RU2844745C1 - Microbial process for improved quality protein concentrate - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and fodder industry. Disclosed is a non-animal protein concentrate, where the concentrate contains a fermented vegetable product containing a. pullulans, containing at least from about 65% to about 75% protein in terms of dry substance, and has an ash content of less than about 2.5%, where said concentrate is an animal protein substitute, and where the non-animal protein is extracted from plant material, including soya beans. Also described is a fodder, in particular for shrimp juveniles, containing said concentrate, and a method for improving the survival rate of shrimp juveniles by feeding them with said fodder.

EFFECT: disclosed is a microbial process for an improved quality protein concentrate.

13 cl, 16 dwg, 9 tbl, 6 ex

Description

AREA OF TECHNOLOGY

The invention relates generally to incubation processes and, in particular, to microbial incubation processes for the production of high-quality protein concentrates, including products made from them, and the use of such products in nutritional feeds.

LEVEL OF TECHNOLOGY

In 2008, approximately 28% of the world's wild marine fish stocks were overexploited and 52% were fully exploited, even though per capita demand for fish and shellfish has increased with population growth. As wild fish stocks decline, commercial aquaculture production has increased dramatically to meet this increased demand. However, one of the main components of aquaculture nutrient formulations, fishmeal protein, is also obtained from wild capture fisheries. It has been estimated that at least 6.7 million tonnes of fishmeal will be needed by 2012 to support commercial aquaculture production. This is clearly unsustainable based on current trends.

Cheaper, more sustainable plant protein sources are being used to partially replace fishmeal in aquaculture diets. Defatted soybean meal (SBM, 42-48% protein) is commonly used to replace up to 20% of the total protein in grower diets for several species, while soy protein concentrate (SPC, 65% protein) has been successfully tested at higher overall protein replacement levels, largely determined by the trophic status of the species. These soy products provide high protein contents and relatively good amino acid profiles, but are still deficient in some essential amino acids (e.g. taurine) required by carnivorous marine fish. SPC can be used in higher quantities than soybean meal, primarily because the solvent extraction process used to produce SPC removes antinutritional factors (e.g. oligosaccharides) and thereby increases protein bioavailability. In addition, a thermal step has been used to inactivate heat-labile antigenic factors. The main limitations of the current solvent extraction process are its cost, lack of use of the oligosaccharides removed in the process, and quality issues that often limit inclusion of 50% of total protein in the diet. In addition, processing of soybean material into soy flour or soy protein concentrates can be environmentally problematic (e.g., chemical waste disposal issues associated with hexane extraction).

Animal protein producers face a range of sustainability risks. By diversifying into sustainable proteins, these companies can hedge these risks and tap into a fast-growing market.

At the same time, innovation in food technology is accelerating and creating opportunities for protein production that could disrupt the existing industry. For intensive animal protein producers, not taking advantage of this innovation is a risk. Diversifying into alternative (i.e. non-animal) proteins is therefore key to both managing resource-constrained supply chain risks and taking advantage of market growth opportunities.

Meat alternatives and broader protein alternatives that can serve as substitutes for traditional animal products are attracting significant financial investment.

Therefore, there is a need for a plant-based protein source that is cost-effective and sustainable, and of sufficiently high quality to completely or substantially replace larger amounts of animal protein in animal diets, including the use of such plant-based sources in meat alternatives in general (e.g. for human consumption).

ESSENCE OF THE INVENTION

The present invention relates to an organic microbial system for converting plant material into a highly digestible concentrated protein source that also contains a binding agent based on microbial gum (exopolysaccharide), including such a concentrated source that is suitable for use as animal feed and food for human consumption.

In embodiments, a composition is described that includes a protein concentrate of non-animal origin, wherein the concentrate comprises a fermented plant product containing A. pullulans with a low yield of pullulan and a protein content of at least about 65% to about 75% (based on dry matter), wherein the protein concentrate exhibits one or more properties including a degree of hydrolysis (DH) of at least about 2%, an ash content of up to about 4%, or a potassium and magnesium content of less than about 0.1 ppm.

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

In another aspect, a non-animal protein concentrate is isolated from plant material, including soybeans, sorghum, peanuts, legumes, rapeseed, oats, barley, rye, lupine, faba beans, canola, peas, sesame seeds, cottonseed, coconut kernels, barley, grape seeds, olives, safflower, sunflowers, copra, corn, coconuts, flaxseed, hazelnuts, wheat, rice, potatoes, cassava, legumes, camelina seeds, watercress seeds, mustard seeds, germ meal, corn gluten meal, corn gluten feed, distillery/brewery by-products, and combinations thereof.

In a related aspect, the plant material is obtained from soybeans in the form of soy flakes or soy flour.

In one aspect, a feed or food product comprising the above composition is described.

In a related aspect, the composition is combined with one or more meat substitutes. In a further related aspect, the meat substitute comprises defrosted and sliced frozen tofu, oncom, tempeh, tofu, soy turkey, artificial turkey, paneer, glamorgan, breadfruit, sapal, eggplant, jackfruit, falafel, ganmodoki and combinations thereof. In a further related aspect, the concentrate improves one or more organoleptic characteristics, including texture, aroma, mouthfeel, bite, crunch, taste, appearance or combinations thereof, of said one or more meat substitutes compared to the same meat substitutes that lack said concentrate. In another related aspect, the food product is intended for human consumption.

In one aspect, the feed is formulated for animals including fin fish, mollusks, crustaceans, pets, farm animals, and combinations thereof.

In another aspect, A. pullulans is NRRL-Y-2311-1.

In one aspect, there is a significant shift toward lower values in the raw NIR spectra between 4664 cm ^-1 and 4836 cm ^-1 for the final product compared to the starting material. In a related aspect, the shift toward lower values is from about at least 10% to about 20%.

In embodiments, a method for processing a plant-based material is described, comprising: a) transferring the plant-based material to a first mixing tank, where the plant-based material is mixed with one or more first solvents to obtain a washed slurry; b) separating the washed slurry into at least one centrifuge and a washed cake; c) moving the washed cake to one or more second mixing tanks, where the one or more second solvents are mixed with the washed cake to obtain a washed cake slurry; o!) transferring the washed cake slurry to one or more fermenters, where the transferred washed cake slurry is inoculated with at least one microbe, and where the inoculated washed cake slurry is incubated for a time sufficient to obtain a fermented mixture; e) heating the fermented mixture for a time sufficient to achieve a degree of hydrolysis (DH) of from about 2% to about 80% of the proteins contained therein; f) separating the heated fermented mixture into a fermented centrifugate and a fermented cake; g) transferring the fermented centrifugate to:

(i) a first mixing tank and/or

(ii) one or more second mixing tanks, wherein the mixing tank contains the plant-based material or the washed cake sludge, and wherein steps (c)-(f) and h) are repeated at least once (1) time for sub-steps (i) or (ii); and h) drying the fermented cake when the at least one microorganism does not form sufficient exopolysaccharides to produce a viscous fermented cake during drying, and wherein the resulting dry fermented cake has a higher protein content and/or has substantially reduced anti-nutritional factors compared to the transferred plant material.

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

In another aspect, the method further comprises transferring at least one centrifugate obtained in step (b) to one or more mixing tanks prior to inoculation.

In one aspect, recirculating the centrifugates: a) reduces the amount of fresh solvent added to the first mixing tank and/or one or more second mixing tanks, and/or b) increases the yield and recovery of protein products.

In another aspect, the method does not include adding cellulose-degrading enzymes.

In one aspect, the method further comprises heating the washed cake slurry prior to transferring to one or more fermenters. In a related aspect, the washed cake slurry is heated to a temperature above 100°C.

In another aspect, the fermentation centrifuge is transferred to a first mixing tank.

In a related aspect, the centrifugates and cakes are obtained by hydrodynamic force, and if the method includes a system of four (4) mixing tanks and four (4) centrifuges arranged in series, where the fermentation centrifuge from the mixing tank 4 is transferred to the mixing tank 3, the centrifuge from the mixing tank 3 is transferred to the mixing tank 2, and the centrifuge from the mixing tank 2 is transferred to the mixing tank 1 before the second fermentation.

In one aspect, the solvent comprises water, an acid, an aqueous enzyme mixture, an antifoam, or a combination thereof, wherein the aqueous enzyme mixture comprises phytase.

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

In one aspect, the non-animal protein concentrate is isolated from plant material, including soybeans, sorghum, peanuts, legumes, rapeseed, oats, barley, rye, lupine, faba beans, canola, peas, sesame seeds, cottonseed, coconut kernels, barley, grape seeds, olives, safflower, sunflowers, copra, corn, coconuts, flaxseed, hazelnuts, wheat, rice, potatoes, cassava, legumes, camelina seeds, mustard seeds, germ meal, corn gluten meal, corn gluten feed, distillery/brewery by-products, and combinations thereof.

In another aspect, the drying is carried out at a temperature above 100°C, and the moisture content of the dried fermented cake is less than about 7%.

In one aspect, at least one microbe is NRRL Y-2311-1. In another aspect, the microbe can be identified by targeting the presence of the amplification products of SEQ ID NO: 1 as a template using PCR. In a related aspect, the amplification products can be used to identify the source of HQPC, as described herein.

In one aspect, there is a significant shift toward lower values in the raw NIR spectra between 4664 cm ^-1 and 4836 cm ^-1 for the final product compared to the starting material. In a related aspect, the shift toward lower values is from about at least 10% to about 20%.

In another aspect, the treatment does not include adding one or more cellulose-degrading enzymes.

In embodiments, a composition is described comprising a solid protein concentrate obtained by the methods described above.

In embodiments, a feed or food product is described that includes the compositions described above.

In a related aspect, the feed is formulated for animals including finfish, mollusks, crustaceans, domestic animals, farm animals, and combinations thereof. In yet another related aspect, the composition is intended for human consumption.

In embodiments, a method for improving the survival of juvenile shrimp is described, comprising feeding juvenile shrimp a feed as described above, wherein the degree of hydrolysis (DH) of 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%.

In embodiments, a feed for juvenile shrimp is described, comprising the composition described above, wherein the feed has 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 DRAWINGS

Figure 1 shows a flow chart of the HQSPC conversion process.

Figure 2 shows the flow chart of the HQSPC conversion process for aquaculture feed.

Figure 3 shows laboratory scale incubation tests to evaluate the composition and yield of HQSPC.

Figure 4 shows the flow chart of the HP-DDGS conversion process for aquaculture feed.

Figure 5 shows the effect of moisture content and extrusion speed on glucose recovery after extrusion of HP-DDGS at 100°C.

Figure 6 shows a schematic diagram of a single HQPC process described in this document.

Figure 7 shows the details for centrifugate 1.

Figure 8 shows the details for centrifugate 4.

Figure 9 shows the value-added products for the scheme in Figure 6.

FIG. 10 shows the raw spectra for various SBM feedstock samples. The X-axis is the wavenumber, and the Y-axis is the intensity in absorbance units (AU).

FIG. 11 shows the raw spectra for the various HQPC products obtained from the SBM samples of FIG. 10. The X-axis is wavenumber, and the Y-axis is intensity in AU.

FIG. 12: Final weight (g) of rainbow trout diet containing HQPC (25%) compared to fishmeal diet.

FIG. 13a: Weekly growth rate (g) of performance of coho salmon fed HQPC.

FIG. 13b: Feed conversion ratio (FCR) of coho salmon fed HQPC.

FIG. 14: Survival of L. vannamei (Z3-PL13) larvae at first feeding fed with artificial diets containing HQPC.

FIG. 15: Average body weight (g) of juvenile L. vannamei fed artificial diets containing HQPC.

FIG. 16: Survival (%) of L. vannamei juveniles fed with HQPC-containing artificial diets at day 10 after EMS challenge. Mean values with the same superscript are not significantly different from each other (Tukey's HSD, P<0.05).

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present composition, methods and techniques, it should be understood that the present invention is not limited to the specific compositions, methods and experimental conditions described, since such compositions, methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

As used in the specification and appended claims, the singular forms "a", "an" and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, references to "a composition" include one or more compositions and/or formulations of the type described herein that will become apparent to those skilled in the art after reading this specification, and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, so long as it is understood that modifications and variations are within the spirit and scope of the present description.

As used herein, the terms "about," "approximately," "substantially," and "significantly" will be understood by those skilled in the art and will vary to some extent depending on the context in which they are used. If there are uses of a term that are not understood by those skilled in the art given the context in which it is used, "about" and "approximately" will mean plus or minus <10% of the particular term, and "substantially" and "substantially" will mean plus or minus >10% of the particular term.

Internal studies developing practical diets for RAS operations using microbially enriched protein as described have shown that the HQPC described herein is a promising solution for the production of environmentally friendly aquaculture feeds. In addition to having more than 70% crude protein content and highly available phosphorus, the HQPC described herein can be produced from non-modified soybeans, making it an ideal product for the European aquaculture industry.

Results from numerous in-house feed trials have shown that the HQPC described herein can support fish and shrimp health, high growth performance and feed efficiency at super dose inclusion levels of up to 70% of total diet ingredients. Feed trials using rainbow trout, barramundi and coho salmon have shown that fish fed HQPC based diets as described consistently utilised feed more efficiently than fish fed a control diet. Trials using these species reared in a RAS system have shown good growth rates when fed diets containing up to 25% HQPC as described and reduced feed conversion rates.

The central challenge facing sustainable RAS is two interrelated constraints that operators must balance to run successful aquaculture operations. The first constraint is related to understanding modern water reuse systems, which limit fish or shrimp production. The second constraint is related to sustainable fish feeds, where the use of soy feeds in such modern water reuse systems makes them difficult to manage. The resulting situation requires modern aquaculture farmers to balance the risks and benefits of each constraint to achieve profitability while also satisfying their customers.

The formulation of low fishmeal aquaculture feeds for RAS systems requires the use of combinations of several ingredients, as most feeds have been shown to have significant nutritional and functional limitations. Fermentation of plant ingredients can reduce anti-nutritional factors and improve digestibility. Although not bound by theory, the significant differences observed in the growth performance of fish and shrimp fed HQPC in the present invention are likely due to the elimination of anti-nutritional factors and the increased digestibility of the ingredient. The results presented herein demonstrate that at least 80% of dietary fish meal can be directly replaced by HQPC, as described in commercial feeds for, among others, shrimp larvae and several species of juvenile and adult fish.

As used herein, the term "animal" means any organism belonging to the kingdom Animalia and includes, without limitation, humans, birds (e.g., poultry), mammals (e.g., humans, cattle, pigs, goats, sheep, cats, dogs, mice, and horses), and aquaculture organisms such as fish (e.g., trout, salmon, bass), shellfish (e.g., edible clams), and crustaceans (e.g., crabs, lobsters, shrimp, and prawns).

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

As used herein, the term "non-animal protein" means a substance that contains at least 0.81 g crude fiber/100 g of composition (calculated on a dry matter basis), this crude fiber being mainly cellulose and lignin obtained as a residue in the chemical analysis of plant substances.

As used herein, the term "incubation process" means providing appropriate conditions for the growth and development of bacteria or cells, when such bacteria or cells use biosynthetic pathways to metabolize various starting materials. In embodiments, the incubation process can be carried out, for example, under aerobic conditions. In other embodiments, the incubation process can include fermentation.

As used herein, the term "incubation products" means any residual materials directly resulting from the incubation process/reaction. In some cases, the incubation product contains microorganisms such that it has an increased nutrient content compared to an incubation product that does not contain such microorganisms. Incubation products may comprise suitable components from the incubation broth. For example, incubation products may include dissolved and/or suspended components of the incubation broth. Suspended components may include undissolved soluble components (e.g., when the solution is supersaturated with one or more components, such as proteins) and/or insoluble materials present in the incubation broth. Incubation products may include substantially all or a portion of the dry solids present at the end of incubation (e.g., from spray drying of the incubation broth and biomass resulting from the incubation). Incubation products may include crude material from incubation where the microorganism/solids/centrifugates/pellets may be fractionated and/or partially purified to increase the nutrient content of the material.

As used herein, the term "conversion culture" means a culture of microorganisms maintained in a medium containing material sufficient for the growth of the microorganisms, such as water and nutrients. The term "nutrient" means any substance that has nutritional value. It may be part of an animal feed, food product, or food additive for animals. Examples of nutrients include, but are not limited to, proteins, peptides, fats, fatty acids, nucleic acids, lipids, water- and fat-soluble vitamins, essential amino acids, carbohydrates, sterols, enzymes, and trace elements such as phosphorus, iron, copper, zinc, manganese, magnesium, cobalt, iodine, selenium, molybdenum, nickel, fluorine, vanadium, tin, silicon, and combinations thereof.

Conversion is a process of culturing microorganisms in a conversion culture under conditions suitable for converting protein/carbohydrate/polysaccharide materials, such as soy components, into a high-quality protein concentrate. Sufficient conversion includes, but is not limited to, utilization of 90% or more of certain carbohydrates to produce microbial cell mass and/or exopolysaccharide, enzymes and microbial metabolites, specific reduction in oligosaccharide concentration, achievement of a selected degree of protein hydrolysis, visibility of a specific % change in NIR spectra between 4664 cm ^-1 and 4836 cm ^-1 , or a combination thereof. In embodiments, the conversion can be aerobic or anaerobic, or a combination thereof.

As used herein, the term "flocculant" or "cleaning agent" is a chemical that promotes the release of colloids from suspension through aggregation and includes, but is not limited to, a multivalent ion and a polymer. In embodiments, such a flocculant/cleaning agent may include bioflocculants such as exopolysaccharides (e.g., pullulan).

As used herein, the term "composition" means a material or mixture prepared according to a specific recipe.

As used herein, the term "food" means a substance suitable for consumption as a nutritious composition that humans or animals eat or drink, or that plants absorb, to maintain life and growth.

As used herein, the term "concentrate" means the liquid remaining after the application of hydrodynamic force after the removal of most of the solid matter, the resulting dry product being referred to as a "cake".

As used herein, the term "suspension" means a heterogeneous mixture that contains solid particles large enough to settle.

As used in this document, the term evaporation refers to the process of converting a liquid into 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 separation process. Both processes can be used for different purposes. While evaporation in evaporation occurs below the boiling point, evaporation in distillation occurs at the boiling point.

As used herein, the term "exopolysaccharides" means high molecular weight polymers containing sugar residues that are produced by a microorganism and released into the environment, where the high molecular weight polymers include products of anabolism and metabolism.

As used herein, the term "degree of hydrolysis (DH)" refers to the proportion of cleaved peptide bonds in a protein hydrolysate. There are several methods for determining DH; the most commonly used include pH-stat, trinitrobenzene sulfonic acid (TNBS), o-phthaldialdehyde (OPA), soluble nitrogen in trichloroacetic acid (SN-TCA), and formal titration methods.

As used herein, the term "trypsin inhibitor unit (TIU)" refers to the amount of trypsin inhibitor in a sample. For example, a method may utilize N-benzoyl-DL-arginine p-nitroanilide as a chromogenic substrate for trypsin, and the ability of aliquots of soybean flour extract to inhibit trypsin activity with respect to this substrate is used to estimate the amount of trypsin inhibitor in the soybean flour 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 soybean extract are used in calculations that yield the number of trypsin inhibitor units (TIU) per gram of original soybean sample.

As used herein, the term "meat substitute" or "meat analogue" means a composition that approximates certain aesthetic qualities (primarily texture, taste, and appearance) or chemical characteristics of a particular meat. In embodiments, such substitutes or analogues include, but are not limited to, dairy-based: paneer cheese, glamorgan sausage, paneer; mushroom-based: edible mushrooms, mycoprotein, fistulina hepatica, lyophyllum decastes; fruit-based: tempeh, breadfruit, coconut burger, green jackfruit, eggplant, jackfruit; legumes: Burmese tofu, falafel, ganmodoki, koya dofu, oncom (red oncom and black oncom), tempeh burger, textured vegetable protein, soy turkey or artificial turkey, vegetarian bacon, vegetarian hot dog, vegetarian sausage and vegetarian burger. In one aspect, the protein concentrate as described herein is combined with meat substitutes.

As used in this document, the term "NIR (near infrared) spectroscopy" is a non-invasive detection method for determining protein content.

As used herein, the term "hydrodynamic force" means the energy exerted on solid bodies immersed in liquids and in motion relative to said liquids. In a related aspect, such force may be applied by processes including, but not limited to, centrifugation and filtration.

As used herein, the term "cellulose-degrading enzymes" means enzymes that act by hydrolyzing, among other things, the glycosidic bonds of linear β-1,4-linked glucose polymers to form glucose and other simple or complex sugars.

As used herein, the terms "antifoam" or "defoamer", including grammatical variations thereof, are a chemical additive that reduces and inhibits foam formation in industrial process fluids. In a related aspect, such chemicals include, but are not limited to, oil-based defoamers; powder defoamers; water-based defoamers; silicone-based defoamers; EO/PO-based defoamers; and alkyl polyacrylates. In a related aspect, such an antifoam includes a water-based food emulsion intended to control foaming in water-based food preservation processes, a non-aqueous, silicone-free antifoam that uses antifoam polymers and biodegradable oils, and a food grade 100% active food grade kosher antifoam intended to destroy foam in an aqueous environment, including food production, fermentation, agricultural and industrial processes.

As used in this document, the term "predicted apparent protein digestibility (PPD)" is a measure of the calculated regression between the apparent protein digestibility (APD) in vivo and the in vitro protein digestibility by digestive enzymes (e.g. degree of hydrolysis) of various feed ingredients.

As used in this document, the term "room temperature" is approximately 25°C at standard pressure.

As used in this document, the term "ARB" is a measure of the ratio of the difference between absorbed and faecal nitrogen to absorbed nitrogen, expressed as a percentage.

As used herein, the term "HQPC" means a high quality protein concentrate from one or more fermented plant materials. Such HQPC can be used as a feed, a raw material alone or in combination with another feed or feedstock components, a probiotic or a component thereof, including as a means of delivering nutraceuticals and/or pharmaceuticals to animals. In embodiments, the protein content of the HQPC can be from about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75% or more (on a dry matter basis (dmb)).

As used herein, the term "solvent" means a substance, usually a liquid, in which other materials dissolve to form a solution. Polar solvents (e.g., water, aqueous solutions) promote the formation of ions; non-polar solvents (e.g., hydrocarbons) do not. Solvents may be predominantly acidic, predominantly basic, amphoteric, or aprotic. Organic compounds useful as solvents include, but are not limited to, aromatic compounds and other hydrocarbons, alcohols, esters, ethers, ketones, amines, and nitrated and halogenated hydrocarbons.

Sources of plant protein

A large number of plant protein sources can be used in connection with the present invention as feedstock 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 when feeding animal proteins to animals of the same species.

Examples of plant protein sources include, but are not limited to, protein from the Fabaceae plant family represented by soybeans and peanuts, from the Brassiciaceae plant family represented by canola, cottonseed, from the Asteraceae plant family represented by but not limited to sunflower, and from the Arecaceae plant family represented by copra. These protein sources, also commonly referred to as oilseed proteins, may be fed whole, but are more commonly fed as a by-product after the oil has been removed. Other plant protein sources include plant protein sources from the Poaceae family, also known as Gramineae, such as cereals and grain crops, especially corn, wheat, and rice, or other staple crops such as potatoes, cassava, and legumes (peas and beans), certain milling by-products including germ meal or corn gluten meal, or distillery/brewery by-products. In embodiments, protein starting materials include, but are not limited to, plant materials from soybeans, sorghum, peanuts, legumes, rapeseed, oats, barley, rye, canola, sesame seeds, cottonseed, coconut kernels, grape seeds, olives, safflower, sunflower, copra, corn, coconuts, flaxseed, hazelnuts, wheat, rice, potatoes, cassava, legumes, pennycress, camelina seeds, mustard seeds, germ meal, corn gluten meal, corn gluten feed, distillery/brewery byproducts, and combinations thereof.

In agriculture, the main plant proteins that are thought to be used include, but are not limited to, soybean meal (SBM), corn gluten meal, rapeseed/canola (Brassica sp.) meal, lupins (Lupinus sp.) such as grain flour proteins from hulled white (Lupinus albus), sweet (L. angustifolius) and yellow (L. luteus) lupins, sunflower (Helianthus annuus) meal, crystalline amino acids; as well as pea meal (Pisum sativum), cottonseed meal (Gossypium sp.), Lemnoidae (duckweed or water lentil), peanut (groundnut; Arachis hypogaea) meal and cake, soy protein concentrate (SPC), soy protein isolate (SPI), corn (Zea mays) gluten meal and wheat (Triticum aestivum) gluten, potato protein concentrate (Solanum tuberosum L.), as well as other plant foods such as moringa leaves (Moringa oleifera Lam.), all in varying concentrations and combinations.

Protein sources can be in the form of unprocessed plant materials and processed and/or extracted plant proteins. For example, thermally processed soy products have high protein digestibility.

The proteinaceous material includes any type of protein or peptide. In embodiments, soybean material or the like can be used, such as whole soybeans. Whole soybeans can be standard commercial soybeans; soybeans that have been genetically modified (GM) in some way; or non-GM identity preserved (IP) soybeans. Examples of GM soybeans include, for example, soybeans engineered to produce carbohydrates other than stachyose and raffinose. Examples of non-GM soybeans include, for example, Schillinger varieties, which are bred from low carbohydrate and low trypsin inhibition lines. High protein varieties include, but are not limited to, N2358 (Benson Hill Inc., St. Louis, MO).

Other types of soybean material include soy protein meal, soy protein concentrate, soy flour, and soy protein isolate, or mixtures thereof. Traditional processing of whole soybeans into other forms of soy protein, such as soy protein meal, soy protein concentrate, soy flour, and soy protein isolate, involves breaking the peeled raw whole soybeans into several pieces, typically six (6) to eight (8), to produce soy chips and the hulls, which are then removed. The soy chips are then conditioned at approximately 140°F (60°C) and ground to a thickness of approximately 0.25 millimeters. The resulting flakes are then extracted with an inert solvent, such as a hydrocarbon solvent, typically hexane, in one of several types of counter-current extraction systems to remove the soybean oil. For soy protein flours, soy protein concentrates and soy protein isolates, it is important that the flakes are desolventized in a manner that minimizes the amount of cooking or roasting of the soy protein in order to maintain the high water-soluble soy protein content. This is typically achieved using steam desolventizers or flash desolventizers. The flakes produced by this process are commonly referred to as "edible defatted flakes" or "white soybean flakes".

White soybean flakes, which are the starting material for soy protein meal, soy protein concentrate and soy protein isolate, have a protein content of approximately 50%. The white soybean flakes are then ground, usually in an open-circuit milling method, using a hammer mill, mill separator, roller mill or impact mill, first into grits and, with further grinding, into soybean meal of the desired particle size. Screening is commonly used to classify the product into uniform particle size ranges and can be accomplished using vibrating screens or cylindrical centrifugal screens. Other oilseeds can be processed in a similar manner.

Soybeans contain a small but very important storage protein, 2S albumin, in addition to glycinin and beta-conglycinin. Soybeans also contain biologically active or metabolic proteins such as enzymes, trypsin inhibitors, hemagglutinins, and cysteine proteases, very similar to papain.

While soy products have high protein digestibility, the upper inclusion level of full-fat or defatted soybean meal in carnivorous fish diets, for example, is 20 to 30%, even when heat-labile antinutrients are excluded. In fish, soy protein has been shown to cause intestinal damage and overall reduced growth performance in a variety of fish species when fed at levels greater than 30%. In fact, most farmers are reluctant to use more than 10% plant protein in the total diet because of these effects.

The present invention solves this problem and allows levels of inclusion of vegetable protein up to 40 or even 50%, depending, among other things, on the species of animals fed, the origin of the vegetable protein source, the ratio of the different vegetable protein sources, the protein concentration and amount, origin, molecular structure and concentration of glucan and/or mannan. In embodiments, the levels of inclusion of vegetable protein are up to 40%, preferably up to 20 or 30%. Typically, the vegetable protein present in the diet is from 5 to 40%, preferably from 10 or 15 to 30%. These percentages determine the proportion of the total vegetable protein source in the feed or diet of the animals, including fat, ash, etc. In embodiments, the levels of pure protein are up to 50%, typically up to 45%, in embodiments, 5-95%.

The ratio of plant protein to other protein in the total feed or ration can be from 5:95 to 95:5, from 15:85 to 50:50, or from 25:75 to 45:55.

In addition to feed rations, the HQPC described herein can be formulated so that it can be used with meat substitutes. In embodiments, the HQPC can be combined with fermented soy products. Examples of fermented soy products include, but are not limited to, thawed and sliced frozen tofu; oncom, a traditional staple of West Javanese (Sudanese) cuisine in Indonesia, comes in two types: red oncom and black oncom (oncom is closely related to tempeh; both products are fermented using mold); soy protein; soy pulp used for veggie burgers and croquettes); tempeh, a traditional Indonesian soy product made from fermented soybeans; textured vegetable protein, a defatted soy flour product that is a by-product of soybean oil extraction (it is often used as a meat analog or meat extender, with a protein content comparable to some types of meat); tofu (although not traditionally considered a meat substitute in Asia, it is widely used for that purpose in the Western Hemisphere); and soy turkey, a faux turkey, a meat substitute in the form of a loaf or casserole made from vegetarian protein, typically made from tofu (soy protein) or seitan (wheat protein) with a grain or bread filling, flavored with broth, and seasoned with herbs and spices; paneer cheese; glamorgan sausage; mushrooms, including but not limited to Fistulina hepatica, Laetiporus, and Lyophyllum decastes; breadfruit; coconut burger (made from sapala, a by-product of the coconut meat from the traditional extraction of coconut milk); eggplant; jackfruit; falafel; and ganmodoki.

In embodiments, the fermented soy product combined with HQPC as described can be used as a meat substitute alone, or can be combined with other meat substitutes or meat analogs to produce various products for human consumption, including various combinations including the examples given above. In one aspect, the addition of HQPC is combined with various meat substitutes and/or meat analogs to improve the texture, aroma, feel, taste, crunch, flavor, or appearance of said meat substitute and/or meat analog. Such aesthetics can be determined, for example, using the Caswell food quality classification (see, for example, Caswell, J Agr Res Econ (1998) 42:409-474).

Microorganisms

The described microorganisms must be capable

convert carbohydrates and other nutrients into a high-quality protein concentrate in the method described herein. In embodiments, the microorganism is a yeast-like fungus. An example of a yeast-like fungus is Aurobasidium pullulans. Other examples of microorganisms include yeasts such as Kluyveromyces and Pichia spp, lactic acid bacteria, Trichoderma reesel, Pleurotus ostreatus, Rhizopus spp, and many types of lignocellulose-degrading microbes. Typical microbes include those that can metabolize stachyose, raffinose, starch, glucose, fructose, lactose, sucrose, xylose, and other sugars. However, one skilled in the art can select other suitable microorganisms based on the described methods without undue experimentation.

In embodiments, the microorganisms exhibit low production of exopolysaccharide (e.g., pullulan), e.g., low pullulan yields of less than about 3.0 g/L are considered when grown in a medium containing yeast extract (YE), wherein YE (as a nitrogen source) is present in an amount of 0.35 to 0.5 g/L (see, e.g., Leathers et al., J Indus Micro (1988) 3:231-239; on page 232, column 2, third paragraph and Table 3, incorporated herein by reference). Without being bound by theory, high exopolysaccharide producers produce end products that are difficult to dry (e.g., drying time is increased) and/or produce viscous products after drying. The methods for determining pullulan content described in Leathers et al. can be used. ((1988), p. 232, col. 1, third paragraph, joining col. 2, first paragraph; incorporated herein by reference), however, one skilled in the art will appreciate that alternative methods are available.

In embodiments, the A. pullulans is adapted to various environmental conditions/stressors encountered during conversion. In one aspect, the A. pullulans strain is selected from NRRL Deposit Nos. 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 designated by NRRL Deposit No. Y-2311-1 can be used, as described herein.

In a related aspect, the microorganism can be identified using PCR. In embodiments, the organism can be targeted by directing primers to the following nucleic acid sequence encoding alpha-arabinofuranosidase (Genbank Accession No.: AY495375):

(SEQ ID NO: 1):

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

Without being bound by theory, it has been observed that the viscosity of the final product(s) appears to be dependent on new materials produced by fermentation, such as exopolysaccharides, which can only exist where the plant material contains a substrate that is metabolized by microorganisms that produce a highly viscous plant-based substrate-producing, fermentation-dependent product (viscosity-increasing exopolysaccharide) that is not pullulan. Thus, the exopolysaccharide/pullulan-producing organism can alternatively be replaced by an organism that does not produce the new viscosity-increasing exopolysaccharide.

Manufacturing process

In exemplary embodiments, after optional pre-treatment (e.g., to increase nutrient availability to cells, remove sugars, and the like), the plant-based material can be mixed with water and/or centrifuged to form a slurry in one or more mixing tanks at a solids loading of at least 5%, with a pH adjusted to 4.5-4.9. In one aspect, the pH is 4.8. The slurry can be treated with sulfuric acid and/or defoamers before applying hydrodynamic force to separate the suspension into a cake and a centrifugate. The cake can be further washed with one or more solvents (e.g., water or centrifugate) before being transferred to the tandem cooker-cooler device(s), where the residence time in the cooker tube can vary. The temperature in the cooking 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.

After cooling to about 30°C, the cooled slurry is transferred to one or more fermenter vessels and an A. pullulans inoculum may be added to the slurry, wherein the resulting inoculated slurry may be incubated for about 7 to about 10 hours, about 10 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% protein is achieved. Although not bound by theory, DH is largely the result of enzymatic hydrolysis rather than pyrolysis, including that DH may be higher with additional recirculation of centrifugate from the broth tank. The inoculation volume (e.g., from a 60-hour seed culture, from about 1 x 10 ¹ to about 100 x 10 ⁹ CFU/mL) can be about 1% of the working volume of one or more fermenter vessels. In a related aspect, the inoculated cells can have a substantially single-cell morphology, and without being bound by theory, the use of cells with a substantially filamentous morphology can result in reduced access to oxygen and other nutrients. During incubation, sterile air can be supplied to the reactor at a rate of 0.5-1 L/L/hr. In embodiments, the conversion culture is converted by incubation with plant material for less than about 12 hours. In 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.

In embodiments, the pH of the conversion culture during fermentation may be from about 4.5 to about 5.5. In embodiments, the pH of the conversion culture may be about 4.8. In embodiments, the conversion culture is actively aerated.

The fermented plant material is transferred from one or more incubation vessels to a broth tank where it can be heated to about 60°C for about 30 minutes to 2 hours. Hydrodynamic force is applied to the heated fermented plant material, resulting in the formation of a fermented cake and a fermented centrifugate. The fermented cake is then dried to a moisture content of less than about 7% at a temperature of about 37.8°C to about 149°C. The fermented centrifugate can be transferred to one or more previously operated mixing tanks (e.g., for water conversion and recycling, increasing protein yield and content, including soluble proteins), for mixing with the incoming plant-based material, including that the fermented centrifugate can be transferred to an evaporator to produce a liquid protein concentrate.

The fermented centrifuge to be evaporated may be subjected to two or more evaporation stages, wherein the evaporation is carried out for a time and temperature sufficient to produce a liquid protein concentrate with a solids % of from about 10 to about 60%. Alternatively, the liquid protein concentrate may be transferred back to one or more mixing tanks to ultimately form one or more additional concentrates/cakes for processing. In one aspect, the cakes/concentrates may be washed and/or precipitated with ethanol.

In embodiments, the recovery of the solid matter with final protein concentrations can be modulated by the plant-based feedstock, incubation conditions, pH, drying time and temperature. For example, about 70% protein or more can be achieved for the final dried cake with a 14-hour incubation, where a solid content of from about 76.59% to about 99.65% is achieved. The protein content of said cake can be from about 65% to about 70%, from about 70% to about 75%, or from about 75% to about 80% (dmb).

In embodiments, 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 with the converting microbe to avoid, for example, contamination with unwanted bacterial strains.

During incubation, samples can be collected at regular intervals during processing (e.g., amino acids, DH, oligosaccharide concentration, pullulan content, and the like). For example, samples for HPLC analysis can be boiled, centrifuged, filtered (e.g., through 0.22 μm filters), placed in autosampler vials, and frozen until analysis. In embodiments, samples can be analyzed for carbohydrates and organic solvents using a WATERS HPLC system, although other HPLC systems can be used. One skilled in the art will understand that other methods (e.g., UPLC) can be used. Samples can be quantified by plate counting or hemocytometric analysis to assess microbial populations. One skilled in the art will understand that other methods (e.g., fluorescence microscopy, flow cytometry, and the like) can be used. Samples can also be analyzed for cellulose, hemicellulose, lignin, starch, and pectin using National Renewable Energy Laboratory procedures.

FIG. 6-9 illustrate manufacturing processes 100, 100(a), 100(b), 100(c) that can be used to produce HQPC as described herein. Referring to FIG. 6, for embodiments, the plant-based material is first placed in a grinding 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 can comprise acids, bases, enzymes, an antifoam, and/or a centrifuge from a downstream separation step (e.g., centrifuge 2 during continuous cycling). Enzymes include non-cellulose-degrading enzymes (e.g., phytases, proteases, and the like). The resulting slurry is separated from the one or more first solvents using a hydrodynamic force device (e.g., a decanter centrifuge) 103(a) to produce a first centrifugate and a first cake. The first centrifugate may be sent to the evaporator 107 to obtain soy soluble substances 108 (i.e., liquid protein concentrate). The first cake is transferred to the second mixing tank 102(b), where the first cake is washed with one or more second solvents and/or a subsequent centrifugate (for example, centrifugate 3, for continuous cycling). The washed first cake is separated from the one or more second solvents by means of a hydrodynamic force device 103(b) to obtain a second centrifugate and a washed second cake. The second centrifugate may be transferred to the first mixing tank 102(a) during a continuous cycle. The washed second cake is transferred to the third mixing tank 102(c), where the washed second cake is washed with one or more third solvents and/or a subsequent centrifugate (for example, product 3, for continuous cycling). The washed second cake is separated from one or more third solvents by a hydrodynamic force device 103(c) to obtain a third centrifugate and a washed third cake. The third centrifugate may be transferred to a 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 slurry, which is transferred to one or more fermenters 104 for inoculation with a system of seed fermenters 105. Prior to incubation, the slurry may be heated and cooled in tandem cooker/cooler devices (not shown), where the residence time and temperature in said cooker may be adjusted to change the characteristics of the final product (e.g., increase protein content). In embodiments, the residence time can be from 0 to about 5 seconds, from about 5 seconds to about 10 seconds, from about 10 seconds to about 15 seconds, from about 20 seconds to about 30 seconds, or from about 30 seconds to 1 minute. In a related aspect, the slurry is heated in a cooking 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, wherein the slurry is cooled to a temperature of about 30(to 32°C) before incubation in the seed fermentor system 105.

Continuing with FIG. 6, after incubation, the resulting fermented slurry is separated into a fourth centrifuge and a final cake using a hydrodynamic force device (e.g., a decanter/disc centrifuge

centrifuge) 103(d). The suspension may be heated in a broth tank (not shown) to at least about 60°C for about 30 minutes to about 2 hours prior to applying the hydrodynamic force. The resulting cake is transferred from 103(d) and/or the broth tank (not shown) to a dryer 106. Drying is accomplished by heating the dryer 106 to about 150°C until the final cake has a moisture content of less than about 7%.

Referring to FIG. 7, the drawing shows in detail the first stages of the production process 100(a) (centrifugate 1). The plant material is first placed in a grinding device 101, and then transferred to a first mixing tank 102(a), where it is mixed with one or more first solvents. The resulting slurry is separated from the one or more first solvents by a hydrodynamic force device 103(a) to obtain a first centrifugate and a first cake. The first centrifugate can be sent to an evaporator 107 to obtain soy soluble substances 108.

Referring to FIG. 8, the drawing shows in detail the production process for the centrifugate 4 100(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 slurry, which is transferred to one or more fermenters 104 for inoculation. After incubation, the resulting fermented slurry is separated into a fourth centrifugate and a final cake by a hydrodynamic force device 103(d), where the centrifugate 4 is transferred to an upstream mixing tank (e.g., 102(c)).

Referring to FIG. 9, the manufacturing process 100(c) offers detailed information regarding the centrifugates 4 and 1, where the centrifugate 4 is subjected to centrifugation in a disc separator 109 and/or ultrafiltration 110 before being transferred to the mixing tank 3 102(c), including that the centrifugate 1 can be subjected to sludge removal and deoiling, ultrafiltration 110 for protein separation, and/or nanofiltration 111 for sugar separation. In embodiments, such separation methods can be used to remove microbes used in fermentation.

In embodiments with reference to FIGS. 6-9, the manufacturing process 100, 100(a), 100(b), 100(c) may be batch or continuous and comprise at least one washing cycle (from the mill 101 to the dryer 106) and at least two centrifuges, where one centrifuge is further processed to concentrate the soy solubles 108.

In embodiments, the feedstock prior to milling and the final product (the final dried cake) are analyzed using NIR spectroscopy. In one aspect, there should be a significant downward shift in the raw spectra between 4664 cm ^-1 and 4836 cm ^-1 for the final product (i.e., the "valley") compared to the starting feedstock (i.e., substantially "flat") (see FIGS. 10 and 11). In a related aspect, the downward shift should be at least about a 10% change, from about 10% to about 15% change, or from about 15% to about 20% change, or more, calculated using the equation:

For example, as shown in FIGS. 10 and 11, the downward shift rate was approximately 16.7% for the SBM sample and the final ME-PRO ⁽ HSPQ product) sample from which it was derived. Without being bound by theory, such a shift indicates increased 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 obtained using the methods described by Fan et al. ((2016); incorporated herein by reference in its entirety), but one of skill in the art will recognize that other suitable methods are available, including any associated hardware and software.

Food compositions

In exemplary embodiments, HQPC recovered from a conversion culture that has undergone conversion is used in food formulations. In embodiments, the recovered HQPC may be the sole protein source in the food formulation. The proportion of the protein source in the food formulations is not limiting and may include from 24 to 80% of the protein concentrate. In embodiments, the HQPC will comprise more than about 50%, more than about 60%, or more than about 70% of the total protein source of the food formulation. The recovered HQPC may replace/supplement protein sources such as fish meal, soybean meal, wheat and corn meal, gluten and concentrates, and animal by-products such as blood, poultry, meat substitutes, meat analogs, and feather meal. Food formulations using HQPC may also include additives such as mineral and vitamin premixes to meet remaining nutrient requirements, if desired.

In some embodiments, the effectiveness of the HQPC can be measured by comparing the growth, feed conversion, protein efficiency, DH, APD, PPD and survival of animals fed a high-quality protein concentrate dietary formulation with animals fed control dietary formulations, including aesthetics for human food products. In embodiments, the test formulations have a constant protein, fat and caloric content. For example, if the animal is a fish, the characteristics of internal organs (fat deposition) and organs (liver and spleen), evisceration rate and rapid fillet analysis, as well as intestinal histology (enteritis) can be measured to assess the nutritional response. In embodiments, for juvenile shrimp formulations, DH, PPD and ADP can be measured in vitro to assess the nutritional response. In embodiments, for piglets, intestinal infections characterized by diarrhea are a major cause of decreased growth performance and increased morbidity and mortality in piglets after weaning, thus a reduction in diarrhea can be measured to assess nutritional response.

It is understood that individual food compositions containing reconstituted HQPC can be optimized for various animal species. In embodiments, the animals include, but are not limited to, fin fish, crustaceans (e.g., shrimp, crabs, prawns, and lobsters), pets (e.g., dogs, cats, birds), and farm animals (e.g., cattle, pigs, and chickens). In a related aspect, the animals are fin fish and crustaceans that are raised in commercial aquaculture. In another related aspect, the crustaceans include juvenile shrimp. Methods for optimizing food compositions are well known and can be readily determined by one skilled in the art without undue experimentation.

Complete feeds for producers can be formulated using HQPC in accordance with the known nutrient requirements of various animal species. In embodiments, the composition can be used for yellow perch (e.g., 42% protein, 8% fat). In embodiments, the composition can be used for rainbow trout (35% protein, 16% fat). In embodiments, the composition can be used for any of the above animals.

Base premixes of minerals and vitamins for plant-based diets may be used to ensure that micronutrient requirements are met. Any additives (deemed necessary by analysis) may be assessed by comparison to an identical formulation without additives; thus, feeding trials may be conducted using a factorial design to account for the effect of additives. In embodiments, feeding trials may include a fishmeal-based control diet and ESPC and LSPC-based reference diets [traditional SPC (TSPC) is produced from soybean flakes that are solvent washed to remove soluble carbohydrates; textured SPC (ESPC) is produced by extruding TSPC in a wet environment at high temperature; and low antigen SPC (LSPC) is produced from TSPC by varying the solvent wash and temperature during processing]. Feed pellets can be produced using a single screw extruder (e.g. BRABENDERPLASTI-CORDER EXTRUDER Model PL2000).

Experiments on fattening

In embodiments, four experimental units per treatment (i.e., each mixture of experimental and control diet) may be replicated (e.g., approximately 60 to 120 days each). Experiments may be conducted in 110-liter round tanks (20 fish/tank) connected in parallel to a closed-loop system driven by a centrifugal pump and consisting of a solids settling tank, a bioreactor, filters (100 μm bag, carbon, and ultraviolet). If necessary, heat pumps may be used to maintain optimal temperatures for the growth of specific species. Water quality (e.g., dissolved oxygen, pH, temperature, ammonia, and nitrite) may be monitored in all systems.

In embodiments, experimental diets can be delivered according to the size of the fish and divided into two to five daily feedings. Growth performance can be determined from total weight measurements taken after one to four weeks (depending on the size of the fish and the duration of the experiment); diets can be adjusted according to growth to ensure satiation feeding and reduce waste streams. Intake can be assessed biweekly by collecting uneaten food from individual aquariums. Uneaten food can be dried to a constant temperature, cooled, and weighed to assess feed conversion efficiency. Protein and caloric digestibility can be determined from fecal material collected manually at the middle of each experiment or by dissecting the lower intestinal tract at the end of the fattening experiment. Survival, weight gain, growth rate, health indices, feed conversion, protein and caloric digestibility, and protein utilization efficiency can be compared between experimental groups. Rapid analysis of dissected fish can be performed to compare fillet composition across different dietary treatments. Amino acid and fatty acid analysis can be performed as needed on fillet components consistent with the purpose of the feeding trial. Responses to feeding trials by food treatment can be compared to the response to a control diet (e.g., fishmeal) to determine whether the performance of the HQPC diets meets or exceeds the control responses.

For shrimp, experiments can be conducted in either 900-gallon tanks or 190-L half-square tanks, each equipped with a recirculating drain that drains water from below the surface and a sludge drain that is attached to the lowest point on the bottom in the center of the tank. The RAS test system can consist of Cornell-type dual-drain tanks, solids settling tanks, mechanical drum filtration, a moving bed bioreactor (MBBR), UV sterilization, cooling, and oxygen supply. Water quality can be monitored daily to ensure that all parameters are within acceptable limits (see, e.g., White et al., Aqua Mar Bio Eco (2020) JAMBE:105).

In addition, shrimp can be analyzed to determine the direct effects of HQPC, such as providing significant amounts of bioactive factors that can increase gut microbiota, reduce intestinal inflammation, and enhance metabolic processes to improve animal health, which may depend, among other things, on the conversion of feed dry matter to mass, where determining digestibility plays a key role. In a related aspect, in vitro protein digestion with standardized shrimp-derived digestive enzymes can be used, including the use of such analysis data to determine DH, PPD, and APD, as well as overall survival.

Statistical analysis of diets and responses to fattening trials can be performed with a priori value of α=0.05. Analysis of treatment effect parameters can be performed with appropriate analysis of variance or covariance (Proc Mixed) and post hoc multiple comparisons, if appropriate. Animal performance and tissue responses can be assessed using nonlinear models.

In embodiments, the present disclosure provides for converting fiber and other carbohydrates in plant materials into additional protein using, for example, a microbe with GRAS status. Microbial exopolysaccharide (e.g., glucans) can also be formed, which can contribute to the formation of extruded feed pellets, eliminating the need for binders. This microbial gum can also provide immunostimulatory activity to activate innate defense mechanisms that protect animals from common pathogens resulting from stress factors. Immunopreventive substances such as β-glucans, bacterial products and plant components are increasingly used in commercial feeds to reduce economic losses due to infectious diseases and minimize the use of antibiotics. The microbes of the present disclosure also produce extracellular peptidases, which should increase the digestibility and absorption of proteins during metabolism, providing higher feed efficiency and yields. As described herein, this microbial incubation process provides a valuable, sustainable plant protein feed that is cheaper per unit of protein than SBM, SPC, and animal feed. In embodiments, the centrifugate components may be subjected to a subsequent evaporation step, in which soluble by-products such as sugars, glycerol, proteins, peptides, and amino acids may be concentrated into a product called syrup or condensed soluble botanical substances (PBS).

As described, the present microbes can metabolize individual carbohydrates in plant materials, producing both cellular mass (protein) and microbial gum (e.g., pullulan), produce enzymes, produce microbial metabolites and probiotics. Different strains of these microbes also enhance fiber breakdown. The microbes of the present invention can also convert proteins in plant material into more digestible peptides and amino acids (electro herein, FIG. 12). In embodiments, the following steps can be performed: 1) determining the efficiency of using selected microbes of the present disclosure to convert plant materials to produce a high-quality protein concentrate (HQPC) with a protein concentration of at least about 65% to about 70% or more, and 2) evaluating the efficiency of HQPC in replacing animal proteins. In embodiments, process/conversion conditions can be optimized to improve microbial efficiency and stability, the resulting feeds can be tested for growing a range of commercially important animals, and process costs and energy requirements can be validated for commercialization. In embodiments, the HQPC of the present invention can replace at least 50% of animal proteins while providing increased growth rates and conversion efficiency. For example, production costs should be lower than commercial soy protein concentrate (SPC) and significantly lower than fish meal.

Figures 1, 2, and 6-9 illustrate various approaches of the present disclosure to processing the plant-based product, converting sugars into cell mass (protein) and gum, recovering HQPC and producing aquaculture feeds, and testing the resulting aquaculture feeds in fish fattening experiments.

Although not required for all processes described herein, cellulose degrading enzymes/pullulanases can be evaluated to produce sugars that the microbes described herein can convert to protein, exopolysaccharides, and gum. In embodiments, sequential exclusion of these enzymes and co-culture evaluation with cellulolytic microbes can be used. Ethanol can be evaluated to wash various pellets, precipitate gum, and improve the recovered protein solutes, which can be suspended in various centrifugates by processing to produce HQPC. Once dried, the HQPC can be incorporated into practical food formulations. In embodiments, experimental growout diets (with mineral and vitamin premixes) can be formulated and compared to control animal protein and commercial plant protein concentrate/isolate based diets in finishing trials with commercially important animals. Performance (e.g. growth, feed conversion, protein efficiency, survival), internal organ characteristics, gut effects, and gut histology can be assessed to assess response.

In other embodiments, the HQPC production process may be optimized by determining optimal conversion conditions while minimizing process costs, improving the efficiency and stability of the microbe, testing the resulting feeds for growing a range of commercially important animals, and checking/updating production costs and energy requirements.

In recent years, several plants have installed dry mills that remove corn husks and germ prior to the ethanol production process. This dry fractionation process produces DDGS with a protein content of up to 42% (hereinafter referred to as dry fractionation DDGS). In some embodiments, low-oil DDGS can be used as a substrate for conversion, where such low-oil DDGS has a higher protein level than regular DDGS. In a related aspect, low-oil DDGS increases the growth rate of A. pullulans compared to regular DDGS.

Several groups have evaluated partial replacement of animal proteins with plant proteins. However, lower protein content, inadequate amino acid balance, and the presence of anti-nutritional factors limit substitution levels to 20-40%. For example, preliminary growth trials indicate that none of the existing DDGS or SPC-based diets provide performance similar to fishmeal-based control diets. Several deficiencies have been identified among commercially produced DDGS and SPC, primarily in protein and amino acid composition, which contribute to variability in growth performance. However, diets containing plant protein as described herein, supplemented with nutritional supplements (formulated to meet or exceed all requirements), provide growth results similar to or superior to animal protein-based control formulations. Thus, the processes described herein and the products developed from them provide higher quality HQPC (relative to nutrient requirements) and support growth performance equivalent to or superior to diets containing animal protein, including diets containing various SPC/SPI.

In embodiments, fish that can be fed with the fish food composition of the present disclosure include, but are not limited to, Siberian sturgeon, sterlet, stellate sturgeon, beluga, arapaima, Japanese eel, American eel, Australian river eel, New Zealand longfin river eel, European eel, milkfish, milkfish, longfin sunfish, green sunfish, white crappie, black crappie, asp, catlya, goldfish, crucian carp, crucian carp, golden cirrina, mrigala, white amur, common carp, silver carp, bighead carp, kalbasa, rohu, Haven's leptobarbus, Uchansky bream, black amur, golden blue bream, Ostiochilus Hasseltii, white Amur bream, Thai silver barb, java barb, roach, tench, eastern loach, prochilodont, gilthead sea bream, black pacu, black pomacanthus, paco, black catfish, channel catfish, blue orca, blue catfish, catfish, pangasius (Swai, Tra, Basa), eeltail catfish, mudfish, Philippine sharptooth catfish, sharptooth catfish, African sharptooth catfish, bighead catfish, vundu, striped trachycorrist, blue catfish, northern pike, ayu, vendace, whitefish, pink salmon, chum salmon, coho salmon, masu salmon, rainbow trout, sockeye salmon, chinook salmon, atlantic salmon, sea trout, arctic char, brook trout, lake trout, atlantic cod, peherei, barge, white bass, barramundi/Asian grouper, Nile perch, Murray cod, golden snapper, striped bass, white bass, European bass, Hong Kong grouper, areola grouper, towheena grouper, blackspotted coral grouper, sand croaker, American white bass, jade grouper, largemouth bass, smallmouth bass, bass, walleye (perch), yellow perch, sand bass, walleye, bluefish, Chinese yellowtail, yellowtail, snubnose pompano, Florida pompano, West Indian trachonot, Japanese horse mackerel, nitrite, red snapper, yellowtail snapper, dusky sea bass grouper, white gar, crucian carp, redfin puffer, red puffer, yellowfin spar, gilthead bream, red croaker, cichlid, red throat cichlid, managu cichlid, mexican mojarra, ceylon etroplus, three-spotted tilapia, blue tilapia, longfin tilapia, mozambique tilapia, nile tilapia, tilapia, rufiji tilapia, blackfin tilapia, pink tilapia, redbelly tilapia, golden mullet, golden mullet, ramada mullet, razorback, rock mullet, striped mullet, white mullet, lebranche mullet, pacific spotted dormitor, marbled Elaeotris, Oramine parr, Yellowfin parr, Goldenrod, Southern Bluefin Tuna, Northern Bluefin Tuna, Crawfish, Brown Gourami, Kissing Gourami, True Gourami, Snakehead, Red Snakehead, Spotted Snakehead, Striped Snakehead, Turbot, Turbot (False Halibut), Summer Flounder, Pacific Flounder, Winter Flounder, Atlantic Halibut, Greenside Rockfish, Sole, and combinations thereof.

In embodiments, crustaceans that can be fed with the food composition of the present disclosure include, but are not limited to, kadal shrimp, swift shrimp, sand shrimp, speckled shrimp, northern brown shrimp, eastern shrimp, brown tiger shrimp, Indian white shrimp, kurumu shrimp, striped shrimp, shrimp, giant tiger shrimp, southern pink shrimp, Sao Paulo shrimp, red tail shrimp, southern white shrimp, green tiger shrimp, northern white shrimp, blue shrimp, southern brown shrimp, white leg shrimp, Atlantic shrimp, small akiami shrimp, monsoon river shrimp, giant river shrimp, common shrimp, American lobster, European lobster, broad-fingered crayfish, crayfish Longclaw crayfish, signal crayfish, red swamp crayfish, Australian freshwater crayfish, red claw crayfish, Maron crayfish, long-legged spiny lobster, gazami crab, Indo-Pacific swamp crab, Chinese river crab and combinations thereof.

In embodiments, farm animals that can be fed with the feed composition of the present disclosure include, but are not limited to, cattle, sheep, goats, deer, horses, chickens, pigs, rabbits, ducks, alpacas, emus, turkeys, bison, and camels.

In embodiments, pets that can be fed the food compositions of the present disclosure include, but are not limited to, dogs, cats, parrots, goldfish, turtles, parrots, hamsters, laboratory rats, guinea pigs, and laboratory mice.

One skilled in the art will appreciate that the feed composition of the present disclosure can be used as a convenient carrier for pharmaceutically active substances, including, but not limited to, antibiotics, chemotherapeutic agents, anti-inflammatory agents, NSAIDs, antimicrobials and immunologically active substances, including vaccines against bacterial or viral infections and any combination thereof.

The feed composition according to the present description can be presented in the form of a liquid flowable emulsion, or in the form of a paste, or in a dry form, for example, in the form of a granulate, powder or flakes. When the feed composition is offered in the form of an emulsion, a fat-in-water emulsion, it can be in a relatively concentrated form. Such a concentrated form of emulsion can also be called a pre-emulsion, since it can be diluted in one or more stages in an aqueous medium to obtain the final medium for enriching organisms.

In embodiments, the cellulose-containing feedstock for the microbial process as described is corn. Corn is approximately two-thirds starch, which is converted into ethanol and carbon dioxide during fermentation and distillation. The remaining nutrients or fermentation products can be converted into condensed soluble substances after distillation or granules after distillation, such as DDGS, which can be used in feed products. Typically, the method includes an initial preparation step of dry milling or grinding corn. The processed corn is then hydrolyzed and enzymes are added to break down the main starch component in a saccharification step. The next fermentation step is carried out by adding a microorganism (e.g., yeast) provided in an embodiment of the present disclosure to produce gaseous products such as carbon dioxide. Fermentation is carried out to produce ethanol, which can be distilled from the fermentation broth. The remaining fermentation medium can then be dried to obtain fermentation products, including DDGS. This step typically involves a solid-liquid separation process by centrifugation, where the solid phase component can be collected. Other methods can be used to accomplish this separation, including filtration and spray drying techniques. The liquid phase components can be further subjected to an evaporation step, which can concentrate soluble by-products such as sugars, glycerol, proteins, peptides and amino acids into a material called syrup or condensed corn solutes (CCS). The CCS can then be recombined with the solid phase component for drying as DDGS. It should be understood that the present compositions can be applied to new or existing dry mill based ethanol production plants to provide an integrated ethanol production process that also produces fermentation products with increased value.

In embodiments, the incubation products obtained in accordance with the present disclosure have a higher commercial value than conventional fermentation products. For example, the incubation products may include improved dry solids with improved amino acid and micronutrient content. Thus, a "golden colored" product may be obtained, which typically indicates higher amino acid digestibility compared to a darker colored HQSP. For example, a light colored HQSP may be obtained with an increased lysine concentration according to embodiments of the present invention compared to relatively darker products, which typically have lower nutritional value. The color of the products may be an important factor or indicator in assessing the quality and digestibility of nutrients in fermentation products or HQSP. Color is used as an indicator of exposure to excess heat during drying, causing caramelization and Millard reactions of free amino groups and sugars, which reduces the quality of certain amino acids.

Another aspect of the present invention is directed to complete feed compositions with an increased concentration of nutrients, which include microorganisms characterized by an increased concentration of nutrients, such as, but not limited to, fats, fatty acids, lipids such as phospholipid, vitamins, essential amino acids, peptides, proteins, carbohydrates, sterols, enzymes and trace elements such as iron, copper, zinc, manganese, cobalt, iodine, selenium, molybdenum, nickel, fluorine, vanadium, tin, silicon and combinations thereof. In one aspect, the method makes more phosphorus available to animals in a manner that does not lead to significant wastewater pollution.

The incubation products obtained after the incubation process usually have a higher commercial value. In embodiments, the incubation products contain microorganisms with increased nutrient content than products with a deficiency of microorganisms. Microorganisms can be present in the incubation system, incubation broth and/or incubation biomass. The incubation broth and/or biomass can be dried (e.g., by spray drying) to obtain incubation products with increased nutrient content.

For example, the spent dried solids recovered from the incubation process are improved as described. These incubation products are generally non-toxic, biodegradable, readily available, inexpensive, and nutrient-rich. The choice of microorganism and incubation conditions are important to obtain a low-toxicity or non-toxic incubation product for use as a feed or food additive, including the selection of a microorganism that does not produce metabolites at concentrations that would otherwise adversely affect the process. Although glucose is the primary sugar formed by the hydrolysis of starch from grain, it is not the only sugar formed in carbohydrates overall. Unlike SPC or DDG produced by the conventional dry process ethanol production process, which contain high levels of non-starch carbohydrates (e.g., at least 35% cellulose and arabinoxylans based on neutral detergent fiber on a dry weight basis), the nutrient-enriched hatchery products produced by enzymatic hydrolysis of non-starch carbohydrates (by microbial metabolism) are more palatable and digestible by non-ruminants.

The nutrient enriched hatchery product of the present disclosure may 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 may depend on the animal to which it is fed, as well as on the context of the rest of the diet and the stage of the animal's life cycle. For example, beef cattle require less histidine than dairy cows. The selection of an appropriate nutrient content for feeding animals is well known to those skilled in the art.

The incubation products 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 products can be further processed to facilitate rumen bypass. In embodiments, the biomass product can be separated from the incubation medium, spray-dried and, optionally, processed to modulate rumen bypass and added to feed as a nutrition source. In addition to producing nutrient-enriched incubation products in an incubation process involving microorganisms, nutrient-enriched incubation products can also be produced in transgenic plant systems. Methods for producing transgenic plant systems are known in the art. Alternatively, when the host microorganism secretes the nutrient contents, the nutrient-enriched broth can be separated from the biomass obtained by incubation, and the clarified broth can be used as an animal feed ingredient, for example, either in liquid or spray-dried form.

The incubation products obtained after the incubation process using microorganisms can be used as animal feed or as a food supplement for humans. The incubation product includes at least one ingredient with increased nutritional content obtained from a non-animal source (e.g., bacteria, yeast and/or plants). In particular, the incubation products are rich in at least one or more of fats, fatty acids, lipids such as phospholipid, vitamins, essential amino acids, peptides, proteins, carbohydrates, sterols, enzymes and microelements such as iron, copper, zinc, manganese, cobalt, iodine, selenium, molybdenum, nickel, fluorine, vanadium, tin and silicon. In embodiments, the peptides contain at least one essential amino acid. In other embodiments, the essential amino acids are encapsulated inside the subject modified microorganism used in the incubation reaction. In embodiments, the essential amino acids are contained in heterologous polypeptides expressed by the microorganism. If desired, the heterologous polypeptides are expressed and stored in inclusion bodies in a suitable microorganism (e.g., fungi).

In embodiments, the hatching products have a high nutrient content. As a result, a higher proportion of the hatching products can be used in a complete animal feed. In embodiments, the feed composition comprises at least about 15% by weight of the hatching product. In a complete feed or diet, this product will be fed with other products. Depending on the nutrient content of the other products and / or the nutritional needs of the animal to which the feed is provided, the modified hatching products can make up from 15% to 100% of the feed. In embodiments, the incubation products in question can provide a lower mixing proportion due to the high nutrient content. In other embodiments, the incubation products in question can provide feed with a very high proportion, such as more than 75%. In suitable embodiments, 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 the subject hatchery products. Typically, the feed composition comprises at least about 20% by weight of the hatchery product. Most often, 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 hatchery product. If desired, the subject hatchery products can be used as the sole source of feed.

In embodiments, the food composition as described herein may have an increased amino acid content with respect to one or more essential amino acids for various purposes, such as increasing weight and improving overall health of the animal. The compositions may have an increased amino acid content due to the presence of free amino acids and/or the presence of proteins or peptides, including an essential amino acid, in the hatchery products. Essential amino acids may include histidine, lysine, methionine, phenylalanine, threonine, taurine, isoleucine and/or tryptophan, which may be present in the preparation as a free amino acid or as part of a protein or peptide rich in the selected amino acid. At least one peptide or protein rich in essential amino acids may contain at least 1% of essential amino acid residues per total amino acid residues in the peptide or protein, at least 5% of essential amino acid residues per total amino acid residues in the peptide or protein, or at least 10% of essential amino acid residues per total amino acid residues in the protein. When animals are fed a nutritionally balanced diet, the nutrient content is utilized to the maximum, requiring less feed to achieve comparable growth rates, milk production, or reducing the nutrient content present in excrement, reducing the bioburden of waste (e.g., reducing phosphorus content in waste streams).

The composition as described herein may contain an increased content of an essential amino acid, may have an essential amino acid content (including a free essential amino acid and an essential amino acid,

present in a protein or peptide), at least 2.0% by weight, based on the weight of the crude protein and the total amino acid content, and more preferably at least 5.0% by weight, based on the weight of the crude protein and the total amino acid content. The feed composition as described herein includes other nutrients derived from microorganisms, including but not limited to fats, fatty acids, lipids such as phospholipid, vitamins, carbohydrates, sterols, enzymes and trace elements.

The feed composition as described herein may comprise a composition in the form of a complete feed, a composition in the form of a concentrate, a composition in the form of a mixture, and a composition in a base form. If the composition is in the form of a complete feed, the percentage nutrient level in which the nutrients are obtained from the microorganism in the hatchery product may be from about 10 to about 25 percent, more preferably from about 14 to about 24 percent; whereas if the composition is in the form of a concentrate, the nutrient level may be from about 30 to about 50 percent, more preferably from about 32 to about 48 percent. If the composition is in the form of a mixture, the nutrient level in the composition may be from about 20 to about 30 percent, more preferably from about 24 to about 26 percent; and if the composition is in the form of a base mixture, the nutrient level in the composition may be from about 55 to about 65 percent. Unless otherwise indicated herein, percentages are indicated in weight percent. If HQPC is high in one nutrient, such as Lys, it can be used as a low dose supplement; if it is balanced in amino acids and vitamins, such as vitamins A and E, it will be a more complete feed and can be fed at a higher rate and supplemented with a low protein, low nutrient feed, such as corn feed.

The feed composition described herein may include a peptide or crude protein fraction present in the hatchery product having an essential amino acid content of at least about 2%. In embodiments, the peptide or crude protein fraction may have an essential amino acid content of at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, and in embodiments, at least about 50%. In embodiments, the peptide may be 100% essential amino acids. For example, a fish meal composition may include a peptide or crude protein fraction present in the hatchery product having an essential amino acid content of up to about 10%. Most commonly, the fish meal composition may include a peptide or crude protein fraction present in the hatchery product, with an essential amino acid content of approximately 2-10%, 3.0-8.0% or 4.0-6.0%.

The composition described herein may include a peptide or a crude protein fraction present in the incubation product having a lysine content of at least about 2%. In embodiments, the peptide or crude protein fraction may 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 some embodiments, at least about 50%. For example, the fish meal composition may include a peptide or crude protein fraction having a lysine content of up to about 10%. Optionally, the fish meal composition may include a peptide or crude protein fraction having a lysine content of about 2-10%, 3.0-8.0%, or 4.0-6.0%.

The composition described herein may include nutrients in the hatchery product from about 1 g/kg dry matter to 900 g/kg dry matter. For example, nutrients in the fish meal composition may include at least about 2 g/kg dry matter, 5 g/kg dry matter, 10 g/kg dry matter, 50 g/kg dry matter, 100 g/kg dry matter, 200 g/kg dry matter, and about 300 g/kg dry matter. In embodiments, the nutrients may be present in an amount of at least about 400 g/kg dry matter, at least about 500 g/kg dry matter, at least about 600 g/kg dry matter, at least about 700 g/kg dry matter, at least about 800 g/kg dry matter, and/or at least about 900 g/kg dry matter.

The composition described herein may include an essential amino acid or a peptide comprising at least one essential amino acid present in the hatchery product, with a content of from about 1 g/kg dry matter to 900 g/kg dry matter. For example, an essential amino acid or a peptide comprising at least one essential amino acid in a fish meal composition may comprise at least about 2 g/kg dry matter, 5 g/kg dry matter, 10 g/kg dry matter, 50 g/kg dry matter, 100 g/kg dry matter, 200 g/kg dry matter, and about 300 g/kg dry matter. In embodiments, the essential amino acid or peptide comprising at least one essential amino acid may comprise at least about 400 g/kg dry matter, at least about 500 g/kg dry matter, at least about 600 g/kg dry matter, at least about 700 g/kg dry matter, at least about 800 g/kg dry matter, and/or at least about 900 g/kg dry matter.

The composition described herein may comprise a nutrient-rich incubation product in the form of biomass formed during incubation and at least one additional nutrient component. In another example, the composition comprises a nutrient-rich incubation product dissolved and suspended in an incubation broth formed during incubation and at least one additional nutrient component. In another embodiment, the composition comprises a crude protein fraction that includes at least one protein rich in essential amino acids. The composition may be prepared in such a way as to provide an improved balance of essential amino acids.

For other formulations, the complete formulation may contain HQPC and one or more ingredients such as wheat semolina ("wheat bran"), corn, soybean meal, corn gluten meal, distillers grains or distillers grains with solubles, salt, macrominerals, trace elements and vitamins, obtained with or without fermentation. Other potential ingredients may typically include, but are not limited to, sunflower meal, malt germ and soybean hulls. The composition of the mixture may contain wheat bran, corn gluten meal, distillers grains or distillers grains with solubles, salt, macrominerals, trace elements and vitamins. Alternative ingredients typically include, but are not limited to, corn, soybean meal, sunflower meal, cottonseed meal, malt germ and soybean hulls. The composition of the basic form may contain wheat bran, corn gluten meal and stillage or stillage with solubles. Alternative ingredients usually include, but are not limited to, soy flour, sunflower flour, malt germ, macrominerals, trace elements and vitamins.

Highly unsaturated fatty acids (HUFA) in microorganisms can be converted to less desirable unsaturated fatty acids or to 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 natural antioxidants such as beta-carotene, vitamin E and vitamin C into the feed. Synthetic antioxidants such as BHT, BHA, TBHQ or ethoxyquin or natural antioxidants such as tocopherols can be included in food or feed products by adding them to the products or they can be included by in situ production in a suitable organism. The amount of antioxidants included in this way depends, for example, on the requirements of subsequent use, such as product composition, packaging methods and desired shelf life.

The incubation products of the present disclosure can also be used as a food supplement for human consumption when the process begins with human-grade raw materials and maintains human food quality standards throughout the process. The incubation product or composition as described herein has a high nutrient content. Nutrients such as protein and fiber are associated with healthy nutrition. Recipes can be developed for using the incubation product or complete feed of the present invention in food products such as cereal, cracker, pie, cookie, cake, pizza crust, summer sausage, meatballs, shakes and any form of edible food products. Another option would be to develop the incubation product as a snack or energy bar, similar to a granola bar, which can be easily eaten and conveniently distributed. The bar may include protein, fiber, germ, vitamins, minerals from the grain, as well as nutraceuticals such as glucosamine, HUFA, or cofactors such as vitamin Q-10.

The formulations containing the hatchery products in question may be further supplemented with flavorings. The choice of a particular flavor will depend on the animal to which the food is provided. Flavors and aromas, both natural and artificial, may be used to make the food more palatable and palatable. These additives may be readily mixed with all ingredients and may be available as a liquid or dry product. Suitable flavors, attractants, and aromas that should be added to animal feeds include, but are not limited to, fish pheromones, fenugreek, banana, cherry, rosemary, caraway, carrot, peppermint, oregano, vanilla, anise, as well as rum, maple syrup, caramel, citrus oils, ethyl butyrate, menthol, apple, cinnamon, and any natural or artificial combinations thereof. Flavors and aromas may vary from animal to animal. Similarly, various fruit flavors, artificial or natural, may be added to food supplements containing the incubation products in question for human consumption.

In embodiments, the HQPC may be part of a kit for preparing various formulations, including the HQPC, a label, at least one container, and instructions for preparing the formulations depending on the animal. In addition, such instructions may be accessible via a web link.

The shelf life of the incubation product or complete feed as described herein may generally be longer than the shelf life of the incubation product that does not contain the microorganism. Shelf life may be affected by factors such as the moisture content of the product, the amount of air that can pass through the feed mass, environmental conditions, and the use of preservatives. A preservative may be added to the complete feed to extend the shelf life to weeks or months. Other methods of extending shelf life include management similar to silage management, such as mixing with other feeds and packaging, polyethylene coating, or bagging. Cool conditions, preservatives, and exclusion of air from the feed mass extend the shelf life of wet by-products. Complete feed may be stored in bins or silo bags. Drying the wet incubation product or complete feed may also extend the shelf life of the product and improve consistency and quality.

The complete feed of the present disclosure can be stored for long periods of time. The shelf life can be extended by ensiling, adding preservatives such as organic acids, or mixing with other feeds such as soybean hulls. Commercial bins or bulk storage sheds can be used to store the finished feeds. In a related aspect, the HQPC described herein can have a shelf life of at least 2 years.

The following examples are illustrative and are not intended to limit the scope of the described subject matter.

EXAMPLES

Example 1. High-quality protein concentrate (HQPC) (precipitation method).

Figure 1 shows an approach to pretreating white flakes, converting sugars into cell mass (proteinaceous material) and gum (e.g., exopolysaccharides), recovering HQSPC and producing aquaculture feeds (FIGS. 2 and 4), and testing the resulting aquaculture feeds in fish feeding trials for the process. The white flakes are first pretreated by extrusion (BRABENDER PLASTI-CORDER SINGEE SCREW EXTRUDER Model PL2000, Hackensack, NJ) at 15% moisture, 50°C, and 75 rpm to break down the structure and provide increased penetration of hydrolytic enzymes during subsequent saccharification. These conditions provide a shear effect against the engineered channels on both sides of the cylinder and have previously been observed to result in a 50-70% increase in sugar release following enzymatic hydrolysis. The extruded white flakes are then crushed through a 3 mm hammer mill screen, mixed with water to achieve 10% solids content and adjusted to a pH of 5. After heating to pasteurize or sterilize the pulp, the pulp is cooled to approximately 50°C and cellulose and oligosaccharide-degrading enzymes (total 15 ml/kg white flakes) are added to hydrolyze the polymers into simple sugars (4-24 h hydrolysis). Specific dosages included are 6% CELLIC STEK (per gram glucan), 0.3% CELLIC HTEK (per gram total dry matter), 0.015% NOVOZYME 960 (per gram dry matter). The resulting slurry is then cooled to 30°C, the pH adjusted to 3-5, inoculated with A. pullulans (1% v/v), and incubated for 4-5 days under agitation at 50-200 rpm and an aeration rate of 0.5 L/L/min to convert sugars to protein and gum. During the incubation, samples are periodically removed and analyzed for sugar content, cell count, and gum formation. After incubation, the pH is increased to 6.5 and ethanol (0.6 L/L broth) is added to precipitate the gum. The protein, pullulan, and microbial mass (HQSPC) are recovered by centrifugation and dried, while the supernatant is distilled to recover the ethanol, and the remaining liquid is subjected to chemical analysis for future recycling to the beginning of the process. FIG. 5 shows the recovery of glucose using the ppt method.

HQSPC using soybean white flakes and microbial conversion with A. pullulans, a pilot system for HQSPC production.

The system contains a 675 L bioreactor, a variable speed progressing cavity pump, a continuous flow centrifuge and a 1 x 4 meter drying table. Inoculum for use in the 675 L bioreactor is prepared in two 5L NEW BRUNSWICK BIOFLO 3 BIOREACTORS. For each trial, 8-10 L of inoculum are prepared by growing A. pullulan as described for 2-3 days. This material is used to inoculate large quantities of extruded and saccharified white flakes prepared in the 675 L bioreactor. Following incubation, ethanol is added and the slurry is centrifuged to recover wet solids, which are then dried and used in fish fattening trials. Several parameters that significantly affect solid recovery are monitored by monitoring conversion process performance, HQSPC yield and composition. In large-scale experiments, the parameters shown in Table 1 vary.

Based on HQSPC yield and protein levels, the following were noted: 1) incubation pH of 3-3.5 and temperature of 30-32°C, along with high aeration maximize A. pullulans growth and minimize pullulan production, 2) incubation time of 4-5 days is optimal for protein content and dry matter recovery, 3) longer incubation times increase protein content but significantly decrease dry matter recovery, 4) shorter incubation times maintain high dry matter recovery but limit protein content, and 5) due to the absence of stachyose and raffinose in the final product, extrusion and/or reduced (absent) enzymatic saccharification are possible.

Preliminary laboratory trials in 5 L bioreactors are conducted to optimize process conditions. Extruded white flakes at 10% solids are used and saccharified for 24 hours, then inoculated with A. pullulans and incubated at pH 5, 0.5 L/L/min aeration, 200 rpm agitation for 10 days. Extended incubation times are tested to establish the optimal harvest window to maximize both dry matter recovery and dry matter protein content. Samples (100 mL) are collected daily and subjected to the following every other day:

Precipitation of all solids with ethanol,

centrifugation and drying of the solid, measurement of residual solid in the resulting supernatant.

Centrifugation of the broth first to recover the solid, drying of the solid, precipitation of pullulan from the resulting supernatant and drying.

The first ethanol precipitation method recovers approximately 97% of the solid (soybean solids, cells and gum) using a laboratory centrifuge (10,000g), where approximately 3% of the solid remains in the liquid phase. The first centrifugation method recovers 81.7% of the solid (soybean solids and cells), where ethanol precipitation of the supernatant recovers approximately 14.8% of the solid (exopolysaccharide) and approximately 3.5% of the solid remains in the liquid.

In these laboratory experiments, the levels of protein, pullulan, and total solids recoverable each day are measured. It is expected that as incubation progresses, protein and pullulan levels will increase, but the total solids recovered will decrease as some nutrients are catabolized into water and _CO2 . The average protein levels in solids from three replicates are shown in FIG. 3. Protein levels reach 70% by days 3-5, while total solids recovered begin to decline by days 5-6. It appears, therefore, that an incubation time of 4-5 days may be optimal.

Example 2. Comparison of products by precipitation method (ppt), 3x wash and 1x wash methods.

HQPC from soybeans was prepared by the methods generally described above and illustrated in FIGS. 6-9, for both 3x wash cycle and 1x wash cycle. A comparison of the resulting compositions for the ppt method (HQSPC run 5 and run 6), 3x wash cycle, and 1x wash cycle (HQPC) is shown in Table 2.

Example 3. Hydrolysis of soy flour by temperature.

A 10% (w/v) soy flour slurry was prepared using soy flour extracted with hexane in water. The pH of the slurry was adjusted to 4.5 and the slurry was stirred to achieve mixing. The soy slurry was heated to 100°C prior to fermentation. The heated slurry was incubated with the fermentative organism. The heated slurry without fermentative organisms was treated with FLAVORZYME® (protease from Aspergillus niger, purchased from Sigma) at a loading rate of 30 mg/g soy flour and used as a control. The sample was incubated at 30°C for 12 hours. After incubation, the samples were heated at 80°C for 2 minutes to inactivate the protease.

The slurry is heated to 100°C for 1.5 min. Samples are centrifuged at 4000 rpm for 10 sec. N-acetylcysteine (3.33% w/v) is prepared in boric acid buffer (0.12 M, pH 10.4). 16.67 μl of sample supernatant is added to 1000 ml of N-acetylcysteine. Absorbance is measured at 340 nm. 6% (w/v) OPA solution is prepared in 96% (v/v) ethanol solution. 10 μl of OPA solution is added to 305 μl of sample. The sample with OPA solution is incubated at room temperature for 15 min. Absorbance is measured at 340 nm.

The following calculation is used to calculate the relative degree of hydrolysis (%) [A340 _test (15) - A340 _test (0)] ×100/ [A340 _control (15) - A340 _control (0)]. See Table 3.

Example 4. Hydrolysis of soy flour suspension by fermentation.

A 10% (w/v) soy flour slurry was prepared using soy flour extracted with hexane in water. The pH of the slurry was adjusted to 4.5 and the slurry was stirred to achieve mixing. The soy slurry was heated to 100°C prior to fermentation. The heated slurry was incubated with the fermentative organism. The heated slurry without the fermentative organism was treated with FLAVORZYME® at a loading rate of 30 mg/g soy flour and used as a control. The sample was incubated at 30°C for 12 hours. Following incubation, the samples were heated at 80°C for 2 minutes to inactivate the protease.

Fermentation samples are collected at 0, 2, 4, 6, 8, 10 and 12 h. Samples are centrifuged at 4000 rpm for 10 s. N-acetylcysteine (3.33% w/v) is prepared in boric acid buffer (0.12 M and pH 10.4). 16.67 μl of sample supernatant is added to 1000 ml of N-acetylcysteine. Absorbance is measured at 340 nm. 6% (w/v) OPA solution is prepared in 96% (v/v) ethanol solution. 10 μl of OPA solution is added to 305 μl of sample. The sample with OPA solution is incubated at room temperature for 15 min. Absorbance is measured at 340 nm. The following calculation is used to calculate the relative degree of hydrolysis (%) [A340 _test (15) - A340 _test (0)] ×100/ [A340 _control (15) - A340 _control (0)]. See Table 4.

Example 5. Hydrolysis of a soy flour suspension obtained in a centrifugate by fermentation.

A 10% (w/v) soy flour slurry was prepared using soy flour extracted with hexane in a centrifugate after solid-liquid separation. The pH of the slurry was adjusted to 4.5 and the slurry was stirred to achieve mixing. The soy flour slurry was heated to 100°C prior to fermentation. The heated slurry was incubated with the fermentative organism. The heated slurry without the fermentative organism was treated with FLAVORZYME® at a loading rate of 30 mg/g soy flour and used as a control. The sample was incubated at 30°C for 12 hours. After incubation, the samples were heated at 80°C for 2 minutes to inactivate the protease.

Fermentation samples are collected at 0, 2, 4, 6, 8, 10 and 12 h. Samples are centrifuged at 4000 rpm for 10 s. N-acetylcysteine (3.34% w/v) is prepared in boric acid buffer (0.12 M and pH 10.4). 16.67 μl of sample supernatant is added to 1000 ml of N-acetylcysteine. Absorbance is measured at 340 nm. 6% (w/v) OPA solution is prepared in 96% (v/v) ethanol solution. 10 μl of OPA solution is added to 305 μl of sample. The sample with OPA solution is incubated at room temperature for 15 min. Absorbance is measured at 340 nm. The following calculation is used to calculate the relative degree of hydrolysis (%) [A340 _test (15) - A340 _test (0)] ×100/ [A340 _control (15) - A340 _control (0)]. See Table 5.

Example 5: Hydrolysis of soy flour suspension with added phytase by fermentation

A 10% (w/v) soy flour slurry was prepared using soy flour extracted with hexane in water. The pH of the slurry was adjusted to 4.5 and the slurry was stirred to achieve mixing. The soy slurry was heated to 100°C prior to fermentation. The heated slurry was incubated with the fermentative organism. The heated slurry without the fermentative organism was treated with FLAVORZYME® at a loading rate of 30 mg/g soy flour and used as a control. The sample was incubated at 30°C for 12 hours. Following incubation, the samples were heated at 80°C for 2 minutes to inactivate the protease.

Fermentation samples are collected at 0, 2, 4, 6, 8, 10 and 12 h. Samples taken at 8, 10 and 12 h are also treated with phytase. Samples are centrifuged at 4000 rpm for 10 s. N-acetylcysteine (3.34% w/v) is prepared in boric acid buffer (0.12 M and pH 10.4). 16.67 μl of sample supernatant is added to 1000 ml of N-acetylcysteine. Absorbance is measured at 340 nm. 6% (w/v) OPA solution is prepared in 96% (v/v) ethanol solution. 10 μl of OPA solution is added to 305 μl of sample. The sample with OPA solution is incubated at room temperature for 15 min. The absorbance is measured at 340 nm. The following calculation is used to calculate the relative degree of hydrolysis (%) [A340 _test (15) - A340 _test (0)] ×100/ [A340 _control (15) - A340 _control (0)]. See Table 6.

Example 6. Experiments on fattening rainbow trout, barramundi and coho salmon.

Long-term and short-term feeding trials were conducted using multiple lots of rainbow trout (Oncorhynchus mykiss), barramundi [bates calcarifer], and coho salmon (Oncorhynchus kisutch). Fish in all trials were fed either a commercial control diet or a high (25-35%) HQPC diet prepared as described in Example 2 (3x wash). All diets were formulated using commercial feed preparation software and manufactured using commercial extrusion processes. All chemical analyses (proximate analysis and mineral composition) of the diets were performed using a third-party laboratory (Midwest Laboratories, Omaha, NE).

Rearing trials (-average individual starting weight 185 g - harvest weight 1000 g) are conducted using a RAS consisting of 3.41 m3 (900 gallons) of tanks. The RAS consists of Cornell-type double drain tanks, solids settling basins, mechanical drum filtration, moving bed bioreactor (MBBR), UV sterilization, cooling and oxygen supply. Water quality is monitored daily to ensure all parameters are within acceptable limits.

On day 0, all fish were weighed by tank group for biomass determination, and a sample of all fish in 1 tank per treatment was anesthetized with 80 mg/L MS-222 and Smith length and weight were measured. Group weighings and identical individual samples were conducted at 3-week intervals throughout the 27-week study. At week 16, 1 fish per tank (six fish per treatment) was sampled for general health assessment, including spleen, liver, visceral fat, and hematocrit.

Experiments on shrimp fattening

Experiments on the development of larvae

Approximately ninety million nauplii (stage 5) of the whiteleg shrimp Litopenaeus vannamei were obtained at the hatchery in Sumacua Choluteca, Honduras, and transferred to aquaria containing 20 metric tons of saltwater (28 ppm) in which all experimental runs were conducted. Shrimp larvae were fed one of four diets containing HQPC (inclusion levels up to 70%), ranging from Zoea 3 to Postlarval 13 (PL13). Survival (%) was determined by extrapolating aliquots in which all surviving larvae were counted at the end of the run.

Growing experiments

Pacific whiteleg shrimp (L. vannamei) (mean weight 1.6 g) were stocked randomly (n = 700) at a density of 20 individuals per tank. Each treatment was randomly assigned to 5 replicate tanks. Experimental diets were offered 3 times daily (08:00, 12:00, and 16:00) for 42 days. All shrimp were fed the same control diet until the start of the trial. At the start of the trial (day 1), the shrimp were offered the experimental diets and fed until apparent satiation. Total feed intake was used to estimate the feed conversion ratio. Tank biomass was recorded when shrimp were stocked (day 0) and again at 3-week intervals until the end of the trial. Measurements of total biomass in the aquarium are used to calculate relative growth (RG), specific growth rate (SGR), and biomass gain over the course of the experiment.

The experiment is conducted in a 8,246 L recirculating aquaculture system (RAS). The system is equipped with 35,190 L half-square tanks, each equipped with a recirculation drain that drains water from below the surface and a sludge drain attached to the lowest point on the bottom in the center of the tank. Each tank has a diffuser with forced air from a blower, an intake rod that controls the direction of flow, and covers that provide darkness for half of the tank and a screen that allows light to enter the other half of the tank. The RAS is also equipped with a centrifugal water pump, a bead filter, a UV filter, a biofilter, 3 solids settling basins, a clarification settling basin, a float valve at the water inlet, and a heater/chiller unit. The replacement water for the RAS is obtained from well water. The water flow into each tank was maintained at 6 to 7 L min ^-1 , and the water temperature was maintained between 28 and 30 °C. The dissolved oxygen content was maintained above 5.0 mg L ^-1 , the pH was maintained between 7 and 8, and the salinity was maintained at 22 ppm throughout the experiment. Temperature, dissolved oxygen, and pH were monitored daily (08:00), while ammonia (NH ₃ ) and nitrite (NO ₂ ) were monitored weekly.

Experiment with challenge infection

An experiment is underway to determine the effectiveness of diets containing HQPC in reducing the severity and impact of early death syndrome/acute hepatopancreatic necrosis

(EMS/AHPND) in shrimp. The test is conducted at the ShrimpVet laboratory in Ho Chi Muhn, Vietnam, and lasts for 33 days, including a one-day adaptation period, a twenty-one-day feeding period, a one-day challenge, and a subsequent ten-day post-challenge period.

The experiments were conducted in 120 L plastic tanks. All tanks were equipped with an activated coral biological filter, aeration, and covered with a plastic lid to reduce the risk of cross-contamination. Brackish water with a salinity of 20 parts per thousand (ppt) was used in each test. The shrimp were fed ad libitum with their respective diets four times daily during the experiment. Feed intake was recorded during the experiment. The tested diets included one basal diet and six treatment diets containing HQPC (inclusion levels of 10-30%). The amount of feed was adjusted depending on the biomass and actual feed intake. Water quality parameters such as dissolved oxygen (DO), pH, and temperature were measured daily. Total ammonia nitrogen, nitrite, and alkalinity were measured twice weekly.

Specific Pathogen Free (SPF) shrimp (Litopenaeus vannamei) are used in this study, with original genetics obtained from Hawaiian broodstock, which are tested for important pathogens including Enterocytozoon hepatopenaeid (EHP), white spot syndrome virus (WSSV), Taura syndrome virus (TSV), infectious myonecrosis virus (IMNV) and EMS/AHPND using PCR method. Nauplii are reared under strict biosecurity conditions. Afterwards, larvae are also tested again for important pathogens including EHP, WSSV, TSV, IMNV and EMS/AHPND using PCR method. Afterwards, larvae are reared under biosecurity conditions. The day before the study, shrimp are weighed as a group to determine the initial weight. The initial average weight of shrimp is 0.56±0.04 grams.

In this experiment, the immersion challenge method is also used. Treatment tanks and positive controls, a total of 28 tanks, are subjected to immersion challenge. Tryptic soy broth +2% sodium chloride (TSB+) inoculated with a consistently virulent strain of Vibrio parahaemolyticus is incubated for 24 hours. Bacterial suspension is added to the tanks to achieve a bacterial density measured by optical absorbance (OD600 nm) at a density expected to kill 90% in the positive control "LD90" for 10 days. Negative controls (a total of 4 tanks) are treated with sterile TSB+ added directly to the tanks. The challenge dose is 3.25 x 105 CFU/mL, which is a lethal dose of 90% (LD ₉₀ ). Standard histopathology, H&F staining is performed on shrimp tissues.

Fish fattening experiment

Results from the multiple trial ANCOVA show that control fish are significantly smaller and are affected by both treatment (P=0.002) and week (P<0.001). Additionally, there is no significant difference in FCR between treatments or weeks (Figure 12). Analysis of covariance (treatment x week) revealed that the vast majority of the variance was due to week rather than diet by treatment. However, there are significant differences in mean weight per fish by both treatment (P=0.046) and week (P<0.001), indicating that fish fed the HQPC-based diet are consistently larger than fish fed the control diet. There are also significant differences in FCR by both treatment (P=0.001) and week (P<0.001), indicating that fish fed the HQPC-based diet consistently utilize feed more efficiently than fish fed the control diet. The overall results of this study are similar to those of other studies feeding rainbow trout with fermented soybean meal. Likewise, no enteritis was observed in these studies, and no adverse effects were found on the distal intestine of rainbow trout fed HQPC-based diets.

In the second and similar experiments, fish fed the HQPC-based diets had significantly and consistently lower FCR (~1.0) than the control diet (~1.25) (Figures 13a, 13b). ANCOVA revealed a significant effect of treatment (P<0.001) as well as week (P<0.001). Differences in mean fish weight were per week (P<0.001) and did not differ between treatments (P=0.305). There were no significant differences in K-value (P=0.758), splenosomatic index (P=0.998), hepatosomatic index (P=0.475), or viscerosomatic index (P=0.411). Fish fed HQPC-based diets had significantly higher viscerosomatic index (P=0.040) and lower hematocrit (P=0.005).

Experiment in shrimp fattening

Experiment on the development of larvae

Inclusion of different proportions of HQPC in hatchery diets showed promising results, improving several performance parameters including larval stage survival (Figure 14).

Experience in growing

The weekly growth rates of shrimp fed HQPC at levels up to 50% were not significantly different from those fed commercial feeds (Figure 15). Although many other factors such as water quality and genetics can influence shrimp growth, based on the observed response of shrimp during the experiment, no differences were observed between the HQPC treatments, supporting the use of HQPC in commercial feed formulations. The results of the growth experiment showed that the higher apparent digestibility coefficient of HQPC along with the higher digestibility of amino acids resulted in better animal growth.

Experiment with challenge infection

The results obtained showed that the application of different inclusion levels had a positive effect on improving the survival of shrimp infected with EMS. Based on the results, the improvement in survival was observed in all treatments of diets containing HQPC. The trial showed that the application of 30% HQPC had a positive effect on increasing the survival of shrimp infected with EMS (Figure 16).

Water quality

Compound feeds are the main factor enabling the intensification of production, but they are also the main source of nitrogen and phosphorus in RAS. In addition, phosphorus is one of the most important minerals in the development of aquaculture feeds for RAS projects. On average, HQPC contains 0.4% phosphorus (Table 7), and the phytic acid concentration in the final product is on average less than 0.12 (g/100 g).

After 18 months of continuous use of HQPC containing feeds, data from a commercial trout farm showed a 69% reduction in phosphorus excretion, from an initial phosphorus excretion of 0.21 mg/L to a level of 0.065 mg/L. During the shrimp culture and challenge trials, water quality parameters were recorded daily. The water quality parameter values (temperature, DO, pH, TAN, nitrite, and alkalinity) are presented (Table 8).

Comparison of fish tests for HQPC testing and fishmeal water quality measurements showed significant differences. Total dissolved solids in RAS water for HQPC and fishmeal treatments were 774 and 822 ppm, respectively. Turbidity (NTU) measured in system tanks showed HQPC (0.4) slightly better than the fishmeal control (0.6).

The digestibility study is carried out at the Aquaculture Laboratory (LAM) of the Oceanographic Institute of the University of São Paulo (USP; Sao Paulo, Brazil). Samples of two soy products were provided by Prairie AquaTech (South Dakota, USA). The soy product samples - non-GMO soybean meal, SBM (46.8% crude protein, CP) and HQPC (74.6% CP) - have an appropriate particle size (>150 microns) and are analyzed to determine their degree of protein hydrolysis (DH, percent).

These samples are tested for in vitro protein digestion using standardized digestive enzymes isolated from the hepatopancreas of pond-reared Pacific white shrimp (average weight 10 grams). The shrimp enzyme hydrolysis involves suspending 80 mg of ingredient protein in distilled water with the pH of the suspension adjusted to 8.0, after adding hepatopancreas enzyme extract for hydrolysis.

The pH shift and hydrolysis monitoring are performed automatically using commercial potentiometric titrators with software control in temperature-controlled units (30±0.6°C). At this reaction pH (=8.0), the enzymatic destruction of the peptide bonds of the ingredients results in a slight decrease in the reaction pH, which is recorded and automatically neutralized by the titrator upon addition of sodium hydroxide, NaOH. At the end of the reaction, the amount of titrant (NaOH) consumed is proportional to the number of peptide bonds cleaved and the quantitative value obtained.

No buffers or other chemicals are used in the assay. If they are determined to be significant, blank DH values are calculated to calculate the net DH of the ingredient protein.

The results of the study show a significant difference in the degree of hydrolysis (DH) of the tested ingredients by shrimp digestive enzymes (Table 9), with the DH value obtained for HQPC demonstrating 42 percent more hydrolyzable protein than soybean flour and 33 percent more than soy protein concentrate.

Typical DH values of SBM range from 4.0% to 4.2% for dehulled and unhulled samples, respectively. Results of comparable statistical pH studies on a set of more than 40 SBM samples from major producing countries (India, Argentina, USA and Brazil) showed DH values from 3.74 to 4.43 percent.

Appropriate screening of new ingredients is needed to assess their potential nutritional value and variability. Various studies have shown that in vitro digestion of ingredients by enzymes from target shrimp or fish species is related to apparent protein digestibility (APD). For almost half a century, the pH-stat method has been used to monitor the effect of heat treatment on the initial rate of tryptic proteolysis of soy protein. Previous studies have also demonstrated a relationship between apparent protein digestibility (APD) and DH in vitro in juvenile Pacific white shrimp.

The average apparent digestibility coefficient in vivo for the crude protein of L. vannamei is between 85 and 90 percent. In the present study, the degree of hydrolysis (DH) of HQPC was estimated to be 7.18 percent and the predicted apparent protein digestibility (PPD) was 93.1 percent. Compared with other studies, the DH and PPD values of HQPC are the highest among the more than 150 ingredients tested, higher than various fish meals, soy protein concentrates, and non-GM soybean flours (Table 9).

In addition to numerous previous studies, this evaluation demonstrated the potential of the commercial HQPC product as a fish meal replacement ingredient in aquaculture diets with respect to its in vitro digestibility. The results show that the product with this ingredient has significantly higher levels of hydrolysable protein as well as higher predicted apparent protein digestibility than various soybean meal ingredients.

All references cited in this document are incorporated by reference in their entirety.

From the above discussion, a person skilled in the art can ascertain the essential characteristics of the invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments to adapt them to various applications and conditions. Thus, various modifications of the embodiments, in addition to those shown and described herein, will be apparent to those skilled in the art from the above description. Such modifications also fall within the scope of the appended claims.

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SEQUENCE LIST

<110> Prairie AquaTech LLC

Harstad, Dennis

<120> MICROBIAL PROCESS FOR IMPROVED QUALITY PROTEIN CONCENTRATE

<130> PRAT-A-GEN2.1-US

<150> US 62/932,684

<151> 2019-11-08

<150> US 63/059,745

<151> 2020-07-16

<150> US 62039,694

<151> 2020-06-16

<150> US 63/036,274

<151> 2020-06-08

<150> US 63/035,797

<151> 2020-06-07

<150> US 17/093,557

<151> 2020-11-09

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 1876

<212> DNA

<213> Aureobasidium pullulans

<400> 1

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atgtgctaca tccacagctc gaacataaat agagaagaca atgccgcctg gctgtccaac 120

atcaactcct ctcatatccg caagcttcct gtcaaccctc ctcacagttc gctcatcact 180

caaacatgcg ttccaggacg aacatcgctc ttggcctagc tgccactggt tccctagtcg 240

ctgccgcgcc ttgcgatatc tatcagaatg gcggtactcc ttgcgtagct gctcacggca 300

caactcgcgc attgtatgat tcctacactg gtcctctcta ccaacttaag agaggctcag 360

atggcactac gaccgatatt tctcctttgt ctgctggtgg tgttgccaat gctgctgctc 420

aggactcttt ctgcaagggt actacctgtc ttatcagtat tatctacgat cagtctgggc 480

gtgcaaacca tctttatcag gcccagaaag gtgctttcag cggaccagat gtcaacggaa 540

acgacaactt ggcaggcgct attggagcac cagtgacttt gaatggcaag aaggcatatg 600

gcgtgttcat ctcgcccggc actgggtaca gaaacgacga agtcagcggc acggccactg 660

gaaacgaacc tgaggcatg tatgctgttc ttgacggcac tcattacaac gatgcttgct 720

gctttgacta cggaaacgcg gaaatcagca acacggatac tggtaacgga catatggagg 780

ccgtctacta tggtaacaac acgatttggg gcagtggctc tggcagcggt ccttggctca 840

tggccgacct tgagaacggt ttgttctctg gccagggtac caagcagaac actgcagacc 900

cttcaatctc caacagattc ttcaccggaa tggtcaaggg agagcctaac cagtgggcgc 960

ttcgcggtag caatgccgcg tccggttcct tgtcgaccta ctacagtggc gctcgtccca 1020

ccgtcggcgg ttacaacccc atgagcctcg agggcgccat cattcttggc atcggtggcg 1080

ataacagcaa tggcgctcag ggcactttct atgagggggt catgacctcg ggctacccgt 1140

ctgatgccac tgaagcctcg gtgcaggcca acattgtggc tgcgaagtac gctaccacat 1200

ctttgaacac agcaccactc actgtcggca acaagatttc gatcaaggtg accacccccg 1260

gctacgacac ccgctatctg gcacacaccg gagccaccgt caacacgcag gttgtctctt 1320

catctagcgc gactagcctc aagcagcagg ccagctggac tgttcgcaca ggcctcggta 1380

acagcggctg ttactctttc gagtcggttg atacacctgg aagcttcatc agacactaca 1440

acttccagct ccagctcaac gcgaatgaca acaccaaggc tttcaaggaa gacgcgactt 1500

tctgctctca gaccggtctt gttaccggca acactttcaa ctcgtggagc taccctgcca 1560

agttcatccg tcactacaac aatgttggat acatcgccag caacggtggt gttcacgact 1620

ttgactctgc tacaggcttc aacaacgatg tcagctttgt ggttggaagc agctttgctt 1680

agatgtaaaa ggtcaggatg aatatgatgg atgtttatga caaaagaagt tatgagtttg 1740

tagttatgga atcttagctg tagcttttga aagcctttgg gatatcagat gtttgtctct 1800

tgttcatgtg ccgttgcaaa gaagaaaaga aggagcagca agcagtgagg ctcttatcgg 1860

gcgatagggc tagatc 1876

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 2

tgactacgga aacgcggaaa 20

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 3

cattgctgtt atcgccaccg 20

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 4

gcttcctgtc aaccctcctc 20

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 5

cacgccatat gccttcttgc 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 6

ttgactacgg aaacgcggaa 20

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 7

aggcttcagt ggcatcagac 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 8

tcagcggacc agatgtcaac 20

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 9

tttccgcgtt tccgtagtca 20

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 10

caacacgatt tggggcagtg 20

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 11

gagcgccatt gctgttatcg 20

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 12

cggaaacgac aacttggcag 20

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 13

gtgttgctga tttccgcgtt 20

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 14

cgataacagc aatggcgctc 20

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 15

gaggctagtc gcgctagatg 20

<210> 16

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<213> Artificial sequence

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gagcgccact gtagtaggtc 20

<---

Claims (13)

1. A non-animal protein concentrate, wherein the concentrate comprises a fermented plant product containing A. pullulans , containing at least about 65% to about 75% protein on a dry matter basis, and has an ash content of less than about 2.5%, wherein said concentrate is a substitute for animal protein, and wherein the non-animal protein is isolated from plant material, including soybeans. 2. The concentrate of claim 1, wherein A. pullulans produces less than about 3.0 g/L pullulan when grown in a medium containing 0.35 to 0.5 g/L yeast extract. 3. The concentrate according to item 1, characterized in that the soybeans are in the form of soy flakes or soy flour. 4. Feed containing the concentrate according to item 1. 5. The feed according to item 4, characterized in that the feed is formulated for animals selected from the group consisting of fin fish, mollusks, crustaceans, domestic animals, farm animals and combinations thereof. 6. The concentrate according to claim 1, characterized in that A. pullulans is NRRL-Y-2311-1. 7. The concentrate according to claim 1, characterized in that there is a significant shift towards lower values in the raw NIR spectra between 4664 cm ^-1 and 4836 cm ^-1 for the final product compared to the starting material. 8. The concentrate according to claim 7, characterized in that the shift toward lower values is from about at least 10% to about 20%. 9. The concentrate according to item 8, characterized in that there is a significant shift towards lower values of the initial NIR spectra from 4664 cm ^-1 to 4836 cm ^-1 for the final product compared to the initial raw material. 10. The concentrate according to claim 9, characterized in that the shift toward lower values is from about at least 10% to about 20%. 11. A method for improving the survival of juvenile shrimp, comprising: feeding juvenile shrimp with the feed of claim 4, wherein the degree of hydrolysis (DH) of protein by shrimp enzymes in said feed is at least 7%. 12. The method according to claim 11, characterized in that the predicted apparent protein digestibility (PPD) of the feed is at least 90%. 13. A feed for juvenile shrimp comprising a concentrate according to claim 1, characterized in that the feed is characterized by a degree of hydrolysis (DH) of at least 7%, a predicted apparent protein digestibility (PPD) of at least 90%, or a combination thereof.