation products into snacks or snack bars like granola bars that are easy to eat and convenient for distribution. The snack bars may contain, in addition to protein, fiber, germ, vitamins, and minerals derived from grains, nutritional supplements such as glucosamine, HUFA, or cofactors such as vitamin Q-10.

[0152] The flavor of the formulations containing the present incubation products can also be supplemented. The choice of a specific flavor depends on the animal to which the feed is given. Flavors and aromas, whether natural or artificial, can be used to make the feed more acceptable and improve the taste. These nutritional supplements can be well formulated with all the ingredients and can be used in the form of liquid or dry products. Suitable flavors, attractants, and aromas added to animal feed include, but are not limited to, fish pheromones, coriander, banana, cherry, rosemary, cumin, carrot, peppermint, oregano, vanilla, anise, in addition to rum, maple, caramel, citrus oils, ethyl butyrate, menthol, apple, cinnamon, and any natural or artificial combination thereof. Flavors and scents may vary between different animals. Similarly, various artificial or natural fruit flavors can also be added to nutritional supplements containing the present incubation products for human consumption.

[0153] In multiple embodiments, the HQPC may be part of a kit for generating various formulations, including the HQPC, a label, at least one container, and instructions regarding the generation of formulations according to the animal. Further, such instructions may be available through a web link.

[0154] The shelf life of the incubation products or the finished feed of the present disclosure may typically be longer than that of incubation products lacking microorganisms. The shelf life may depend on factors such as the moisture content of the product, the amount of air flowing through the feed mass, environmental conditions, and the use of preservatives. Preservatives can be added to the finished feed to extend the shelf life by several weeks to several months. Other methods for extending the shelf life include management similar to silage management, such as mixing and packing with other feeds, covering with plastic, or bagging. Cooling conditions, preservatives, and the exclusion of air from the feed mass all extend the shelf life of wet by-products. The finished feed can be stored in a storage warehouse or a silo bag. Also, drying the wet incubation products or the finished feed can extend the shelf life of the product and improve consistency and quality.

[0155] The finished feed of the present disclosure can be stored for a long time. The shelf life can be extended by storing in a silo, adding preservatives such as organic acids, or blending with other feeds such as soybean hulls. Commercial containers or bulk storage warehouses can be used to store the finished feed. In a related aspect, the HQPC disclosed herein can have a shelf life of at least two years.

[0156] The following examples are illustrative and are not intended to limit the scope of the disclosed subject matter. [Examples] [Example 1] High-quality protein concentrate (HQPC) (sedimentation method)

[0157] Figure 1 shows an approach where white flakes are pre-treated, sugars are converted into cell populations (proteinaceous materials) and gums (e.g., exopolysaccharides), HQSPC is recovered, and aquafeed is produced (Figures 2 and 4), and the resulting aquafeed is tested in a fish feeding trial with respect to the process. First, the white flakes were subjected to extrusion pretreatment (BRABENDER PLASTI-CORDER SINGLE SCREW EXTRUDER Model PL2000, Hackensack, NJ) at a moisture content of 15%, 50 °C, and 75 rpm to disrupt the structure and enable an increased input of hydrolytic enzymes during subsequent saccharification. These conditions result in a shearing effect on the ridge channels on both sides of the barrel, which has previously been observed to increase the release of sugars after enzymatic hydrolysis by 50 - 70%. The extruded white flakes were then powdered through a 3 mm hammer mill screen, mixed with water to a solid loading rate of 10%, and adjusted to pH 5. After heating to sterilize or pasteurize the mash, the mash was cooled to approximately 50 °C, and cellulase and oligosaccharide-degrading enzymes (a total of 15 ml / kg of white flakes) were added to hydrolyze the polymer into monosaccharides (4 - 24 hours of hydrolysis). The specific dosages were 6% CELLIC CTEK (per gram of glucan), 0.3% CELLIC HTEK (per gram of total solids), and 0.015% NOVOZYME 960 (per gram of solids). The resulting mash was then cooled to 30 °C, the pH was adjusted to 3 - 5, A. pullans (1% v / v) was inoculated, mixed at 50 - 200 rpm, and incubated for 4 - 5 days at an aeration rate of 0.5 L / L / min to convert the sugars into proteins and gums. During incubation, samples were periodically removed and analyzed for sugars, cell count, 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 gums. The proteins, pullulan, and microbial mass (HQSPC) were recovered by centrifugation and dried, the supernatant was distilled to recover ethanol, and the remaining liquid was chemically assayed for reuse at the start of future processes. Figure 5 shows glucose recovery using the ppt method.

[0158] HQSPC Using Soybean White Flakes and Microbial Conversion by A. pullans, Pilot-Scale System for HQSPC Production This system included a 675L bioreactor, a variable speed progressive cavity pump, a continuous flow centrifuge, and a 1×4 meter drying table. The inoculum for use in the 675L bioreactor was prepared in two 5L NEW BRUNSWICK BIOFLO 3 bioreactors. For each test, the described A. pullans was grown for 2 - 3 days to prepare an inoculum in an amount of 8 - 10L. Using this material, a larger amount of white flakes prepared in the 675L bioreactor and subjected to extrusion and saccharification was inoculated. After incubation, ethanol was added, the mash was centrifuged to recover the wet solids, which were subsequently dried for use in the fish feed test. By monitoring the performance of the conversion process, the yield and composition of HQSPC, several parameters that had a significant impact on solid recovery were observed. In larger-scale tests, the parameters shown in Table 1 were varied.

Table 2

[0159] From the yield and protein level of HQSPC, the following were observed: 1) Incubation with a pH of 3 - 3.5, a temperature of 30 - 32°C, and a high level of aeration maximized the growth of A. pullans and minimized pullulan production. 2) An incubation time of 4 - 5 days was optimal for protein content and solid recovery. 3) Longer incubation times increased the protein content but substantially decreased solid recovery. 4) Shorter incubation times maintained high solid recovery but limited the protein content. 5) Since the final product does not contain stachyose and raffinose, it may be possible to reduce (omit) extrusion and / or enzymatic saccharification.

[0160] To optimize the process conditions, preliminary bench-scale tests were carried out in a 5 L bioreactor. Using a 10% solid loading rate for the extruded white flakes, after saccharification for 24 hours, A. pullans was inoculated and incubated for 10 days under a pH of 5, aeration of 0.5 L / L / min, and agitation of 200 rpm. To establish the optimal harvesting period that maximizes both the solid recovery rate and the protein content in the solids, tests were conducted on the extension of the incubation time. Samples (100 ml) were taken out daily and the following treatments were carried out every other day: Precipitate all solids with ethanol, centrifuge the solids to dry them, and measure the residual solids in the resulting supernatant. First, centrifuge the broth to recover the solids, dry the solids, and precipitate and dry pullulan from the resulting supernatant.

[0161] In the method where ethanol precipitation is carried out first, by using a laboratory centrifuge (10,000 g), approximately 97% of the solids (soybean solids, cells, and gums) were recovered, and approximately 3% of the solids remained in the liquid phase. In the method where centrifugation is carried out first, approximately 81.7% of the solids (soybean solids and cells) were recovered, approximately 14.8% of the solids (exopolysaccharides outside the cells) were recovered by ethanol precipitation of the supernatant, and the remaining solids in the fluid were approximately 3.5%.

[0162] Through these bench-scale tests, the levels of protein, pullulan, and total solids that could be recovered each day were measured. As the incubation progressed, the levels of protein and pullulan increased, but it was expected that the total solids recovered would decrease because some nutrients were dissimilated into water and CO2. The average protein level of the solids from three repeated tests is shown in Figure 3. The protein level reached 70% by the 3rd to 5th day, but the total solids recovered had begun to decrease by the 5th to 6th day. Therefore, an incubation period of 4 - 5 days is considered optimal. [Example 2] Comparison of products of the precipitation (ppt) method, three - wash method, and one - wash method

[0163] HQPC from soybeans was obtained for both 3 - wash cycles and 1 - wash cycle by the method substantially described above and shown in Figures 6 - 9. A comparison of the compositions obtained for the ppt method (HQSPC Tests 5 and 6), 3 - wash and 1 - (HQPC) - wash is shown in Table 2. [Table 3] [Table 4] [Example 3] Hydrolysis of Soybean Meal by Temperature

[0164] Soybean meal extracted with hexane was used in water to prepare a 10% (w / v) soybean meal slurry. The pH of the slurry was set to 4.5 and the slurry was stirred to obtain a mixture. Before fermentation, the soybean slurry was heated to a temperature of 100 °C. The heated mash was incubated with fermentative organisms. The heated mash without fermentative organisms was treated with FLAVORZYME® (a protease derived from Aspergillus niger purchased from Sigma) at a loading rate of 30 mg / g of soybean meal and used as a control. The samples were incubated at 30 °C for 12 hours. After incubation, the samples were heated at 80 °C for 2 minutes to inactivate the protease.

[0165] The mash was heated at 100 °C for 1.5 minutes. The samples were centrifuged at 4000 rpm for 10 seconds. N - acetylcysteine (3.33% w / v) was prepared in boric acid buffer (0.12 M, pH 10.4). 16.67 μL of the sample supernatant was added to 1000 mL of N - acetylcysteine. The absorbance was measured at 340 nm. A 6% (w / v) OPA solution was prepared in a 96% (v / v) ethanol solution. 10 μL of the OPA solution was added to 305 μL of the sample. The sample with the added OPA solution was incubated at room temperature for 15 minutes. The absorbance was measured at 340 nm. The relative degree of hydrolysis (%) was calculated using the following calculation. [A340test (15) - A340 test (0)] × 100 / [A340 control (15) - A340 control (0)]. Refer to Table 3.

Table 5

[0166] Hexane-extracted soybean meal was used in water to prepare a 10% (w / v) soybean meal slurry. The pH of the slurry was set to 4.5, and the slurry was stirred to obtain a mixture. Before fermentation, the soybean slurry was heated to a temperature of 100 °C. The heated mash was incubated with fermentative organisms. The heated mash without fermentative organisms was treated with FLAVORZYME® at a loading rate of 30 mg / soybean meal g and used as a control. The samples were incubated at 30 °C for 12 hours. After incubation, the samples were heated at 80 °C for 2 minutes to inactivate the protease.

[0167] Fermentation samples were collected at 0, 2, 4, 6, 8, 10, and 12 hours. The samples were centrifuged at 4000 rpm for 10 seconds. N-acetylcysteine (3.33% w / v) was prepared in boric acid buffer (0.12 M, pH 10.4). 16.67 μL of the sample supernatant was added to 1000 mL of N-acetylcysteine. The absorbance was measured at 340 nm. A 6% (w / v) OPA solution was prepared in a 96% (v / v) ethanol solution. 10 μL of the OPA solution was added to 305 μL of the sample. The sample with the OPA solution added was incubated at room temperature for 15 minutes. The absorbance was measured at 340 nm. The relative degree of hydrolysis (%) was calculated using the following calculation. [A340 test (15) - A340 test (0)] × 100 / [A340 control (15) - A340 control (0)]. Refer to Table 4.

Table 6

[0168] Using soybean meal extracted with hexane in the centrifuged liquid by solid-liquid - liquid separation, a 10% (w / v) soybean meal slurry was prepared. The pH of the slurry was set to 4.5 and the slurry was stirred to obtain a mixture. Before fermentation, the soybean slurry was heated to a temperature of 100 °C. The heated mash was incubated with fermentative organisms. The heated mash without fermentative organisms was treated with FLAVORZYME® at a loading rate of 30 mg / g of soybean meal and used as a control. The samples were incubated at 30 °C for 12 hours. After incubation, the samples were heated at 80 °C for 2 minutes to inactivate the protease.

[0169] Fermentation samples were taken at 0, 2, 4, 6, 8, 10, 12 hours. The samples were centrifuged at 4000 rpm for 10 seconds. N-acetylcysteine (3.34% w / v) was prepared in boric acid buffer (0.12 M and pH 10.4). 16.67 μL of the sample supernatant was added to 1000 mL of N-acetylcysteine. The absorbance was measured at 340 nm. A 6% (w / v) OPA solution was prepared in a 96% (v / v) ethanol solution. 10 μL of the OPA solution was added to 305 μL of solution A. The sample to which the OPA solution was added was incubated at room temperature for 15 minutes. The absorbance was measured at 340 nm. The relative degree of hydrolysis (%) was calculated using the following calculation. [A340 test (15) - A340 test (0)] × 100 / [A340 control (15) - A340 control (0)]. See Table 5. [Table 7] [Example 5] Hydrolysis of phytase-added soybean meal slurry by fermentation

[0170] Soybean meal extracted with hexane was used in water to prepare a 10% (w / v) soybean meal slurry. The pH of the slurry was set to 4.5 and the slurry was stirred to obtain a mixture. Before fermentation, the soybean slurry was heated to a temperature of 100 °C. The heated mash was incubated with fermentative organisms. The heated mash without fermentative organisms was treated with FLAVORZYME® at a loading rate of 30 mg / g of soybean meal and used as a control. The samples were incubated at 30 °C for 12 hours. After incubation, the samples were heated at 80 °C for 2 minutes to inactivate the protease.

[0171] Fermentation samples were collected at 0, 2, 4, 6, 8, 10, and 12 hour time points. Samples at 8, 10, and 12 hours were also treated with phytase. The samples were centrifuged at 4000 rpm for 10 seconds. N-acetylcysteine (3.34% w / v) was prepared in boric acid buffer (0.12 M, pH 10.4). 16.67 μL of the sample supernatant was added to 1000 mL of N-acetylcysteine. The absorbance was measured at 340 nm. A 6% (w / v) OPA solution was prepared in a 96% (v / v) ethanol solution. 10 μL of the OPA solution was added to 305 μL of solution A. The sample to which the OPA solution was added was incubated at room temperature for 15 minutes. The absorbance was measured at 340 nm. The relative degree of hydrolysis (%) was calculated using the following calculation. [A340 test (15)-A340 test (0)]×100 / [A340 control (15)-A340 control (0)]. See Table 6. [Table 8] [Example 6] Feeding tests of rainbow trout, barramundi, and ginza salmon

[0172] Long-term and short-term feeding trials were completed using multiple lots of rainbow trout (Oncorhynchus mykiss), barramundi (Lates calcarifer), and coho salmon (Oncorhynchus kisutch). In all trials, the fish were fed either a commercially available control diet or a diet utilizing high levels (25 - 35%) of HQPC prepared as described in Example 2 (washed three times). All diets were formulated using commercially available feed formulation software and manufactured using a commercially available extrusion method. All chemical analyses of the diets (proximate analysis and mineral composition) were analyzed using a third-party laboratory (Midwest Laboratories, Omaha, NE).

[0173] 3.41m 3 Growth trials (average individual initial weight of approximately 185 g to harvest weight of 1000 g) were conducted using a RAS consisting of (900 gal) tanks. The RAS consisted of a Cornell-style dual-drain tank, solid settling tank, mechanical drum filtration, moving bed biofilm reactor (MBBR), UV sterilization, cooling, and oxygen injection. Water quality was monitored daily to confirm that all parameters were within acceptable ranges.

[0174] On day 0 starting samples, all fish were weighed by tank group to measure biomass, and one sample of all fish in one tank per treatment was anesthetized using 80 mg / L of MS-222, and fork length and weight were measured. Group body weight and sampling of individual fish of the same identity were completed at 3-week intervals throughout the 27-week test period. At the 16-week time point, one fish per tank (6 fish per treatment) was sampled for a general health assessment including spleen, liver, visceral fat, and hematocrit.

[0175] Shrimp Feeding Trial Larval Development Trial At the Sumacua hatchery in Choluteca, Honduras, approximately 90 million nauplii (stage 5) of Litopenaeus vannamei were produced and transferred to tanks containing 20 metric tons of seawater (28 ppt) for all experiments. Shrimp larvae from Zoea 3 to Postlarval 13 (PL 13) were fed one of four diets containing HQPC (up to 70% content). Survival rate (%) was determined by extrapolation of aliquots that counted all larvae surviving at the end of the test.

[0176] Growth test Whiteleg shrimp (Litopenaeus vannamei) (average 1.6 g) were randomly stocked (n = 700) at 20 shrimp per tank. Each treatment was randomly assigned to five replicate test tanks. Experimental diets were fed three times a day for 42 days (0800, 1200, and 1600 hours). Prior to the start of the test, all shrimp were fed at the same rate as the control diet. From the start of the test (day 1), shrimp were fed the experimental diet until they appeared full. Feed conversion ratio was estimated using total feed consumption. Tank biomass was recorded at the time of shrimp storage (day 0) and at three-week intervals until the test was completed. Relative growth (RG), specific growth rate (SGR), and biomass increase during the test period were calculated using total tank biomass measurements.

[0177] The experiment was conducted in an 8,246 L recirculating aquaculture system (RAS). The system was equipped with a 35,190 L semi-square tank, and each tank was equipped with a circulating drain pipe that draws water from below the surface and a drain pipe for sludge attached at the lowest position at the bottom of the center of the tank. Each tank had a forced blower blown by a blower, a flow valve at the inlet to control the direction of the flow, a cover that darkens half of the tank, and a net that allows light to pass through the other half of the tank. The RAS was also equipped with a centrifugal water pump, a bead filter, a UV filter, a biofilter, three solid sedimentation samples, a purification sample, an inlet float valve, and a heater / cooler unit. The makeup water for the RAS was sourced from well water. The water flow to each tank was maintained at 6 - 7 L·min -1 and the water temperature was maintained at 28 - 30 °C. The dissolved oxygen was maintained at 5.0 mg·L -1 or higher, the pH was maintained at 7 - 8, and the salinity concentration was maintained at 22 ppt throughout the test period. The temperature, dissolved oxygen, and pH were monitored daily (at 0800 hours), and ammonia (NH3) and nitrite (NO2) were monitored weekly.

[0178] Challenge test To investigate the effectiveness of a diet containing HQPC in reducing the severity and impact of early mortality syndrome / acute hepatopancreatic necrosis disease (EMS / AHPND) in shrimp, a test was conducted at the ShrimpVet Research Institute in Ho Chi Minh City, Vietnam. The test was continued over 33 days, including a 1-day adaptation period, a 21-day feeding period, a 1-day challenge, and a 10-day post-challenge follow-up period.

[0179] The tests were conducted in 120 L plastic tanks. All tanks were equipped with activated coral biofilters and aeration, and covered with plastic caps to reduce the risk of cross - contamination. For each test, brackish water with a salinity of 20 parts per thousand (ppt) was used. During the test period, the shrimp were fed 4 times a day and allowed to feed freely. The feed consumption during the test was recorded. The test diets included 1 type of basal diet and 6 treatment diets containing HQPC (content 10 - 30%). The feeding amount was adjusted according to the biomass and actual feed consumption. Water quality parameters, such as dissolved oxygen (DO), pH, and temperature, were measured daily. Total ammonia nitrogen, nitrite, and alkalinity were measured twice a week.

[0180] Specific pathogen - free (SPF) shrimp (Litopenaeus vannamei) with the original genetic properties obtained from Hawaiian broodstock, which were tested using the PCR method for important pathogens including Enterocytozoon hepatopenaeid (EHP), white spot syndrome virus (WSSV), Taura syndrome virus (TSV), infectious myonecrosis virus (IMNV), and EMS / AHPND disease, were used in this test. Nauplius larvae were reared in a strict biosecurity facility. Post - larvae were also retested using the PCR method for important pathogens including EHP, WSSV, TSV, IMNV, and EMS / AHPND disease. The post - larvae were grown under biosecurity conditions. One day before the start of the test, they were weighed within the group to measure the initial body weight of the shrimp. The average initial body weight of the shrimp was 0.56 ± 0.04 grams.

[0181] In this test, the immersion loading method was also used. A total of 28 tanks, including treatment tanks and positive controls, were subjected to immersion loading. Pathogenic Vibrio parahaemolyticus was consistently inoculated into Tryptic Soy Broth + 2% Sodium Chloride (TSB+), and incubated for 24 hours. This bacterial suspension was added to the tanks to achieve a bacterial density measured by optical density absorbance (OD 600nm), which is the density expected to kill 90% of the positive controls within 10 days, the "LD90". Negative controls (a total of 4 tanks) were treated with sterilized TSB+ added directly to the tanks. The dosing amount was 3.25×105 CFU / mL, which was the 90% lethal dose (LD90). As a standard histological examination, the tissues of the shrimp were stained with H&F.

[0182] Fish feeding test The results of ANCOVA for multiple tests showed that the control fish were significantly smaller and were affected by both treatment (P = 0.002) and weeks (P < 0.001). In addition, no significant difference in FCR was observed between treatments or weeks (Figure 12). Analysis of covariance (treatment × weeks) revealed that most of the differences were due to weeks rather than the treatment feed. However, there were significant differences in the average weight per shrimp for both treatment (P = 0.046) and weeks (P < 0.001), indicating that shrimp fed the HQPC-based feed were consistently larger than those fed the control feed. Also, significant differences in FCR were found for both treatment (P = 0.001) and weeks (P < 0.001), showing that shrimp fed the HQPC-based feed utilized the feed more efficiently and consistently than those fed the control feed. The overall results of this test are similar to other experiments where rainbow trout were fed fermented soybean meal. Similarly, no enteritis was observed in these tests, and no adverse effects were seen in the distal intestine of rainbow trout fed the HQPC-based feed.

[0183] In the second similar test, the shrimp individuals fed with the HQPC-based feed showed significantly and consistently lower FCR (about 1.0) than those fed with the control feed (about 1.25) (Figure 13a, Figure 13b). By ANCOVA, significant effects of weeks (P<0.001) and treatment (P<0.001) were revealed. The difference in average weight per shrimp individual was due to weeks (P<0.001), and there was no difference due to treatment (P=0.305). There were no significant differences in K value (P=0.758), spleen somatic index (P=0.998), hepatosomatic index (P=0.475), and visceral somatic index (P=0.411). The shrimp individuals fed with the HQPC-based feed had a significantly larger visceral somatic index (P=0.040) and a lower hematocrit (P=0.005).

[0184] Shrimp feeding test Larval growth test When HQPC was included in the hatchery diet at various ratios, promising results were obtained and several productivity parameters including larval survival rate were improved (Figure 14).

[0185] Growth test The weekly growth rate of shrimp reared with HQPC up to a level of 50% was not significantly different from that of shrimp fed with commercial feed (Figure 15). Although shrimp growth can be affected by many other factors such as water quality conditions and genetic properties, based on the shrimp responses observed in the test, there was no difference during the HQPC treatment, and it was confirmed that HQPC was used in the commercial feed formulation. As a result of the growth test, it was revealed that the higher the apparent digestibility coefficient of HQPC, the higher the digestibility of amino acids and the better the growth of animals.

[0186] Challenge test From the results obtained, it was shown that applying various inclusion levels had a positive effect on improving the survival rate of EMS-infected shrimp. Based on these results, an improvement in survival rate was observed in all treatments with diets containing HQPC. These tests showed that applying 30% of HQPC had a positive effect on improving the survival rate of EMS-infected shrimp (Figure 16).

[0187] Water quality The formulated feed is a major driving force enabling production intensification and is also a major source of N and P in RAS. In addition, phosphorus is one of the most important minerals when formulating aquaculture feeds for RAS projects. On average, HQPC contains 0.4% phosphorus (Table 7), and the phytic acid concentration in the final product is less than 0.12 (g / 100g) on average.

Table 9

[0188] After continuously using the feed containing HQPC data collected from commercial mass operations for 18 months, a 69% reduction in phosphorus emissions was observed, from an initial level of 0.21 mg / liter to 0.065 mg / liter. During the shrimp growth test and loading test, water quality parameters were recorded daily. The values of water quality parameters (temperature, DO, pH, TAN, nitrite, and alkalinity) are presented (Table 8).

Table 10

[0189] When comparing the test of shrimp tested for HQPC with the water quality measurements of fish meal, no particular differences were found. The total dissolved solids in the RAS water for HQPC and fish meal treatments were 774 and 822 ppm, respectively. Turbidity (NTU) measured in the system tank showed that HQPC (0.4) was slightly superior to the fish meal control (0.6).

[0190] The digestibility tests were carried out at the Aquaculture Laboratory (LAM), Institute of Oceanography, University of São Paulo (USP; São Paulo, Brazil). Samples of two soybean products were provided by Prairie AquaTech (South Dakota, USA). Samples of soybean products, non-GMO soybean meal, SBM (crude protein 46.8%, CP) and HQPC (74.6%, CP), were of appropriate particle size (above 150 microns) and were analyzed to measure the degree of protein hydrolysis (DH, %).

[0191] These samples were tested for in vitro protein digestion with standardized digestive enzymes recovered from the hepatopancreas of farmed white shrimp (average weight 10 grams) in ponds. Hydrolysis by shrimp enzymes was carried out by suspending 80 mg of the component protein in distilled water, setting the pH of the suspension to 8.0, and then adding the hepatopancreas enzyme extract for hydrolysis.

[0192] Monitoring of pH shift and hydrolysis was automatically performed by a commercially available software-controlled potentiometric titrator in a temperature-controlled device (30 ± 0.6 °C). At this reaction pH (= 8.0), the reaction pH decreased slightly due to the enzymatic cleavage of the component peptide bonds, which was registered and automatically neutralized by the titrator adding sodium hydroxide NaOH. The amount of titrant (NaOH) consumed at the end of the reaction is proportional to the number of cleaved peptide bonds and the quantitative value provided.

[0193] Buffers and other chemicals were not used in the analysis. Blank DH values were calculated to calculate the net DH of the component protein if judged significant.

[0194] The results of the tests showed significant differences in the degree of hydrolysis (DH) of the test components by shrimp digestive enzymes (Table 9), and from the DH values obtained for HQPC, it was shown that the hydrolyzable protein was 42% more than soybean meal and 33% more than soybean protein concentrate.

[0195] Typical DH values of SBM were in the range of 4.0% - 4.2% for skinned samples or unskinned samples, respectively. Results of comparative pH statistical tests on a sample set of more than 40 SBMs from major producing countries (India, Argentina, the United States, and Brazil) indicate that the DH values are between 3.74% and 4.43%.

[0196] For novel ingredients, appropriate screening is necessary to evaluate potential nutritional value and variability. Various tests have shown that in vitro digestion of ingredients by enzymes from target shrimp or fish species is related to apparent protein digestibility (APD). For nearly half a century, the pH-stat method has been used to monitor the effect of heat treatment on the initial rate of trypsin proteolysis of soy proteins. Previous tests have also shown the relationship between apparent protein digestibility (APD) and in vitro DH in juvenile Litopenaeus vannamei.

[0197] The average in vivo apparent digestibility coefficient for crude protein in Litopenaeus vannamei is 85 - 90%. In the tests conducted, the degree of hydrolysis (DH) of HQPC was 7.18%, and the predicted apparent protein digestibility (PPD) was estimated to be 93.1%. Compared with other tests, the HQPC values for DH and PPD are the highest among those determined for more than 150 ingredients tested, and are higher than various fish meals, soy protein concentrates, and non-GM soy flours (Table 9).

Table 11

[0198] In addition to multiple previous tests, this evaluation also showed the potential of the commercial product HQPC as an alternative ingredient to fish meal in aquaculture feeds regarding its in vitro digestibility. These results demonstrated that this ingredient product has a significantly higher level of hydrolyzed protein and a higher predicted apparent protein digestibility than various soy meal components.

[0199] All references cited in this specification are hereby incorporated by reference in their entirety.

[0200] From the above considerations, those skilled in the art can identify the essential characteristics of the present invention and make various changes and modifications to the embodiments to adapt to various applications and conditions without departing from the spirit and scope of the present invention. Therefore, in addition to what is shown and described in the drawings and this specification, various modified forms of the embodiments will be apparent to those skilled in the art from the above description. Such embodiments are also intended to be included within the scope of the appended claims.

Claims

1. 1. A method for treating plant material comprising the steps of: a) transferring the plant material to a first mixing tank, where the plant material is mixed with one or more first solvents to generate a washed mash; b) separating the washed mash into at least one centrate and a washed cake; c) transferring the washed cake to one or more second mixing tanks, wherein the one or more second solvents are mixed with the washed cake to produce a washed cake suspension; d) transferring the washed cake suspension to one or more fermentors, wherein the transferred washed cake suspension is inoculated with at least one microorganism and the inoculated washed cake suspension is incubated for a sufficient time to produce a fermented mixture; e) heating the fermented mixture for a time sufficient to achieve a degree of hydrolysis (DH) of at least about 10% for the protein therein; f) separating the heated fermented mixture into a fermented centrate and a fermented cake; g) removing the fermented centrate; (i) a first mixing tank and / or (ii) one or more second mixing tanks wherein a mixing tank contains said plant material or said washed cake, and steps (c) through (f) and h) are repeated at least once for sub-steps (i) or (ii); and h) drying the fermented cake; wherein the at least one microorganism does not produce sufficient exopolysaccharides to produce a sticky fermented cake during drying, and the resulting dried fermented cake has a higher protein content and / or substantially reduced anti-nutritional factors compared to the transferred plant material.

2. 10. The method of claim 1, wherein the at least one microorganism produces less than about 3 g / L of pullulan when grown in a medium containing 0.35 to 0.5 g / L of yeast extract.

3. 10. The method of claim 1, further comprising transferring the at least one centrate of step (b) to one or more of the mixing tanks prior to inoculation.

4. 4. The method of claim 3, wherein recycling the centrate a) reduces the amount of fresh solvent added to the first mixing tank and / or the one or more second mixing tanks, and / or b) increases protein yield and recovery.

5. The method of claim 1 , which does not include the addition of cellulolytic enzymes.

6. 10. The method of claim 1, further comprising heating the washed cake suspension prior to transferring to one or more fermentors.

7. 7. The method of claim 6, wherein the washed cake suspension is heated to greater than 100°C.

8. 4. The method of claim 3, wherein the fermentation centrate is transferred to the first mixing tank.

9. 4. The method according to claim 3, wherein the centrate and cake are produced by hydrodynamic forces, the method comprising a system of four mixing tanks and four centrifuges in series, and prior to the second fermentation, the fermentation centrate of mixing tank 4 is transferred to mixing tank 3, the centrate of mixing tank 3 is transferred to mixing tank 2, and the centrate of mixing tank 2 is transferred to mixing tank 1.

10. 2. The method of claim 1, wherein the solvent is selected from one or more of water, an acid, an aqueous enzyme mixture, an antifoam agent, or combinations thereof, and the aqueous enzyme mixture comprises a phytase.

11. 10. The method of claim 1, wherein the centrate from the first mixing tank, the fermented centrate, or both, is transferred to at least one evaporator that produces a liquid protein concentrate.

12. 12. The method of claim 11, wherein the centrate is evaporated at a temperature of about 60° C. to 90° C. and / or at about 1 psia to 6 psia.

13. 10. The method of claim 1, wherein the non-animal protein concentrate is isolated from plant material from the group consisting of soybean, sorghum, peanut, legumes, rapeseed, oats, barley, rye, lupin, faba bean, canola, pea, sesame, cottonseed, palm kernel, barley, grape seed, olive, safflower, sunflower, copra, corn, coconut, flaxseed, hazelnut, wheat, rice, potato, cassava, legumes, camelina seed, mustard seed, germ meal, corn gluten meal, distillery / brewery by-products, and combinations thereof.

14. 10. The method of claim 1, wherein drying is carried out at a temperature above 100°C and the dried fermented cake exhibits a moisture content of less than about 7%.

15. 2. The method of claim 1, wherein said at least one microorganism is NRRL Y-2311-1.

16. The method of claim 1 , wherein the treatment does not include adding one or more cellulolytic enzymes.

17. The raw NIR spectrum of the final product shows a peak at 4664 cm －１ ~4836cm －１ 2. The composition of claim 1, wherein the composition exhibits a significant downward shift in relative to the feedstock.

18. 18. The composition of claim 17, wherein the downward shift is at least about 10% to about 20%.

19. 13. A composition comprising a solid protein concentrate produced by the method of claim 1.

20. 12. A composition comprising a protein condensate produced by the method of claim 11.

21. 21. A feed or foodstuff comprising a composition according to claim 19 or 20.

22. 16. The feed of claim 15, formulated for an animal selected from the group consisting of fish, shellfish, crustaceans, farm animals, livestock, and combinations thereof.

23. 16. The foodstuff of claim 15, wherein the composition is for human consumption.

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