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Title:
OMEGA-3 FATTY ACID ENHANCED DDGS FOR AQUACULTURE FEEDS
Document Type and Number:
WIPO Patent Application WO/2015/051322
Kind Code:
A1
Abstract:
The -present invention describes fermentation methods for producing animal feeds enriched with omega.-3 fatty acids from celIulosic feedstock.

Inventors:
BOOTSMA JASON (US)
GIBBONS WILLIAM (US)
RINEHART MICHAEL (US)
CHEN LIYAN (US)
Application Number:
PCT/US2014/059165
Publication Date:
April 09, 2015
Filing Date:
October 03, 2014
Export Citation:
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Assignee:
PRAIRIE AQUA TECH (US)
International Classes:
A23J1/12; A23K1/18; A23K10/38
Domestic Patent References:
WO2013148415A12013-10-03
Foreign References:
US20100248320A12010-09-30
US20110269185A12011-11-03
US20010000151A12001-04-05
US20130224333A12013-08-29
US20090004219A12009-01-01
Attorney, Agent or Firm:
NAKAMURA, Dean (Post Office Box 2630Montgomery Village, MD, US)
Download PDF:
Claims:
We claim herein:

1 , A method of increasing the omega-3 Fatly acid content of a non-animal based protein concentrate comprising:

a) optionally pce-treating a first celluiosic feedstock;

b) mixing the celluiosic feedstock with water and feeding the resulting mixture into a reactor;

c) sterilizing the mixture;

d) cooling the sterilized mixture and adding a consortiura of saccharifying enzymes under conditions to form a hydrosylate;

e) cooling the hydrosylate and inoculating the hydrosylate with a biocatalyst that metabolizes sugars in the hydrosylate;

f) incubating the inoculated hydrosylate; and

g) optionally adding a second celluiosic feedstock to the inoculated hydrosylate and continuing incubation until the sugars are depleied to form a slurry, wherein the slurry comprises protein, the biocatalyst and omega-3 fatty acids.

2, The method of claim I , wherein the first celluiosic feedstock is DOGS,

3, The method of claim L wherein the second celluiosic feeds tock is CCS.

4, The method of claim 1 , wherein the protein content is between about 30 to abou 55% on a dry matter basis.

5, The method of claim } , wherein the omega-3 fatty acid is DHA.

6, The method of claim 5, wherein the DHA content is about 0.5 to about 3% on a dry matter basis.

7, The method of claim 1, wherein the biocatalyst is 5'. limacmum.

8. The method of claim 1, wherein the a second celluiosic Feedstock is added, afte 4S hours incubation at step (f).

9. A method of increasing the oniega-3 fatty acid content of a non-animal based protein concentrate comprising:

a) mixing a celiuiosic feedstock with water and feeding the resulting mixture into a reactor;

b) sterilizing the mixture;

c) cooling the sterilized mixture and adding a consortium of saccharifying enzymes under conditions to form a hydrosyiate;

d) cooling the hydros iate and inoculating the hydrosyiate with a biocatalyst thai metabolizes sugars in the hydrosyiate;

c) incubating the inoculated hydrosyiate; and

f) optionally adding a second celiuiosic feedstock to the inoculated hydrosyiate and continuing incubation until the sugars are depleted to form a slurry, wherein the slurr comprises protein, the biocatalyst and. omega- 3 fatty acids.

10. The method of claim 9, wherein the first celiuiosic feedstock and the second celi uiosic feedstock are CCS .

1 1. The method of claim 9, wherein the omega-3 fatty acid is EPA.

12. The method of claim 9, and the protein content is between about 30 to about 55% on a dry matter basis.

13. The method of claim 1.1, wherein the EPA content is about 0.8 to greater than about 3% on a dry matter basis.

1 . The method of claim 9, wherein the biocatalyst is P, irregulars.

15. A protein concen trate produced by the method of claim 6, wherein the protein content is between about 30 to about 55% (dry matter basis).

16. A protein concentrate produced by the method of claim .13, wherein the protein content is between about 30 to about 55% (dry matter basis). 4!

17. A protein concentrate containing a non-animal based protein enriched in omega-3 fatty acid content comprising a first shiny and a second slurry, wherein the first shiny is produced by:

a) optionally pre-treating a first celluiosic feedstock by extrusion; b) mixing the first celluiosic feedstock with, water and feeding the resulting first mixture into a first reactor;

c) sterilizing the first mixture;

d) cooling the sterilized first mixture and adding a first consortium of saccharifying enzymes under conditions to form a first hydrosyiate; e) cooling the first hvdrosylate and inoculating the first hydrosykte with a .first biocatalyst that metabolizes sugars in the first hydrosyiate;

i) incubating the inoculated first hvdrosylate; and

g) optionally adding a second celluiosic feedstock, to the inoculated first hydrosyiate and contumiag incubation until the sugars are depleted to form said first slurry,

and wherein the second slurry is produced by:

h) mixing the second ceiiulosic feedstock with water and feeding the resulting second mixture into a second reactor;

i) sterilizing the second mixture;

j) cooling the second sterilized mixture and adding a second consortium of saccharifying enzymes under conditions to form a second hydrosyiate; k) cooling the second hydrosyiate and inoculating the second hydrosyiate with a second biocatalyst that metabolizes sugars in the second, hydrosyiate; 1) incubating the second inoculated hydrosyiate; and

ra) optionally adding additional second ceiiulosic feedstock to the second inoculated hydrosyiate and continuing incubation until the sugars are depleted io form said second slurry',

wherein the concentrate is made by mixing the first and second slurries.

1 . The composition of claim 17. which composition, comprises protein, a biocatalyst, DBA and EPA,

1 . The composition of claim 17, wherein said composition has a protein content of between about. 30 to about. 55% on a dry matter basis (drab), a DMA conteni of between about 0.5 to about 3% dmb, and an EPA content of between about 0,8 to abou t 3% drab.

20. A feed composition containing the composition of claim 17.

Description:
OMEGA-3 FATTY ACID ENHANCED .DPGS FOR AOUACULTURE FEEDS B AC K.G O U D OF THE INVENTION

[0001} This work was made with Governmental support from USD A, National Institute of Food and Agriculture, under contract No. 2014-33610-21950. The Government has certain rights in this invention.

FIELD OF THE INVENTION

[0002} The present invention relates generally to fermentation processes, and specifically to fermentation processes to produce protein concentrates and lipids containing omega-3 fatty acids, products made therefrom, and use of such produc ts in the formulation of nutrient, feeds.

BACKGROUND INFORMATION

[0003] Both fish oil and fish meal are ke ingredients in aquacuiture feeds, but unfortunately their production from wild-caught fishes cannot meet world demand. The iimited avaiiabiiiiy and high prices of fish oil and meal have increased aquacuiiure feed costs, reducing profitability, and thus, inhibit expansion of the U.S. aquacuiiure industry.

(00043 Each year over the past 20, wild fish harvest has decreased by -0.7 metric tons per year, and t he FAO recently reported t hat 53% of the world's wild fish stocks are no w fully exploited, where 32% are over-exploited, depleted or recovering from depletion. This, coupled with rising demand for fish and shellfish products, has caused aquacuiiure to grow at a consistent rate of 9% annually over the past decade. This has created a similar increase in demand for fish meal and fish oil, the primary ingredients in aquacuiture and early-life stages livestock feeds. Unfortun tely, wild harvest of species used for fish meal and fish oil has been similarly exploited, resulting in price spikes and shortages for both. Currently 70-80% offish meal, and 80-90% of fish oil are used for aquacuiture, resulting in competition .for livestock and human food uses, respectively. Prices of fish meal and fish oil have more than doubled in the last five years. High prices are driving up production costs and thereby limiting continued expansion of aquacuiture. [0005} in aquaculture diets (particular carnivorous species) fish meal supplies much of the required 40-50% protein content, while fish oil supplies the required 2-4% omega-3 fatty acid levels. A variety of plant-based protein sources, such as soybean meal and distiller's grains have been used to replace up to 30% of fish meal in aquaculture diets. Unfortunately; anti-nutritional or non-digestible components i these feedstufFs limit inclusion rates.

[00061 Replacement of fish oils remains a challenge. Trials using flax-seed oil (rich in omega-

3 fatty acids) as an alternative to fish oil hav ' had limited success because flax seed oil does not contain DBA or EPA, which are specif ic omega-3 fatty acids required by fish. Moreover, flax seed oil contains increased levels of omega-6 fatty acids, which actually reduce the effectiveness of omega-3 fa tty acids in non-salmonids. Consequently fish fed with fla seed oil have lower levels of omega-3 fatty acids in their tissues. Algal oil represents a separate alternative.

Unfortunately, the Sow volumetric productivity and yield of these systems, combined with expensive oil recovery and dewatering steps, have resulted in prohibitively high costs for feed applications, such that algal oil is only feasible for the high value human consumption market.

[0007] Therefore, there remains- a need tor replacement of omega-3 fatly acids sources derived from fish oils.

SUMMARY OF THE INVENTION

[0008] The present invention describes fermentation methods that result in non-animal protein concentrates enriched with omega-3 fatty acids, which concentrates ma be used to prepare feeds for animals and fish.

[Θ009] h . embodiments, a method of increasing the omega-3 fatty acid content of a non- animal based protein concentrate is disclosed including optionally pre-treating a first cellulosic feedstock; mixing the cellulosic feedstock with water and feeding the resulting mixture into a reactor; sterilizing the mixture; cooling the sterilized mixture and adding a consortium of saccharifying enzymes under conditions to form a hydrosylate; cooling the hydrosylate and inoculating the hydrosylate with a biocatalyst that metabolizes sugars in the hydrosylate;

incubating the inoculated hydrosyiaie; and optionally adding a second cellulosic feedstock to the inoculated hydrosylate and continuing incubation, until the sugars are depleted to form a slurry, where the shiny includes protein, the biocataiyst and omega-3 fatty acids. In a related aspect, the a second celliilosic ieedstock is added, after 48 hours incubation.

[Θ01.0] hi. one aspect, the first ceHulosic feedstock is DOGS. In a related aspect, the second celialosic feedstock is CCS.

[0011 In another aspect, the protein content is between about 30 to about 50% on a dry matter basis.

[0012] In one aspect, the omega-3 fatty acid is DHA. In a related aspect, the DHA content is about 0.5 to about 0.7% on a dry matter basis. In a .farther related aspect, the biocataiyst is S. limacinu .

[0013] In another embodiment, a method of increasing the omega-3 fatty acid content of a non-animal based protein concentrate is disclosed including mixing a ceHulosic ieedstock with water and feeding the resulting mixture into a reactor; sterilizing the mixture; cooling the sterilized mixture and. adding a consortium of saccharifying enzymes under conditions to form a hydrosyiate; cooling the hydrosyiate and inoculating the hydrosyiate with a biocataiyst that .metabolizes sugars in the hydrosyiate; incubating the inoculated hydrosyiate; and optionally adding a second ceHulosic feedstock to die inoculated hydrosyiate and continuing incubation until the sugars are depleted to form a slurry, where the slurry comprises protein, the biocataiyst and omega-3 fatty acids, in a related aspect, the biocataiyst is P. irregulars.

[Θ0Ι4] hi one aspect, the first ceHulosic feedstock and the second ceHulosic feedstock are CCS.

[001.5] in anothe aspect the omega-;? fatty acid is EPA. I a related aspect, the EPA content is about 0.8 to about 1 ,0% on a dry matter basis,

[0016] in. one aspect, the protein content is between about 30 to about 50% on a dry matter basis.

[0017 In one embodiment, a protein concentrate containing a non-animal based protein enriched in omega-3 fatty acid, content including a .first slurry and a second slum' is disclosed, where the first slurry is produced by optionally pre-treating a first ceHulosic feedstock by extrusion; mixing the first ceHulosic feedstock with water and feeding the resulting first mixture into a first reactor; sterilizing the first mixture; cooling the sterilized first mixture and adding a first consortium of saccharifying enzymes under conditions to form a first hydrosylate; cooling the first hydrosviate and inoculating the first hydrosylate with a first biocatalyst that metabolizes sugars in the first hydrosylate; incubating the inoculated first hydrosylate; and optionally adding a second celluiosie feedstock to the inoculated first hydrosylate and. continuing incubation until the sugars are depleted to form said first slurry, and the second slimy is produced by mixing the second celluiosie feedstock -with water and feeding the resulting second mixture into a second reactor; sterilizing the second mixture; cooling the second sterilized mixture and adding a second consortium of saccharifying enzymes under conditions to form a second hydrosyiate; cooling the second hydrosylate and inoculating the second hydrosylate with a second biocatalyst that metabolizes sugars in the second hydrosyiate; incubating the second inoculated hydrosylate; optionally adding additional second celluiosie feedstock to the second inoculated hydrosylate and continuing incubation until the sugars are depleted, to form, said second slurry; where the concentrate is made by mixing the first and second slurries.

[0018] In a related aspect, the concentrate includes protein, a. biocatalyst, DHA and EPA. in a further related aspect, the concentrate has a protein content of between about 30 to abou t 50% on a dry matter basis (dmb), a DMA content of between about 0.5 to about 0.7% dmh, and an EPA content of between about 0.8 to about 1 .0 dmb.

In another embodiment, a feed composition is disclosed containing a protein content of between about 30 to about 50% on a dry matter basis (dmb), a DHA content of between about 0.5 to about 0.7% dmb, and an EPA content of between about 0.8 to about 1 .0% dmb.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG, 1 shows the performance of protein concentrate without enriched fatty acids. (A) Growth performance (Fish Meal v. Protei Concentrate (PC)); (B) Protein Digestibility (Fish Meal v. PC 1.0 and PC 2.0). 602J J FIG. 2 shows a flow diagram for EtO ' H/DDGS production.

10022) FIG. 3 shows omega-3 DDGS production process from com ethanol byproducts (flow diagram). [0023} FIG, 4 shows a table listing the supplement variation from a Plackett-Burman design to detennrae the effects of minerals and micro-nntrients on. DHA production by S. Um cinum using 5% DDGS substrate.

DETAILED DESCRIPTION OF THE INVENTION

(0024] Before the present composition, methods, and raethodologies are described, it is to be understood that this invention is not limited to particular compositions, methods, and

experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[09251 As used in this specification and the appended claims, the singular forms "a". "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "a nucleic acid" includes one or more nucleic acids, and/or compositions of the type described herein which will become apparent to those persons skilled in the art upon reading this, disclosure and. so forth.

(0026J 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 belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, as it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure.

(0027] As used herein, "about," "approximately," "substantially" and "significantly" will be understood by a person of ordinary skill in the art and will vary in some extent depending on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art. given the context in. which it is used, "about" and "approximately" will mean plus or minus <10% of particular term and "substantially" and "si nificantly" will mean plus or minus >! 0% of the particular term.

[Θ028] As used herein, "consisting essentially of means, the particular component and may include other components, which other components do not change the novel properties or aspecis of the particular component. For example, slurries should consist essential of protein, omega- 3 fatty acids, water and biocatalyst, where concentrates should consist essentially of protein omega-3 fatty acids, and biocatalyst.

[Θ029] 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. cattle, swine, goal, sheep, cat, dog. mouse and horse) as well as aquaeulture organisms such as fish (e.g. trout, salmon, perch), molhisks (e.g., clams) and crustaceans (e.g. lobster and shrimp).

[0030] Use of the term "fish" includes ail vertebrate fishes, which may be bony (teleosts) or cartilaginous (cfeondrichthyes) fish species.

[0031 j As used herein "non-animal based protein" means that the protein concentrate comprises at least 0.83 g of crude fiber/lOOg of composition (dry matter basis), which crude fiber is chiefly cellulose, hermceilutose, and iignin materia! obtained as a residue in the chemical analysi s of vegetable substances,

[0032] As used herein, "incubation process" means the provi sion of proper conditions for growth and development of bacteria or ceils, where such bacteria or cells use biosynthetk pathways to metabolize various feed stocks, in embodiments, the incubation process may be carried out, for example, under aerobic conditions, hi other embodiments, the incubation process may include anaerobic fermentation,

[0033] As used herein, a "conversion culture" means a culture of microorganisms which are contained in a medium that comprises material sufficient for the growth of the microorganisms, e.g., water and nutrients. The term "nutrient" means an substance with nutritional value. It can be part of an animal feed or food supplement for an animal Exemplary nutrients include but are not limited to proteins, peptides, fats, fatty acids., lipids, water and fat soluble vitamins, essential amino acids, carbohydrates, sterols, enzymes, functional organic acids and trace minerals, such as, phosphorus, iron, copper, zinc, manganese, magnesium, cobalt, iodine, selenium,

molybdenum, nickel, .fluorine, vanadium, tin, and silicon.

[Θ034] The term "pretreaied " means that a composition has been subjected to a treatment prior to saeehariikaiion..

[0035} The term "celluSosic" means a composition comprising cellulose. Θ36] "Under conditions to form a hydrosylate" means conditions such as pH, composition of medium, and temr^rature under which .saceharification enzymes ar active,

[Θ037] "Hydrosylate" means any product of hydrolysis . Θ038] "Consortium of saccharifying enzymes" means one or more enzymes selected primarily, but not exclusively, from the group "glycosidases" which hydrolyze the ether linkages of di-, oKgo-, and polysaccharides and are found in the enzyme classification EC 3,2.1 .x

(Enzyme Nomenclature 1992, Academic Press, San Diego, Calif, with Supplement 1. (1 9:3), Supplement 2 (1994), Supplement 3 (1995, Supplement 4 (1997) and Supplement 5 {in Eur. J. Biochem. (1994) 223: 1 -5, Eur, j. Biochem, (1995) 232: 1 -6, Eur. j, Biochem, (1996) 237: 1-5, Eur. j. Biochem. (1997) 250: 1-6, and Eur. J. Biochern. (1999) 264:610-650, respectively]) of the general group "hydrolases" (EC 3.). Glycosidases useful in the present method can be categorized by the biomass component thai they hydroiyze. Glycosidases useful for the present method include cellulose-hydrolyzing glycosidases (for example, celluloses, endoglucanases, exoglucanases, ce!lobiohydroiases, β-glncosidases), hemicellolose-hydrolyzitig glycosidases (for example, xylanases, endoxylanases, exoxylanases, β-xylosidases, arahi.noxylanases, mammses, galactases, pectirtases, glucuronidases), and starch-hydroJy ing glycosidases (for example, amylases, a-amy!ases, β-amylases, gSocoamylases, tt-giucosidases, isoaraylases). in addition, it may he useful to add other activities to the saceharification enzyme consortium such as peptidases (EC 3.4,x.y), lipases (EC 3,1.1 ,x and 3.1 ,4.x), ligninases (EC 1, 1 1 ,1.x), and feruloyl esterases (EC 3.1.1.73) to help release polysaccharides from other components of the biomass. It is well known in the art that microorganisms that produce polysaccharide~hydrolyzmg enzymes often, exhibit an activity, such as cellulose degradation, thai is catalyzed by several enzymes or a group of enzymes having different substrate specificities. Thus, a "cellulase" from a

microorganism may compri se a group of enzymes, all of which may contribute to the cellulose- degrading activity. Commercial or non-commercial enzyme preparations, such as cellulase, may comprise numerous enzymes depending on the purification scheme utilized to obtain the enzyme. Thus, the saceharification enzyme consortium of the present method may comprise enzyme activity, such as "eellu!ase", however it is recognized that thi activity may be catalyzed, by more than one enzyme.

[Θ039] "Slurry" means a thick mixture of water and another substance. Θ 4Θ] "Biocatalyst" means a substance thai initiates or increases die rate of a chemical reaction, and includes, but is not limited to, microorganisms selected from bacteria, filamentous fungi and yeast. jiM ! j Conversion is the process of cuhuring microorganisms in a conversion culture under conditions suitable to convert pro ein ca-rbohydraie/poiysaccharide materials, for example, DDGS material into a high-quality protein concentrate. Adequate conversion means utilization of 90% or more of specified carbohydrates to produce microbial cell mass and/or protein or lipid. In embodiments, conversion may be aerobic or anaerobic.

[0042] A large number of plant protein sources may be used in connection with the present disclosure as feed stocks for conversion. The main reason for using plant proteins in the feed industry is to replace more expensive proiein sources, like animal protein sources. Another important factor is the danger of transmitting diseases thorough feeding animal proteins to animals of the same species. Examples for plant protein sources include, but are not limited to, protein from the plant family F bace e as exemplified, by soybean and peanut, from the plant family Brassic/ac ae as exemplified by canoia, cottonseed, the plant family Asterace e including, but not limited to sunflower, and the plant family Ar c ceae including copra. These protei -sources, also ' commonly defined as oilseed proteins may be fed whole, but they are more commonly fed as a by-product after oils have been removed. Other plant protein sources include plant protein sources from the family Poaceae, also known as Gramlmae, like cereals and grains especially corn, wheat and rice or other staple crops such as potato, cassava, and legumes (peas and beans), some milling by-products including germ, meal or corn gluten meal, or

distillery brewery by-products. In embodiments, feed stocks for proteins include, but are not limited to, plant materials from soybeans, peanuts, Rapeseeds, barley, eanola, sesame seeds, cottonseeds, palm kernels, grape seeds, olives, safflowers, sunflowers, copra, corn, coconuts, linseed, hazelnuts, wheat, rice, potatoes, cassavas, legumes, camelina seeds, mustard seeds, germ meal, corn gluten meal, distillery/brewery by-products, and combinations thereof.

(00431, In the fish fanning industry the major fishmeai repiacers with plant origin reportedly used, include, but are not limited to, soybeao meal (SBM), maize gluten meai, apeseed/canola (Brassi a sp.) meal, lupin (Lupimis sp, like the proteins in kernel meals of de-hulled white {Lupinus albiis), sweet (L ngi ifoUm) and yellow CI. hm ) lupins, Sunflower {Helkmth ' s annum) seed meal, crystalline amino acids; as well as pea meal (Pimm sativum), Cottonseed {Gossypmm ψ.) meal. Peanut (groundnut; Araehis hypogaea meai and oilcake, soybean protein concentrate, com. (Zea mays) gluten meal, and wheat (Tritk- m aestivum) gluten, Potato (Salanum tuberosum L.) protein concentrate as well as other plant feedstafls like Moringa (Moringa ohifer Lam.) leaves, all in various concentrations and combinations,

J0044] The protein sources may be in the form of non-treated plain materials and treated and/or extracted plant proteins. As an example, heat treated soy products have high protein digestibility.

[0945) in embodiments, distiller's dried grain solubles (DDGS) may be used. DDGS are currently manufactured by the corn ethanol industry. Traditional DDGS comes from dry grind facilities, in which the entire corn kernel is ground and processed. DDGS in these facilities (e.g., "front end" fermentation) typically contains 28-32% protein and between about to about 13% crude fat. However, in "back end" oil extraction, about 1/3 of the com oil is extracted from, e.g., thin stallage, prior to producing "reduced-oil" DDGS (containing about 5 to about 9% crude fat), which has slightly more protein and .fiber relative to DDGS produced without, oil extraction. n a related aspect, either reduced oil or traditional DDGS may be used,

[0046] The present invention solves this problem and allows for plant protein inclusion levels of up to 40 or even 50%, depending on, amongst other factors, the animal species being fed, the origin of the plant protein source, the ratio of different plant protein sources, the protei

concentration and the amount, origin, molecular structure and concentration of the glucan and/or raannan. In embodiments, the plant protein inclusion levels are up to 40%, preferably up to 20 or 30%. Typically the plant protein present in the diet is between 5 and 40%, preferably between 1 0 or 15 and 30%. These percentages define the percentage amount of a total plant protein .source in the animal feed or diet, this includes fat, ashes etc. In embodiments, ure protein levels are up to 50%, typically up to 45%, in embodiments 5-95%.

[Θ047] The proportion of plant protein to other protein in the total feed or diet may be 5:95 to 95:5, 1.5:85 to 50:50, or 25:75 to 45:55.

Microorganisms

[Θ048] The disclosed microorgan sms must be capable of converting carbohydrates and other nutrients into a high-quality protein concentrate in a conversion culture. In embodiments, the microorganism is a yeast-like fungus. An example of a yeast -like fungus is Aurobasidium ptdhdans. Oilier example microorganisms include yeast such as K! yveromyces and Pichi ,ψρ, Lactic acid bacteria, Trichoderma reesei, Pieurotm streatm, Rhizopus spp, and many types of Jignocel!ttlose degrading microbes. Generally, exemplary microbes include those microbes thai can metabolize stachyose, raffinose, xylose and other sugars, .However, it is within the abilities of a skilled artisan to pick, without undue experimentation., other appropriate microorganisms based on the disclosed methods.

ΪΘ049) in embodiments, the microbial organisms that may be used in the present process include, but are not limited to, Aureohasldium pidhdam, Schizoc-hytrium !imacinum, Pythm ' m irregulare, Fmari tn venenatwti, Sc!erotmm ghtcanlcitm, Sphmgomon paucimohite, .Rafstoma eutropha, RhodospirUhm ntbrwn, issatchenkia spp, Penic/l!imn spp, Kiuyve myees and Pichm spp, Trichoderma ree ei, Pieurotm ostreatm, Rhizopus spp, and combinations thereof. In embodiments, the microbe is S. I m cmum or P, irreg lare.

[00501 in embodiments, the J. puilulam is adapted, to various environmerus/stressors encountered during conversion, in embodiments, an A. puU iam strain denoted by NRRL deposit No. 50793, which was deposiied with the Agricultural Research Culture Collection (NRRL), Peoria, ill., under the terms of the Budapest Treaty on November 30, 2012, exhibits lower gum production and is adapted to DDGS and SBM based media. In embodiments, an A. ptdhdans strain denoted by NRRL deposit No. 50792, which was deposited with the Agricultural Research Culture Collection (NRRL). Peoria, HI., under the terms of the Budapest Treaty on November 30, 2012, is adapted to high levels of the antibiotic tetracycline (e.g., from about 75 pg ml tetracycline to about 200 pg ml tetracycline).. In embodiments, an A . pulhd s ' strain denoted by NRRL deposit No. 50794, which was deposited with the Agricultural Research Culture

Collection (NRRL). Peoria, III, under the terms of the Budapest Treaiy on November 30, 2012, is adapted io high levels of the antibiotic LACTROLi) (e.g.. from about 2 ug/ml virgmiamycm to about 6 .ug ml vii-giniamycin). in embodiments, m A. ptdhdans strain denoted by NRRL deposit No. 50795, which was deposiied with the Agricultural Research Culture Collection (NRRL), Peoria, 111., under the terms of the Budapest Treaty on November 30, 201.2, is acclimated, to condensed corn solubles. 005J ] In other embodiments, an A. pallidums strain may be acclimated to 450-550 ppm LACTROL© (e.g., virgintamycin). In embodiments, an A, puilulam strain may be acclimated to H . ,5- 1.75.. In embodiments,, an A. pitllulam strain may be acclimated to 90-1 10 ppm Isostab, In embodiments, an A. puikiiam strain may be acclimated to 80-100 ppm Betastab. in

embodiments, an A. pirfh a strain may produce ceilulase enzymes and may be acclimated to DDGS. in embodiments, the put!uiam is selected from NRRL 42023, NRRL 58522 or Y- 2311- 1.

[0§52| in other embodiments, a Thermoiolerant Pichia strain may be acclimated to CCS and

DDGS.

[0953) in embodiments, an issafchenkla spp strain may be acclimated to CCS and DDGS.

[0054) In embodiments, .a fusan m venenatwn strain, ma produce ceilulase en¾ymes and may b ac limated to CCS and DDGS.

[9955) In other embodiments, a PeniciUium spp strain may produce ceilulase enzymes nd may be acclimated to CCS and DDGS.

[9956] in. embodiments, Aspergillus onyae strain may be acclimated to CCS and DDGS.

[9957] its. embodiments, & limacinwn strain may be acclimated io CCS and DDGS.

[9958] In embodiments, P. Irregular strain may be acclimated to CCS and DDGS.

[9059] in. embodiments, microorganisms which are capable of producing lipids comprising omega-3 and/or oraega-6 -polyunsaturated fatty acids include those microorganisms which are capable of producing DHA. Io a related aspect, such organisms include marine microorganisms, for example algae, such as Thramiochytrkis of the order Thir stoc yirkiles, more specifically Thraustochyfriales of the genus Thraustoehyirium and Sehizochytrmm, including

Thra toohytrict s which are disclosed in U.S. Pat. Nos. 5,340,594 and 5,340 ,742, the disclosures of which are incorporated herein, by reference in their entireties. It is to be imdersiood, however, that the invention as a whole is not intended to be so limited, and that one skilled in the art will recognize that the concept of the present invention will be applicable to other microorganisms producing a variety of other compounds, including other lipid

compositions, in accordance with the techniques discussed herein. Θ 6Θ] As used herein a "fatty acid" means an aliphatic onocarhoxyiic acid. Lipids are recognized to be fats or oils including the giyceri.de esters of fatty acids alon with, associated phosphatides, sterols, alcohols, hydrocarbons, ketones, and related compounds.

J 006! I A commonly employed, shorthand system is used, m this disclosure to denote the structure of the fatty acids (e.g., Weete, "Lipid Biochemistry o Fungi and Other Organisms". Plenum Press, New York (1980)). This system uses the letter "C" accompanied by a number denoting the number of carbons in the hydrocarbon chain,, followed by a colon and a number indicating the number of double bonds, e.g., C20;5, eicosapentaenoic acid. Fatty acids are numbered starting at the carboxy carbon. Position of the doable bonds is indicated by adding the Greek letter delta ( ' Δ) followed by the carbon number of the double bond; i.e., C20;5omega-3 A !s, ,l J \ The "omega" notation is a shorthand system for unsaturated fatty acids whereby .numbering from the carboxy-tenninal carbon is used. For convenience,, a>3 will be used to symbolize "omega-3.," especially when using the numerical shorthand nomenclature described herein. Omega-3 highly iinsaturated fatty acids are understood to be polyethylenic fatty acids in which the ultimate ethylenic bond is 3 carbons from and including the terminal methyl group of the fatty acid. Thus, the complete nomenclature for eicosapentaenoic acid, an omega-3 highly unsaturated fatty acid, would be 020:5ω3Δ ",ί! "' 1 ι- ί4 · " ' For the sake of brevity, the double bond locations (A 5-8 "* 1 '' 4 '' ') will be omitted. Eicosapentaenoic acid is then designated C20;5w3, ' Docosapentaenoic acid (C22:5co3d ' O> i!> ' 19 ) is C22:5co3, and Docosahexaenoie acid

(C22:6n>3A 4, ,,<0>,->>K>,ii ') is C22:6t 3. The nomenclature "highly unsaturated fatty acid" means a fatty acid with 4 or more double bonds. "Saturated forty acid" means a fatty acid, with 1 to 3 double bonds.

Desirable characteristic s of the organisms tor the production of omega-3 highly unsaturated fatty acids include, but are not limited t those: 1 ) capable of heterotrophic growth; 2) high content of o.raega-3 highly unsaturated fatty acids; 3) unicellular; 4) low content of saturated and omega-6 highly unsaturated fatty acids; 5) themiotoierant (ability to grow at temperatures above 30° C) ; and 6) eur haline (able to grow over a wide range of salinities, including low salinities).

[0063] Lipids may comprise one or more of the .following compounds: lipstatin, statin, TAPS, pimaricine, nystatins., fat-soluble antibiotic (e.g., laidlomycin) fat-soluble anti-oxidanf (e.g., coenzyme Q 10), cholesterol, phytosteroL desmosteroi, tocotrieno tocopherol, earotenoki or xanthophyl!s, for instance beta-carotene, lutein, lycopene, astaxanthin, zeaxanthin, or canihaxanth.in, fatly acids, such as eonjungated linoieic acids or polyunsaturated fatty acid (PUFAs). in embodiment ' s, the lipid comprises at least one of the compounds mentioned above at a concentration of at about 5 wt % or at !east about 10 wt. % (with respect to the weight of the lipid).

[006 1 Lipids may be obtained comprising for example triglyceride, phospholipid, free fatly acid, fatty acid ester (e.g., methyl or ethyl, ester) and/or combinations thereof in embodiments, lipids have a triacytglycerol content of at least about 50%, at least about 70%, or at leas t about 90%.

10 65] In embodiments, a lipid comprises a polyunsaturated fatty acid (PUPA), for instance a PUPA having at least 18 carbon atoms, for instance a C €½ or C 2 PUFA. I.n embodiments, the PUFA is an omega-3 PUFA (to3) or an omega-6 PUFA (<o6). In related aspects, the PUFA has at least 3 double bonds, in embodiments, PUFAs are: docosahexaenoic acid (DBA, 22:6 e>3); y- !inolenic acid (GLA, 18:3 ω6>; α-linolenic acid (ALA, 18:3 c«3); dihomo-y-linolenic acid (DGLA, 20:3 ©6); arachidonic acid (ARA, 20:4 (u6) and eicosapentaenoic acid (EPA, 20:5 <»3).

[ii066j In embodiments, a lipid comprises at least one PUFA (for instance ARA or DHA) at a concentration of at least about 5 wt. %, for instance at least about 10 wt. %, for instance at least about 20 wt. % (with respect to the weight of the lipid).

[0067] The PUFA may be in the form of a (mono-, di, or tri) glyeeri.de, phospholipid, free fatty acid, fatty acid ester (e.g. methyl or ethyl ester) and or combinations thereof. In a related aspect, a lipid is obtained wherein at least about 50% of all PUFAs are in triglyceride form.

(0068] The lipid amy be an oil or fat, for instance an oil comprising a PUF A.

\ϋ&69] In embodiments, the process as disclosed herein increases omega-3 fatty acid content of a non-animal feedstock from about 0% to about 0.24%, from about 0.24% to about 1..5%, from about 1.5% to about 2%, from about 2% to about 3% on a dry matter basis (dmb). in one aspect, limaeinum fermentation on DDGS increases the DHA content from about 0% to about 0.3%, from about 0% to about 0.24%, from about 0% to about 1.5%, or from about 0% to about 3% (dmb). in a related aspect, <S'. limaeinum fermentation on. DDGS increases the protein content to about 36% or to about 50%, or to about 55% (dmb). In another aspect, P. vregidare fermentation oo CCS increase EPA content from about 0% to about 0.94%, about 0% to about 1.5%, from about 0% to about 2%, or from about 0% to about 3% (dmb). In. a related aspect, P. irr gu! re fermentation on CCS increases the protein content to about 34,5% or to about 40%, or to greater tii an about 50% (dmb).

J0070] The cells may be any ceils comprising a lipid. Typically, the cells have produced the lipid. The cells may be whole cells or ruptured cells. The cells may be of any suitable origin. The cells may for instance be plant cells, for instance cells from seeds or cells of a microorganism (microbial cells or microbes). Examples of microbial cells or microbes are yeas t ce ll, bacterial ceils, fungal cells, and alga! cells. In embodiments,, fungi ma be use, fo example, such as the order Miico les, for example MoHiereila, Pky omyce , Blake !&a, Aspergillus,

nmmfoch tri m, Pythium or Entomophthora. in embodiments, a source of arachidonic acid (ARA) may be from Mortierella alpinci, Biahesiea trisp r , Aspergillus lerreus or Pythium insidiosum. Algae may be duiofiagellate and/or include Porpkyridium, Niiszchia, or

Crypthecodmium (e.g. Cr pthec dinh cohnu). Yeasts may include those of the genus Plchi or Saceharomyc.es, such as Plchia ciferii. Bacteria may be of the genus Pmpionibacferium.

Examples of plant cells comprising a lipid are cells from soy beau, rape seed, eanola, sunflower, coconut, flax and palm seed. In em.bodim.ents, the cells are plant cells comprising lipid which lipid comprises ARA. In embodiments, the ceils as disclosed may be used aione or in combination.

10071] in embodiments, the cells are used in fermentation,

10072] Various plant-based protein sources have been used in aquaculture diets, including DDGS (10-35% inclusion) and soy bean meal (5-30% inclusion) as partial replacements for marine-derived fish meal. These protein sources have limitations, particularly with piscivorous species, DDGS cootains high levels of fiber and insufficient levels of essential amino acids (e.g., lysine and methionine), hi embodiments, the methods as disclosed herein, provide DDGS derived products with reduced fiber content and improved amino acid profiles using a micro-fungal process., resulting in a significantly hi her fish digestibility. Soy bean meal (SBM) is limited by an ti -nutritional factors (e.g.. stachyose, raffinose, trypsin inhibitors), in a related aspect, the methods as disclosed herein provide a soy protein concentrate wherein the anti-n iritional factors have been removed, while at the same time said concentrate has an increase in protein levels, enabling a replacement of fish meal ut yellow perch diets (FIG. I ). In another related aspect, the methods as described herein provide for the production of a product which also provides the required omega-3 fatty acids that is normally provided by fish oil.

[0073] The omega-3 fatty acid profile of fish oil is high in EPA and DHA, but low in a- Smolenic acid (ALA). By contrast, flaxseed oil contains hig levels of ALA, lacks DHA. and EPA, arid also contains high titers of omega-6 fatty acids, which reduces the effectiveness of omega-3 fatty acids. Thus, flaxseed oil has not succeeded as a fish oil replacement. Algal oil is much more appropriate due to its proper balance of omega-3 fatty acids and many efforts are underway to exploit this opportunity. Unfortunately, photoautotrophic platforms for algal production have low productivity in relation to capital and or opportunity costs, this limiting these systems to higher value human supplement markets. Heterotrophic alga! systems have higher productivities, can utili e low cost waste materials as feedstocks, and can use lower cost bioreactors. However, downstream processing for cell recovery (centrifugation or filtration) and oil extraction and drying are significant costs that must be overcome. Alternatively, the processes as disclosed herein retain microbiaUy-derived omega-3 fatty acids, along with the microbial mass, in the final omega-3 DDGS product. In embodiments, the microbial conversion of distiller's wet grains and condensed com ' solubles (CCS) is integrated prior to their normal blending and drying in corn ethanol facilities, thus, yielding a DDGS with enhanced levels of DHA, EPA and protein . j W)?4J in embodiments, S. Umacinum and ma be used with distiller's grains to produce DHA. In other embodiments, P. irr gulare may be used with CCS to produce EPA. Both are oleaginous microbes that can produce more than 25% lipid on a dry ceil weight basis. S. Umacinum is classified as a marine protist (Thraustochytrids) that can accumulate >50% of its dry weight as lipids, of which more than. 25% are DHA, Ceils are heterogeneous in sixe, approximately 6-21 urn m diameter, with a granular cytoplasm containing oil micelles. In embodiments, the may serve as a source of carotenoids, such as asiaxanthin, which has good antioxidant properties. Some Thrausiochyirids have been known, to produce proteases, lipases, esterases, acid and alkaline phosphatases, cellulases, and xylanases, thus, in a related aspect, such protists may afford a process with the ability to hydrolyze fibers in DDGS,

}t)t)?5| P. irregulare is a filamentous fungus which has the highest reported EPA production of all fungi, at 25% of total lipids content when grow on lactose in a 14 L reactor. Θ076] Glucose is most commonly used for omega-3 fatty acid production with these microbes, but maltose and starch may also been. used. To reduce production costs, lower cost byproducts may also be used, including but not limited to, crude glycerol, residues from beer and potato processing, sweet sorghum juice, thin siiliage, and sweet whey permeate. Although such a approach may be tempered by the use of costly nitrogen sources (yeast extract, peptone, and tryptone). in a related aspect, use of the dilute byproducts may require that cells be recovered and dried before being used as an omega-3 fatty acid supplement In embodiment, omega-3 production may be integrated into the DDGS recovery section of ethanoi plants to take advantage of these concentrated processing streams and minimize recovery/drying costs. In a related aspect, distiller's wet grains and CCS contain sugars, organic acids, glycerol, corn oil, and other nutrients that will support omege-3 production.

[6077] DDGS is a co-product of the com ethanol industry (FIG. 2) and current U.S.

production capacity is about 40 million tons yr. The large quantities and lower protein content of DDGS have led to a reduced market price for DDGS compared to other protein meals like SBM, canola, and animal derived meals. DDGS has been traditionally fed to ruminant animals, but It does have potential for use as a fish meal repiacer in aquacu!iure diets. Due to the removal of starch in the ethanol process, DDGS contains approximately 3.5- 12.8% fat, 26.8-33.7% protein, 5.4-10.6% fiber, and 2.0-9.8 minerals, DDGS contains none of the anti-nutritional factors (e.g., trypsin inhibitors, oligosaccharides) found in SBM However, DDGS contains fiber and lower amounts of the essential amino acids lysine and. methionine, compared to fish meal. DDGS has excellent potential as a substrate for EPA and DMA production due to its high content of metabolizable carbon sources for microbes (oil, glycerol, and fibers which could be hydrolyzed into sugars). j ( l07S| The combination of omega-3 enhanced DDGS with fermentation methods as disclosed hi, e.g., U.S. Pub. No. 20130142905 arid U.S. Ser. No. 14/453,597 (each of which is incorporated by reference in its entirety) provides a feedstuff that addresses the needs for oil, omega-3 fatty acids, and protein in aquacu!iure diets. An advantage of this process as disclosed includes lower production costs due to the inexpensive feedstock and. minimal processing steps and higher performance in fish due to the nutritional superiorit of omega-3 DDGS product which addresses multiple nutritional needs. [0079} in embodiments, <V. Ifmacinwn in non-preireated DDGS resells in about 0.6! % DHA and about 36% protein. In a related aspect, performance may be increased to the levels shown in Table 1 by pre- treating distiller's wet grains via extrusion, followed by enzymatic

saccharifieat!on to convert fibers into simple sugars. In previous processes with A. puUulam protein levels have been increased from 33 to >50% by applying a similar extrusion and saceharifieation process (see, e.g., U.S. Fob. No, 20130142905). In embodiments, CCS is added in a fed-batch mode to provide additional glycerol, as well as cause a nitrogen limitation to trigger additional DHA production, in a related aspect, a biphasic process in which an inexpensive nitrogen source may be added initially to boost cell mass and thus protein levels is disclosed. CCS may be added to provide glycerol (and cause nitrogen limitation) while aeration may be reduced to stress cells io produce additional DHA. Preliminary work has also established that P. irregular can convert diluted CCS into 0.94% EPA and 34.5% protein, in embodiments, CCS is added in a led batch mode to provide additional glycerol. In certain aspects, a biphasic process may initially boost eel! mass/protein levels, which is followed by a fed-batch process with reduced aeration to increase EPA levels (Table I ).

Table I . Preliminary Performance (dmb).

jOOSO] In embodiments, the distiller's wet grain and CCS processing streams may be blended in different ratios and then centrifuged and dried into omega-3 DDGS. These may then be tested in perch digestibility and feeding trials at various replacement levels for fish oil and fish meal. In one aspect, the omega-3 DDGS performs at least as well as fish meal/οϋ feeds (growth rate, feed conversion, and protein efficiency) at .100% replacement le vels, and. at much, lower costs. Viscera characteristics and intestinal, histology may be examined t assess fish responses.

}ί!ίί81 j In embodiments, additional optimisation steps may include, but are not limited, to 1) optimizin the production process (strain enhancement, omitting cellulases), 2) evaluating the omega-3 DDGS in a range of commercially important fishes, 3) validating process costs and energy requirements, and 4) completing steps for scale-up and commercialization. [Θ082] in embodiments, in addition to generating a protein source to replace fish meal, the process as disclosed herein integrates microbial production of two critical omega-3 fatty acids (DBA. and EPA). The feedstocks may include distiller's wet grain for production of HA and protein from S. Hmacinum and CCS for production of EPA and protein from P. irregulare.

[0083] in embodiments, the method as disclosed may be summarized as shown in FIG. 3, which depicts an approach to converting com ethano? processing by-products into omega-3

DDGS, which product may be used to replace fish meal and fis oil in aquaculture feeds. In a related aspect, distiller's wet grain may be subjected to extrusion pretreatraent and enzymatic hydrolysis (under optimized conditions), followed by incubation with 8. Umacinum .for

production of DMA and si ngle-ceil protein . CCS may be added, to boost DMA production, in one aspect, ceikdases may be omitted sequentially to evaluate if co-culturing with cellulolytic microbes may be used as substitutes for the enzymes. In another aspect, CCS may be mixed with water as needed and then inoculated with P. irregulars for production of EPA and single-cell protein,

(0084) in embodiment, following incubation, the process streams ma be mixed to achieve desired levels of DM A, EPA, and protein. Solids may be recovered by centrimgation and dried, with supernatant evaluated for recycling to the front end of the process. In one aspect, sol ids may be formulated into omega-3 DDGS based feeds and tested in perch feeding trials, with control diets prepared using fish meal and fish oi!. Performance (e.g., digestibility, growth, feed conversion, protein efficiency), viscera characteristics, and intestinal histology maybe examined to assess fish responses. In another aspect, the processes as disclosed herein allows for

optimization, of oniega-3 DDGS production process by determining optimum pretreatment and conversion conditions while minimizing process inputs, improving the performance and robustness of the microbes, testing the resultant grower feeds for a range of commercially important .fishes, validating process costs and energy requirements, and completing initial steps for scale-up and commercialization.

' Dietary Formulations

(0085] in exemplary embodiments, the protein concentrate and lipids recovered are used in dietary formulations. In embodiments, the recovered protein concentrate (PC) will be the only protein source in a dietary formulation. Protein source percentages in dietary formulations are not meant to be limiting and may include 24 to 80% protein. In embodiments, the protein concentrate will be more than about 50%, more than about 60%. or more than about 70% of the total dietary formuJation protein source. Recovered PC/lipid combinations may replace sources such as fish meai, soybean meai, wheat and corn ilours and glutens and concentrates, and animal byproduct such as blood, poultry, and feather meals. Dietary formulations using recovered PC/lipids may also include supplements such as mineral and vitamin premixes to satisfy remaining nutrient requirements as appropriate.

[0086) in certain embodiments, performance of the PC, such as high-quality protein from DDGS or other upgraded plant-based meals alone or in combination with generated lipids, may be measured by comparing the growth, feed conversion, protei efficiency, a d survival of animal on a. high-quality protein concentrate dietary formulation to animals fed control dietary formulations, such as fish-meal. In embodiments, test formulations contain consistent protein, lipid, and energy contents. For example, when the animal is a fish, viscera (fat deposition) and organ (liver and spleen) characteristics, dress-out percentage, and fillet proximate analysis, as well as intestinal histology (enteritis) may be measured to assess dietary response.

[0087) As is understood, individual dietary formulations containing the recovered PC and/or combinations with recovered lipids may be optimized for different kinds of animals. In embodiments, the animals are fish grown in commercial aquacalture. Methods for optimization of dietary formulations are well-known and easily ascertainable by the skilled artisan without undue experimentation.

[0088) Complete grower diets may be formulated using PC in accordance with known nutrient requirements for various animal species. In embodiments, the formulation may be used for yellow perch (e.g., 42% protein, 8% lipid), in embodiments, the formulation may be used, for rainbow trout (35% protein, 1.6% lipid). In embodiments, the formulation may be used for any one of the animals recited supra.

[0089J Basal mineral and vitamin premixes for plant-based diets may be used to ensure that micro-nutrient .requirements will be met. Any supplements (as deemed necessary by analysis) may be evaluated by comparing to an identical formulation without siipplementation; thus, the feeding trial may be done in a factorial design to account for supplementation effects. In embodiments, feeding trials may include a fish meal-based control diet and ESPC- and LSPC- based reference diets [traditional SPC (TSPC) is produced from solvent washing soy Hake to remove soluble carbohydrate; textarked SPC (ESPC) is produced by extruding TSPC under moist, high temperature; and low-antigen SPC (LSPC) is produced from TSPC by altering the solvent wash and temperature during processing]. Pellets for feeding trials may be produced using the lab-scale ingle screw extruder (e.g., BRABENDERPLASTJ-CORDER EXTRUDER Model PL2000).

Feeding Trials

[9090 j In embodiments, a replication of four experimental units per treatment (i.e., each experimental and control diet blend) may be used (e.g. , about 60 to 120 days each). Trials may be carried out in ! !O-L circular tanks (20 fish/tank) connected in parallel to a closed-loop recirculation system driven by a centrifugal pump and consisting of a solids sump, and bioreacior, filters (100 μηι bag, carbon and ultra-violet). Heat pumps may be used as required to maintain optimal temperatures for species-specific growth. Water quality (e.g., dissolved oxygen, pli, temperature, ammonia and nitrite) may be monitored in all systems.

[9991 J In embodiments, experimental diets may be delivered according to fish size and split into two to five daily feedings. Growth performance may be determined by total mass measurements taken at one to four weeks (depending upon fish size and trial duration); rations may be adjusted in accordance with gains to allow satiation feeding and to reduce waste streams. Consumption may be assessed biweekly ftotn collections of uneaten feed from individual tanks. Uneaten feed may be dried to a constant temperature, cooled, and weighed to estimate feed conversion efficiency. Feed protein and energy digestibilities may be determined from fecal material manually stripped during the midpoint of each, experiment or via necropsy from the lower intestinal tract at the conclusion of a feeding trial . Survival, weight gain, growth rate, health indices, feed conversion, protein and energy digestibilities,, and protein efficiency may be compared among treatment groups. Proximate analysis of necropsied fishes may be carried out to compare composition of fillets among dietary treatments. Analysis of amino and fatty acids may be done as needed for fillet constituents, according to the feeding trial objective. Feeding trial responses of dietary treatments may be compared to a control (e.g., fish meals diet response to ascertai n whether performance of PC diets meet or exceed control responses. Θ092) Statistical analyses of diets and feeding trial responses may be completed with an a priori :::: 0.05. Analysis of performance parameters among treatments may be performed with appropriate analysis of variance o covarianee (Proc Mixed) and post hoc multiple comparisons, as needed. Analysts of fish performance and tissue responses may be assessed by nonlinear models.

[00 31 In embodiments, the present disclosure proposes to convert fibers and other carbohydrates in D.DGS into additional protein using, for example, a GRAS-status microbe. A microbial exopalysacchari.de {.i.e., gum) may a!so be produced that may facilitate extruded feed pellet formation, negating the need for binders. This microbial gum may also provide mmunostimukmt activity to activate innate defense mechanisms that protect fish from common pathogens resulting from stressors, immuiioprophylactic substances, such as β-glucans, bacteria! products, and plant constituents, are increasingly used in commercial feeds to reduce economic losses due to infectious diseases and minimize antibiotic use. The microbes of the present disclosure also produce extracellular peptidases, which should increase com protein digestibility and absorption during metabolism, providing higher feed efficiency and yields. As disclosed herein, this microbial incubation process provides a valuable, sustainable aquacukure feed that is less expensive per unit of protein than SBM, SPC, and fish meal.. j W)94J As disclosed, the instant microbes may metabolize the individual carbohydrates in DDGS, producing both ceil mass (protein) and a microbial gum. Various strains of these microbes also enhance fiber deconsiractkm. The microbes of the present invention may also convert soy and cora proteins into more digestible peptides and amino acids. In embodiments, the following actions in may be performed; 1) determining the efficiency of using select microbes of the present disclosure to convert preireated DDGS and the like, yielding a protein concentrate (PC) with a protein concentration of between about 45% and 55% or at least about 50%. and 2) assessing the effectiveness of PC in .replacing fish meal, hi embodiments, optimizing DDGS preireatment and conversion conditions may be carried ou to improve the performance and robustness of the microbes, test the resultant grower feeds for a range of commercially important fishes, validate process costs and energy requirements, and complete steps for scale-up and commercialization . in embodiments, the PC of the present disclosure may be able to replace a t least 50% of fish meal, while providing increased growth rates and conversion efficiencies. Production costs should be less th n commercial soy protein concentrate (SPC) and substantially less than fish meal

[0095] After extrusion pretreatmem, cellulose-deconstructing enzymes may be evaluated to generate sugars, which microbes of the present disclosure may convert to protein and gum. in embodiments, sequential omission of these enzymes and evaluation of co-cultiiring with ceiinloiytie microbes may be used. Ethanol may be evaluated to precipitate the gum and improve centrifugal .recovery of the PC. After drying, the PC may be incorporated into practical diet formulations. I» embodiments, test grower diets may be formulated (with mineral and vitamin premkes) and comparisons to a fish-meal control and commercial SPC (SPC is distinctly different from Soybean meal, as it contains traces of oiigopolysaceharides and antigenic

substances glycinin and b-conglyeinm) diets in feeding trials with a commercially important fish, e.g., yellow perch or rainbow trout, may be performed. Performance (e.g., growth, feed conversion, protein efficiency), viscera characteristics, and intestinal histology may be examined to assess fish responses.

[0096] in other embodiments, optimizing the PC/iipid production process by determining optimum pretreatraem and conversion conditions while minimizing process inputs, improving the performance and robustness of the microbe, testing the resultant grower feeds for a range of commercially important fishes, validating process costs and energy requirements, and completing initial steps for scale-up and commercialization may be carried out,

(0097] i the past few years, a handful of facilities have installed a dry mill capability that removes corn hulls and germ prior to the ethanol production process. This dry fractionation process yields a DDGS with up to 42% protein (hereafter referred to as dtyfrac DDGS). In embodiments, conventional and dryfrae DDGS under conditions previously determined to rapidly generate a sufficient amoun of high protein DDGS (HP-DDGS) for use in perch feeding trials may be compared. In embodiments, careful monitoring of the performance of this conversion (via chemical composition changes) is carried out and parameters with the greatest impact on HP-DDGS quality identified. In some embodiments, low oil DDGS may be used as a substrate for conversion, where such low oil DDGS has a higher protein level than conventional DDGS. In a related aspect, low oil DDGS increase growth rates of A. pulhiiam compared to conventional DDGS. [Θ098] Severn! groups are evaluating partial replacement of fish-meal with plant deri ved proteins, such as soybean meal and DDGS. However, the lower protein content, .inadequate amino acid balance, and presence of ani -nutritional factors have limited the replacement levels to 20-40%. Preliminar growth tna!s indicate that no current DDGS or SPC-based diets provide performance similar to fish-meal control diets. Several deficiencies have been identified among commercially produced DDGS, principally in protein and amino acid composition, which impart variability in growth performance and fish composition. However, HP-DDGS diets as disclosed herein containing nutritional supplements (formulated to meet or exceed all requirements) have provided growth results that are similar to or exceed fish-meal controls. Thus, the processes as disclosed herein and products developed therefrom provide a higher quality HP-DDGS (relative to nutritional requirements) and support growth performance equivalent to or better than diets containing fish meal.

[0099] Fish that can be fed the fish, feed composition of the present disclosure include, but are not limited to, Siberian sturgeon. Sterlet sturgeon, Starry sturgeon, White sturgeon, Arapaima, Japanese eel, American eel. Short-finned eel, Long-finned eel, European eel, Chanos chanos, MOkfish, Bluegill sunfisb. Green sunfish, White erappie, Black erappie. Asp, Catla, Goldfish, Crucian carp. Mud carp, Mriga! carp, Grass carp. Common carp. Silver carp, Bighead carp, Orangefin labeo, Roho labeo, Haven's carp, Wuchang bream, Black carp. Golden shiner, Nilem carp. White amur bream. Thai silver barb, Java, Roach, Tench, Pond loach, Bocachico, Dorada, Caciiama , Cacbania Bianca, Paco, Black bullhead, Channel catfish, Bagrid catfish, Blue catfish, Wels catfish, Pangasius (Swai, Tra, Basa) catfish, Striped catfish, Mudfish, Philippine catfish. Hong Kong catfish, North African catfish, Bighead catfish, Samoa, South American catfish, Aiipa, Northern pike, Ayu sweeifish, Vendace, Whitefisb, Pink salmon, Chum salmon, Coho salmon, Masu salmon. Rainbow trout, Soc ' keye salmon, Chinook salmon, Atlantic salmon, Sea trout, Arctic char. Brook trout, Lake trout, Atlantic cod, Pejerrey, Lai, Common snook,

Barramundi Asian sea bass, Nile perch, Murray cod, Golden perch. Striped bass. White bass, European seabass. Hong Kong grouper, Areoiate grouper. Greasy grouper. Spotted coralgrouper, Silver perch, White perch, Jade perch, Largemouth bass, Smallmooth bass, European perch.. Zander (Pike-perch), Yellow Perch, Sauger, Walleye, Bluefish, Greater amberjack, Japanese amberjack, Snubnose pompano, Florida pompano, Palometa pompano, Japanese jack mackerel, Cobia, Mangrove red snapper, Yello tail snapper, Dark seabream. White seabream. Crimson seabream, Red seabream. Red porgy, Goidlined seabream, Gilthead seabream, Red dram. Green terror, Blackbe.lt cichlid, Jaguar guapote, Mexican raojarra, Pearlspot,. Three spotted tilapia. Blue tilapia, Longfiii tilapia, Mozambique tilapia, Nile tilapia, Tilapia, Wami tilapia, Blackchin tilapia. Redbreast tilapia, Redhelly tilapia, Golden grey mullet, Largescale muilet, Gold-spot mullet, Thinlip grey mullet. Leaping mullet, Tade muliet, Flathead grey mullet. White mullet, Lebranche mullet, Paci fic fat sleeper, Marble goby, White-spotted spinefoot, Goldlined spinefoot. Marbled spinefoot. Southern biueiiii tuna. Northern bluefin tuna. Climbing perch, Snakeskm gourami, Kissing gourami, Giant gourami, Snakehead, Indonesian snakehead, Spotted snakehead. Striped snakehead, Turbot, Bastard halibut (Japanese flounder). Summer Flounder, Southern flounder., Winter flounder, Atlantic Halibut, Greenback flounder, Common sole, and combinations thereof

ΙΘ010Θ1 It xvil be appreciated by the skilled person that the fish feed composition of the present disclosure may be used as a convenient carrier for pharmaceutically active substances. jOO!OlJ The fish feed composition according to present disclosure may be provided as a liquid, pourable emulsion, or in the form of a paste, or in. a dry form, for example as an extrudate, granulate, a powder, or as flakes. When the fish feed composition is provided as an emulsion, a lipid-in-water emulsion, it is may be in a relatively concentrated form. Such a concentrated emulsion form may also be referred to as a pxe-erau!sion as it may be diluted in one or more steps in an aqueous medium to provide the final, enrichment medium for the organisms.

{00202] hi embodiments, cellulosic-containirig starting material for the microbial- based process as disclosed Is com. Corn is about two-thirds starch, which is converted during a fermentation and distilling process in to ethano! and carbon dioxide. The remaining nutrients or fermentation -products may result in condensed distiller's solubles or distiller's grains such as DDGS, which can be used in feed products. In general, the process involves an initial preparation step of dry milling or grinding of the com. The processed corn is then subject to hydrolysis and enzymes added to break down the principal starch component in a sacchariikaiion step. The following step of fermen ation is allowed to proceed upon addition, of a microorganism (e.g., yeast) provided in. accordance with, an embodiment of the disclosure to produce gaseous products such as carbon dioxide. The fermentation is conducted for the production of ethanol which may be distilled from the fermentation broth. The remainder of the fermentation medium may then be dried to produce fermentation products including DDGS. This step usually includes solid/liquid separation process by centrifugation wherein a solid phase component may be collected. Other methods including filtration and spra dry techniques may be employed to effect such separation. The liquid phase components ma be subjected further afterwards to an evaporation step thai can concentrate soluble eoproduets, such as sugars, glycerol and ammo acids, mto a material called syrup or condensed com solubles (CCS). The CCS ma then be recombaied with the solid phase component to be dried as incubation products (DDGS). It shall be understood that the subject compositions and may be applied to new or already existing ethanol plants based on dry milling to provide an integrated ethanol production process that also generates fermentation products with increased value.

[001031 in embodiments, incubation products produced according to the present disclosure have a higher commercial value than the conventional fermentation products. For example, the incubation products may include enhanced dried solids with improved amino acid and

micronutrient content. A "golden colored" product can be thus provided which generally indicates higher amino acid digestibility compared to darker colored SP, For example, a light- colored SP may be produced with an increased lysine concentration in accordance with embodiments herein compared to relatively darker colored products with generally less nutritional value. The color of the products may be an important factor or indicator in the assessing the quality and nutrient digestibility of the fermentation products or SP. Color is used as a indicator of exposure to excess heat during drying causing caranielization and Maillard reactions of the free amino groups and sugars, reducin the quality of some amino acids. j 00104} The basic steps in a dry mill or grind ethanol manufacturing process may be described as follows: milling or grinding of corn or other grain product, saccharification, fermentation, and distillation. For example, selected whole corn kernels may be milled or ground with typicall either hammer mills or roller mills. The particle size can influence cooking hydration and subsequent enzymatic conversion. The milled or ground corn can be Chen mixed with water to make a mash that is cooked and cooled, it may he useful t include enzymes during the initial steps of this conversion, to decrease the viscosity of the gelatinized, starch. The mixture may be then transferred to saccharification reactors, maintained at selected temperatures such as 140° F, where the starch is converted by addition of saccharifying enzymes to fermentable sugars such as glucose o mal ose. The converted mash can be cooled to destred temperatures such as 84° F, and fed to fermentation reactors where fermentable sugars are converted to carbon dioxide by the yse of selected strains of microbes provided in accordance with die disclosure that results in more nutritional fermentation products compared to more traditional ingredients such as Saceharomyces yeasts. The resulting product may be flashed to separate oat carbon dioxide and the resulting liquid m be fed to a recovery system consisting of distillation, columns and a stripping column. The ethanol stream may be directed to a molecular sieve where remaining water is removed using adsorption technology. Purified ethanol, deoatured with a small amount of gasoline, may produce fuel grade ethanol. Another product may be produced by farther purifymg the initial distillate ethanol to remove impurities, resulting in about 99.95% ethanol for non-fuel uses.

[00105} The whole stiliage may be withdrawn from the bottom of the distillation unit and centtifuged to produce distiller's wet grains (DWG) and thin stiliage (liquids). The DWG may leave the centrifuge at 55-65% moisture, and may either be sold wet as cattle feed or dried as enhanced fermentation products provided in accordance with die disclosure. These products include an enhanced end product that may he referred to herein as distiller's dried grains (DDG), Using an evaporator, the thin stiliage (liquid) may be concentrated to form distiller's solubles, which may be added back to and combined with a distiller's grains process stream and. dried. This combined product in accordance with embodiments of the disclosure may be marketed as an enhanced fermentation product having increased, amino acid and micronirtrient content. It shall be understood that various concepts of the disclosure may be applied to other fermentation processes known in the field oilier than those illustrated herein.

(001063 Another aspect of the present invention is directed towards complete fish meal compositions with an enhanced concentration of nutrients which includes microorganistns characterized by an enhanced concentration of nutrients such as. hot not limited to, fats, fatty acids, lipids such as phospholipid, vitamins, essential amino acids, peptides, proteins, carbohydrates, sterols, enzymes, and trace minerals such as, iron, copper, zinc, manganese, cobalt, iodine, selenium, molybdenum, nickel,, fluorine, vanadium, tin, silicon, and combinations thereof.

[001071 i n an incubation process of the present disclosure, a carbon source may be hydro!yzed to its component sugars by microorganisms to produce alcohol and other gaseous products. Gaseous product includes carbon dioxide and alcohol includes ethanol The incubation products obtained after the incubation process are typically of higher commercial value. In embodiments, the incubation products contain microorganisms that have enhanced nutrient content than those products deficient in the microorganisms. The microorganisms may be present in an incubation system, the incubation broth and/or incubation biomass. The inclination broth and/or biomass may be dried (e.g. , spray-dried), to produce the incubation products with an enhanced content of the nutritional contents. j OOlftS} For example, the spent dried solids recovered following the incubation process are enhanced in accordance with the disclosure. These incubation products are generally non-toxic, biodegradable, readily available, inexpensive, and rich in nutrients. The choice of microorganism and the incubation conditions are important to produce a low toxicity or non-toxic incubation product for use as a feed or nutritional supplement. While glucose is the major sugar produced from the hydrolysis of the starch from grains, it is not the only sugar produced in carbohydrates generally. Unlike the SPC or DDG produced from the traditional dry mill ethanol production process, which contains a large amount of non-starch carbohydrates (e.g., as much as 35% percent of cellulose and arabmoxylans-measured as neutral detergent fiber, by dry weight), the subject nutrient enriched incubation products produced by enzymatic hydrolysis of the non-starch carbohydrates are more palatable and digestible to the non-ruminani.

[Θ0Ι09] The nutrient enriched incubation product of this disclosure may have a nutrient content of from at least about 1% to about 95% b weight. The nutrient content is preferably in the range of at least about ί 0%-20%, 20%-30%, 30%- 0%, 40%-50%, S0%-60% ? 60%-70% 5 and 70%- 80% by weight. The available nutrient content may depend upon the animal to which it is fed. and the context of the remainder of the diet, and stage in the animal life cycle. For instance, beef cattle require less histidine than lactating cows. Selection of suitable nutrient content for feeding animals is well known to those skilled in the art.

[00110J The incubation products may be prepared as a spray-dried biomass product.

Optionally, the biomass may be separated by known methods, such as centrifugation, filtration, separation, decanting, a combination of separation and decanting, ultrafiltration or

microftltraiion. The biomass incubation products may be further treated to facilitate rumen, bypass, in embodiments, the biomass product may be separated from the incuba tion medium, spray-dried, and optionally treated io modulate rumen bypass, and added to feed as a nutritional source, hi addition to producing nutritionally enriched incubation products in an incubation process containing microorganisms, the nutritionally enriched incubation products may also be produced in transgenic plant systems. Methods for producing transgenic plant systems are known in the art. Alternatively, where the microorganism host excretes the nutritional contents, the uirtrUional!y-enriched broth may be separated, from the biomass produced by the incubation, and the clarified broth may be used as an animal feed ingredient, e.g., either in liquid .form or in spray dried form.

[0011 1} The incubation products obtained after the incubation process using microorganisms may be used as an animal feed or as food supplement for humans. The incubation product includes ai least one ingredient, that has an enhanced nutritional content thai is derived from a non-animal source (e.g., a bacteria, yeast, and/or plant), in particular, the Incubation products are rich in at least one or more of fats, fatty acids, lipids such as phospholipid, vitamins, essential amino acids, peptides, proteins, carbohydrates, sterols, enzymes, and trace minerals such as, iron, copper, zinc, manganese, cobalt, iodine, selenium, molybdenum, nickel, fluorine, vanadium, tin and silicon. In embodiments, the peptides contain at least one essential amino acid, in other embodiments, the essential amino acids are encapsulated inside subject modified

microorganism used in an incubation reaction. In embodiments, the essential amino acids are contained in heterologous polypeptides expressed, by the microorganism . Where desired, the heterologous polypeptides are expressed and stored in the inclusion bodies in a suitable microorganism (e.g., fungi).

[00132 | In embodiments, the incubation products have a high nutritional, content. As a result, a higher percentage of the incubation products may be used in a complete animal feed, in embodiments, the feed composition comprises at least about 15% of incubation product by weight. In a complete feed, or diet, this material will be fed with other materials. Depending upon the nutritional content of the other materials, and/or the nutritional requirements of the animal to which the feed is provided, the modified incubation products may range from 15% of the feed to 100% of the feed, in embodiments, the subjec t incubation products may provide lower percentage blending due to high nutrient content, in other embodiments, the subject incubation products may provide very high fraction feeding, e.g. over 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%, ai least about 70%, or at least about 75% of the subject incubation products. Common ly, the feed composition comprises at least about 20% of incubation product by weight. More commonly, the feed composition comprises at least about 1 5-25%, 25-20%, 20-25%, 30%-40%, 40%-50%, 50%-60%, or 60%-7O% by weight of incubation product. Where desired, the subjec t incubation products may be used as a sole source of feed.

[00 13| The complete fish meal compositions may have enhanced amino acid content with regard to one or more essential amino acids for a variety of purposes, e.g., for weight increase and overall improvement of the animal's health. The complete fish meal compositions may have enhanced amino acid content because of die presence of free amino acids and/or the presence of proteins or peptides including an essential amino acid, in the incubation products. Essential amino acids may include histidme, lysine, methionine, phenylalanine, threonine, taurine (sulfonic acid), isoteucrae, and/or tryptophan, which may be present in the complete animal feed as a free amino acid or as part, of a protein or peptide that, is rich. in. the selected: amino acid. At least one essential amino acid-rich peptide or protein ma have at least 1% essential amino acid residues per total amino acid residues in the peptide or protein, at least 5% essential amino acid residues per total amino acid residues in the peptide or protein, or at least 10% essential amino acid residues per total, amino acid residues in the protein. By feeding a diet balanced, in nutrients to animals, maximum use is made of the nutritional content, requiring less feed to achieve

comparable rates of growth, milk production, or a reduction in the nutrients present in the excreta reducing bioburden of the wastes. 00114| A complete fish meal composition with an enhanced content of an essential amino acid, may have an essential amino acid content (including free essential amino acid and essential amino acid present in a protein or peptide) of at least 2,0 wi % relative to the weight of the crude protein and total amino acid content, and more suitably at least 5.0 wt % relative to the weight of the crude protein, arid, total amino acid content. The complete fish meal composition includes other nutrients derived from microorganisms including but not limited to, fats, fatty acids, lipids such as phospholipid, vitamins, carbohydr tes, sterols, enzymes, and trace minerals.

[001.15} The complete fish meal composition may include complete feed form composition, concentrate form composition, blender form composition, and base form composition, .if the composition is in the form of a complete feed, the percent nutrient level, where the nutrients are obtained from the microorganism in an incubation product, which may be about 10 to about 25 percent, more suitably about 14 t about 24 percent; whereas, if the composition is in the form of a concentrate, the nutrient level may be about 30 to about 50 percent, more suitably about 32 to about 48 percent. If the composition is in the form of a blender, the nutrient level, in the composition may be about 20 to about 30 percent, more suitably about 24 to aboui 26 percent; and if the composition is in the form of a base mix, the nutrient level, in the composition may be about 55 to about 65 perceiii. Unless otherwise stated herein, percentages are stated on a weight percent basis, if the PC is high in a single nutrient, e.g., Lys, it will be used as a supplement at a low rate; if it is balanced in amino acids and Vitamins, e.g., vitamin A and E, it will be a more complete feed and will be fed at a higher rate and supplemented with a low protein, low nutrient feed stock, like corn stover. 0116} The fish meat composition may include a peptide or a crude protein fraction present in an incubation product having an essential amino acid content of at least about 2%. In

embodiments, a peptide or crude protein fraction may have an essential amino acid con tent 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 1 0% essential amino acids. Commonly, the fish meal, composition may include a peptide or crude protein fraction present in an incubation product having an essential amino acid content of up to about 10%. More commonly, the fish meal composition may include a peptide or a crude protein fraction present in an incubation product having an essential amino acid content of about 2-10%, 3.0-8.0%, or 4.0-6.0%. j 00117} he fish meal composition may include a peptide or a erode protein fraction present in an 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 abou 20%, at least about 30%, ai least about 40%, and in embodiments, at least about 50%, Typically, the fish meal composition may include the peptide or crude protein fraction having a lysine content of up to about 1 %. Where desired, the fish meal composition ma include the peptide or a crude protein fraction having a lysine content of about 2-10%, 3.0-8.0% ; or 4.0-6.0%. jOOI 18} h e fish meal composition may include nutrients in the incubation product from about 1 g/Kg dry solids to 900 g/Kg dry solids. In embodiments, the nutrients in a fish meal

composition may be present to at least about 2 g/Kg dry solids, 5 g/Kg dry solids, 10 g/Kg dry solids, 50 g/Kg dry solids, .100 g/Kg dry solids, 200 g/Kg dry solids, and about 300 g/Kg dry solids, in embodiments, the nutrients may be present to at least about 400 g/Kg dry solids, at least about 500 g/Kg dr solids, at least about 600 g Kg dry solids, at least about 700 g Kg dry solids, at least about 800 g/Kg dry solids and/or at least about 900 g/Kg dry solids.

(Θ01.1 | The fish meal composition may include an essential amino acid or a peptide containing at leas one essential amino acid present in a incubation product having a content of about J g/Kg dry solids to 900 g/Kg dry solids, in embodiments, the essential amino acid or a peptide containing at least one essential amino acid in. a fish meal composition may be present to at least about 2 g/Kg dry solids, 5 g/Kg dry solids, 10 g/Kg dry solids, 50 g/Kg dry solids, 100 g Kg dry solids, 200 g/Kg dry solids, and about 300 g/Kg dry solids. In embodiments, the essential amino acid or a peptide containing at least one essential amino acid may be present to at least about 400 g/Kg dry solids, at leasi about 500 g Kg dry solids, at least about 600 g/Kg dry solids, at least about 700 g/Kg dry solids, at least about 800 g/Kg dry solids and/or at least about 900 g/Kg dry solids.

[00120] The complete fish meal composition may contain a nutrient enriched incubation product in the form of a biomass formed doting incubation and at least one additional nutrient component. In another example, the fish meal composition contains a nutrient enriched incubation product that is dissolved and suspended from an. incubation broth formed during incubation and at least one additional nutrient component, in a further embodiment, the fish meal composition has a crude protein fraction that includes at least one essential amino acid-rich protein. The fish meal composition may be formulated, to deliver an improved balance of essential amino acids.

[00121] For compositions comprising DDGS, the complete composition form may contain one or more ingredients such as wheat middlings ("wheat midds"), corn, soybean meal, corn gluten meal, distiller's grains or distiller's grains with solubles, salt, macro-minerals, trace minerals and vitamins. Other potential ingredients may commonly include, but not be limited to sunflower meal, mail sprouts and soybean hulls. The blender form composition may contain wheat middlings, com gluten meal, distiller's grains or distiller's grains with solubles, salt, macro- minerals, trace minerals and vitamins. Alternative ingredients would commonly include, but not be limited to, corn, soybean meal, sunflower meal cottonseed meal, malt sprouts and soybean hulls. The base form composition may contain wheat middlings, com gluten meal, and distiller's grains or distiller's grains with solubles. Alternative ingredients would commonly include, but are not limited to, soybean meal, sunflower meal, mail sprouts, macro-minerals, trace minerals and vitamins,

|00122] Highly unsaturated fatty acids (HUFAs) in microorganisms, when exposed to oxidizing conditions may be converted to less desirable unsaturated fatty acids or to saturated fatty acids. However, saturation of omega- 3 HUFAs may be reduced or prevented by the introduction of synthetic antioxidants or naiurally-occurrmg antioxidants, such as beia-carotene, vitamin E and vitamin C, into the feed. Synthetic antioxidants, s«cb as BHT, BHA, TBHQ or ethoxyquin, or natural antioxidants such as tocopherols, may be incorporated into the food or feed products by adding them to the products, or they may be incorporated by in situ production in a suitable organism. The amount of antioxidants incorporated in this manner depends, for example, on subsequent use requirements, such as product formulation, packaging methods, and desired shelf life.

(Θ0Ι23] incubation products or complete fish meal containing the incubation products of the present, disclosure, may also be utilized as a nutritional supplement for human consumption if the process begins with human grade input materials, and human food quality standards are observed through out the process. Incubation product or the complete feed as disclosed herein is high in nutritional content. Nutrients such as, protein and fiber are associated ith health diets. Recipes may be developed to utilize incubation product or the complete feed, of the disclosure in foods such as cereal, crackers, pies, cookies, calces, pizza crust, summer sausage, meat balls, shakes, and in any forms of edible food. Another choice may be to develop the incubation product or the complete feed of the disclosure into snacks or a snack bar, similar to a granoia bar that could be easily eaten, convenient to distribute. A snack bar may include protein, fiber, germ, vitamins, minerals, from the grain, as well as nutraceuticals such as glucosamine, HUFAs, or co-factors, such as Vitamin Q-i . θ§124| The fish meal comprising the subject incubation products may be further supplemented with flavors. The choice of a particular fla vor will depend on the animal to which the feed is provided. The flavors and aromas, both natural and artificial, may be used in making feeds more acceptable and palatable. These supplementations may b!end well with all ingredients and may be available as a liquid or dry product, form. Suitable flavors, attractants, and aromas to be supplemented in the animal feeds include but not limited to fish pheromones, fenugreek, banana, cherry, rosemary, cumin, carrot, peppermint oregauo, vanilla, anise, plus rum, maple, caramel citrus oils, ethyl buiytaie, menthol apple, cinnamon, any natural or artificial combinations thereof. The favors and aromas may be interchanged between different animals. Similarly, a variety of fruit flavors, artificial or natural may be added to food supplements comprising tire subject incubation products for human consumption,

[00125] The shelf-life of the incubation product or the complete feed of the present disclosure may typically be longer than the shelf life of an incubation, product that is deficient in ihe microorganism. The shelf-life may depend on factors such as, the moisture content of the product how much air can flow through the feed mass, the environmental conditions and the use of preservatives. A preservat e may be added to the complete .feed to increase the shelf life to weeks and months. Other methods to increase shelf fife include management similar to silage management such as mixing with other feeds and packing, covering with plastic or bagging. Cool conditions ; , preservatives and excluding air from the feed mass all extend shelf life of wet co- products. The complete feed can be stored in bunkers or silo bags. Drying the wet incubation product or coropleie feed may also increase the product's shelf life and improve consistency and quality.

[00126) The complete feed of the present di sclosure may be stored for long periods of time. The shelf life ma be extended by ensiling, adding preservatives such as organic acids, or blending with other feeds such as soy hulls. Commodity bins or bulk storage sheds may be used for storing the complete feeds,

[00127] As used herein, "room temperature" is about 5" C under standard pressure.

[00128] The following examples are illustrative and are not intended to limit the scope of the disclosed subject matter,

[00129] The following examples are intended to illustrate but not limit the invention.

Example 1. DOGS eon version with S. limacinum for EPA and, protein production

[00130] Wet distiller's grains is converted into a DHA and protein enriched product as follows; pretreatme invol ves extrusion at 25% moisture content, 100 °C, and screw speed of 200 rpm. using a single screw extruder. These conditions result in 36% greater sugar release due to fiber disruption. The extruded .material is then mixed w th water so. benchtop o pilot scale bioreactors to achieve a solid loading rate of 10-15%, the pH is adjusted to 5.5, and the slurry is auto laved or pasteurized. After cooling, a cocktail of ovozyrne enzymes (6% Cellic Ctek2 per gm gkican and 0.3% Cellic Htek2 per gm total solids) is added for 24 h saccharification at 50 ,:> C and 1 0 rpm.

(00.131} The temperature is then reduced to 25 °C, the pH is adjusted to 7.0, and the slurry is inoculated with 2% (v/v) of a 24 h culture of & limacin m. The slurry is agitated and aerated under optimal conditions until sugar utilization ceases (about 96-! 20h). Alter 48 h CCS is added in a fed-batch mode to provide additional glycerol, as well as cause a nitrogen limitation to trigger additional. DHA production. During incubation, samples are removed at 6-12 h intervals and assayed for; !} carbohydrates and organic solvents using a Waters HPLC system, 2} microbial populations via plate or hemocytorneter counts, and 3) DHA, EP A, and protein levels via AOAC methods.

.Example 2. CCS.c nygmOT wH^

(00132} CCS is mixed with water to achieve a solid loading rate of 15-20% in benchtop or pilot scale bioreactors and the pH is adjusted to ~7. Following autoclaving or pasteurization the temperature is set at 25 °C and the slurry is inoculated with 2% (v/v) of a 24 h culture of Pyth tm irregulare. The slurry is agitated and aerated under optima! conditions until maximum. EPA levels are achieved (about -!20h). After 48 h, additional CCS is added in a fed-batch mode to provide glycerol and cause nitrogen limitation to trigger further EPA production. This also maximizes EPA and protein titers to minimize product recovery costs. During incubation, samples are be removed and analyzed as described above.

Example 3. Slurry blending and solids recovery

(00133) The converted distiller's grains and CCS slurries are blended to achieve desired levels of DHA, EPA, and protein, and the omega-3 DDGS is recovered and dried. Following analysis, the solids are used to manufacture omega-3 DDGS based feeds that are tested in fish feeding trials. Supernatant is evaluated for re-use at the start of the process. [00.134} The omega-3 DDGS compositions are analyzed for niriritional competencies in view of yellow perch .requirement. Samples are subjected to chemical analyses: proximate analysis, insoluble carbohydrates, amino acids, and fatty acids. This ensures that nutritional benchmarks are satisfied in treatment diets. Anti-nutritional concentrations (e.g., phytate) determined in input products are compared with omega-3 DDGS and. provide a basis for future process modifications. Yellow perch feeding trials are performed under IACUC approval i 1-070A,

[00135} Initially, a reference diet formulation is blended with each omega-3 DDGS product at a 70:30 ratio, each diet containing an. inert marker. A minimum of 25 fish per test diet are fed to satiation twice daily for 10 days prior to the first fecal collection. Additional fecal collection is made every 10 days alter initiation of feeding until adequate sample sizes are obtained. Fecal samples are collected from anesthetized fish (tricam methanesul bnate) 12-16 h post-feeding by abdominal palpation strippin of distal digesta and then flash frozen. Sampled fish are allowed to recover in an oxygenated tank. Individual feca! collections from each tank are pooled to ensure adequate dry sample in each replicate for analysis, if needed, diets are rotated among the tanks during the digestibility trials so that three replicate fecal sainpies for each diet are obtained from fish in different tanks over time. Apparent digestibility coefficients (ADC) are determined by standard methods for each .nutrient in the test diets.

[ 0136) The feeding perfomiance trial uses complete diets formulated in accordance with known nutrient requirements for yellow perch (e.g., 45% protein, 9% lipid). Lot ingredient analyses are used in diet formulation to ensure that nutritional benchmarks are satisfied and to allow diet blends to be prepared on an isonitrogenous and iso!ipkiic basis for direct performance comparisons between ' fish meal/oil control, DDGS and. omega-3 DDGS treatment diets... DDGS and omega-3 DDGS amino acid (e.g., lysine) and fatty acid (e.g., HUFA n.-3-s) concentrations are examined for deficiencies in diet formulations and. supplemented (e.g., flaxseed and fish, oils) for comparison to ionn.uiat.ions prepared wiihoot supplemen ts (neo-sy.nihesis potential). (Follow-up factorial studies incorporating incremental omega-3 DDGS inclusions are done with refined products). Mineral and vitamin premises designed for plant-based diets are included at fixed minimum levels in ail diets, including control, to ensure that micro-nutrient requirements are met across all diets. Macro-minerals are similarly supplemented as necessary. Pellets for feeding trials are produced using a single scre extruder (Extru-Tech Model 325, Sabetna, S) with optimized extruder conditions (temperature, moisture, and speed). [001.37} Feed extrudates are analyzed according to approved procedures for dry matter at 105 ' -'C for 3 h, crude protein using a I.ECO combustion analyzer (Method 990.03), ether extract (i.e., crude fat) (Method 920.39A), crude fiber (Method 978.10), and ash (Method 942.05). Amino acids are analyzed by HPLC with post column mnhydrin derivaiization according to Method 982.30 E (a, b, c). Extracted lipids are esterified by boron trifluor e reagent (Method 969.33) and then methyl esters of fatty acids are separated fay capillary GLC (Method 996.06). The gross energy of the diets was determined by bomb calorimetry.

[00138} Replication of four experimental units per dietary treatment are used in 105 d feeding trials (15-20 g fish; 20-25 fish/tank. Type Ϊ error probability < 0,05). Trials are completed in 110- L circular tanks connected in parallel to a closed-loop recirculation system, (solids sump, bioreactor, filters [100 pm bag, carbon and ultra-violet], and a centrifugal pump). Inline heat pumps are used to maintain optimal temperature. Water quality (e.g., dissolved oxygen, pH. temperature, ammonia and nitrite) is monitored daily. Flow rates (2 L m.in) are monitored with fixed mouometers and the dissolved oxygen concentration is maintained at saturation (rotary blower and diffusers).

[00139} Experimental diets are delivered twice daily. Growth performance is determined by total tank mass measurements at four week intervals; feed ra ons are adjusted in accordance with gains to allow satiation feeding and to reduce waste streams. Consumption is assessed biweekly from collections of uneaten pellets from individual tanks. Uneaten feed pellets are counted and dried to a constant weight to estimate conversion efficiency. Feed digestibilities (ADC protein and energy) are determined from fecal material, manually stripped during the midpoint of each experiment or via necropsy from the lower intestinal tract at the conclusion.- Survival (%), weight, gain (g), specific growth rate, health indices, feed conversion, and protein and energy

digestibilities are compared among treatments.

[001.40} Measurements of whole body, total viscera, visceral fat, gonads. livers, and .fillet weights are done a the end of the trial. Samples of fillets, visceral fat, and li ver are pooled (n=5) to yield one sample per tank and stored in polyethylene bags at -20 °C. Fillet samples are analyzed for proximate composition, while fatty acid profiles are determined for fillets, livers, and visceral fat using methods described earlier (and. applied to duplicates). Color of fillets and livers are determined using a spectrophotometer (LabScan XE, Hunter Associates Laboratories, Inc., Reston, VA). where: £ refers to brightness/darkness, a refers to redness/greenness, and h refers to yeilowness bfueness. Analysis of performance parameters and compositions among treatments are analyzed as a completely randomized design using individual tanks as an experimental unit. Analyses are performed using SAS v. 9.3 (Cary, NC). Post-hoc Duncan's multiple range tests compare treatment means with significance declared at P< 0.05.

[00141] A preliminary mass balance is performed on the conversion process, and product yield is calculated. Process energy requirements are calculated based on a full scale model.. This information is used to generate an. economic model to project the processing costs.

Example 4: Effect of different concentrations of MgSQ4.7H?.Q on DHA production by S.

Umactmm

[001421 Various concentrations of gS0 4 .7¾0 (i.e., 0%, 0.061%, 0. 122%, 0.244%, 0.305%, 0.488%) were added to 5% DDGS (2.5g DDGS in 47.5 ml distilled water + 2.5 raj of inoculum). The pH of the DDGS slurry was adjusted to 7 and sterilized prior to inoculation with 5% microaJgae. The inoculated mixture was incubated at 25 "C for 5 days. After the incubation period, the material was freexe-dried and nulled. The milled material was then assayed for protein and DHA content. Based on preliminary studies, optimal M S0 .7¾0 concentration should increase DHA values from 0.24% to between 1.5 to 3%, and protein content from 36% to between about > 50 to about 55%.

Example 5; Plackett-Burman design for different minerals and micro-nutrients supplemented in DDGS fermented by £. Limacmum

[00143] In order to further optimize DHA production by ' . ! ^ . · different minerals and micro-nutrients were used to supplement DDGS. The minerals and micro-nutrients investigated are listed in Table 2.

Table 2. Different minerals and micro-nutrients supplemented to DDGS fermented by

Schiza hytriu

(69] 441 Design Expert software was used to generate the PJackett-Burman design for this trial.

The different runs from the Plackett-Burman design ate listed in the table in FIG, 4,

(091451 As above for the MgSO,}.7HjO example, the substrate was 5% DDGS. For the 20 different runs, DDGS was supplemented with various concentrations of nutrients, A to , added to the substrate, including varying the pH ( ). The substrates were autoclaved at 121 °C for 20 mill prior to inoculation. After cooling down the substrate, 5% Schizochytrium was used to inoculate the substrates, where subsequently, the supplemented DDGS samples were incubated at room temperature (25 °C) for five (5) days. After incubation, the samples were freexe-dried and milled for DMA analysis by GC. Based on preliminary studies* optimal mineral and micro- nutrient concentrations should increase DBA values from 0.24% to between 1 .5 to 3%.

(001461 All of the references cited herein are incorporated by reference i their entireties.

[U01471 From the above discussion, one skilled in the art can ascertain the essentia! characteristics of the invention, and without departing from the spirit and scope thereof, can make various changes and. modifications of the embodiments to adapt to variou uses 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 foregoing description. Such modifications are also intended to fall within the scope of the appended claims.