WO2006127836A2

WO2006127836A2 - Animal feed compositions with enhanced amino acid content

Info

Publication number: WO2006127836A2
Application number: PCT/US2006/020126
Authority: WO
Inventors: Mervyn Desouza; Michael Messman
Original assignee: Can Technologies, Inc.; Cargill, Incorporated
Priority date: 2005-05-26
Filing date: 2006-05-24
Publication date: 2006-11-30
Also published as: WO2006127836A3

Abstract

Disclosed are compositions and methods for supplementing ruminant feeds. The compositions include at least one ingredient that has an enhanced leucine and/or threonine content. This ingredient commonly is rumen-protected. The methods include feeding ruminants feed compositions that include the ingredient to improve milk production.

Description

ANIMAL FEED COMPOSITIONS WITH ENHANCED AMINO ACID CONTENT

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional application no. 60/685,017, filed on May 26, 2005, the disclosures of which is incorporated by reference herein in its entirety.

BACKGROUND

[0002] All animals require amino acids (AA), the building blocks of proteins necessary for optimal growth, reproduction, lactation, and maintenance. Amino acids absorbed in the cow's small intestine are derived from microbial protein and from dietary proteins that are undegraded in the rumen. Proteins digested in the small intestine must supply 10 essential amino acids (EAA), which cannot be manufactured by the cow, including arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. Ideally, the relative proportions of each of the EAA absorbed would exactly match the cow's requirements, because a shortage of one can limit the utilization of others.

[0003] Ruminants (cattle, sheep) complicate protein nutrition because they have pre-stomach chambers where digestion occurs. In the first two chambers, the rumen and the reticulum, a population of symbiotic bacteria and protozoa ferment the feeds and grow from non-protein nitrogen sources like ammonia or urea. These bacteria can digest fiber in plants enabling cattle to obtain energy from these feeds. They also synthesize protein from inexpensive byproducts. Microbial protein production is directly related to microbial growth, which is largely determined by the presence of carbohydrates such as starch, non-detergent fiber (NDF), sugars, and residual non- fiber carbohydrates (e.g., pectin and beta-glucans). The microbial population continuously washes out of the rumen to the true stomach (i.e., abomasum) where it is digested to supply amino acids to the cow. [0004] In addition to obtaining amino acids from microbial produced protein, ruminants also obtain amino acids from undegraded essential amino acids (UEAA) that pass from the rumen to the abomasum. Lactating ruminants excrete more of certain amino acids in milk than are consumed in the diet and appear at the small intestine of the cow. These amino acids that are in deficit are called limiting amino acids. Supplementation of limiting amino acids to the animal can improve milk production and milk component composition. Limiting amino acids may be provided in the form of UEAA.

SUMMARY

[0005] Compositions and methods directed generally to increasing milk production in dairy cattle and other ruminants are provided herein. Feeding ruminant animals for optimum production of animal products involves understanding amino acid, fatty acid, and carbohydrate nutrition. Compositions and methods of improving the nutrition of ruminant animals are provided herein, in particular amino acid nutrition. Also provided herein is a method to alleviate amino acid limitation and improve milk production and milk component composition of lactating ruminants by feeding ruminants a feedstuff that has an enhanced content of one or more limiting amino acids.

[0006] By feeding a dairy cow a particular feed composition which delivers an improved balance of the ten essential amino acids, the cow's milk production may be increased. In particular, the feed composition may have an enhanced content of one or more limiting amino acids, as determined by the cow's amino acid requirements for maintenance, growth, and milk production. Limiting amino acids may include leucine, threonine, histidine, lysine, methionine, and/or phenylalanine. The feed composition may be formulated to deliver an improved balance of essential amino acids post-ruminally.

[0007] The feed composition typically includes at least one ingredient that has an enhanced content of leucine, threonine, or both. The ingredient is typically derived from a non-animal source (e.g., a bacteria, yeast, and/or plant). For example, the composition may include an amino acid source which includes L-Leu, L-Thr, or both. The amino acid source may include fermentation constituents formed during fermentation of a leucine-producing microorganism, a threonine-producing microorganism, and/or a microorganism that produces both leucine and threonine. In another example, the feed composition includes a amino acid source which may include L-Leu, L-Thr, or both, and dissolved and suspended constituents from a fermentation broth formed during fermentation of a leucine-producing microorganism, a threonine-producing microorganism, and/or a microorganism that produces both leucine and threonine. In other embodiments, the feed composition may have a crude protein fraction which includes at least one leucine-rich protein and/or threonine-rich protein of non-animal origin, {i.e., an animal or non-animal leucine-rich protein and/or threonine-rich protein produced by bacteria, yeast, and/or plants). In addition, the feed composition may include an animal or non-animal leucine-rich protein and/or threonine-rich protein produced by recombinant bacteria, yeast, and/or plants, (e.g., by fermentation of recombinant bacteria). For example, the bacteria, yeast, and/or plants may be engineered to produce a leucine-rich protein and/or threonine-rich protein that is present in a feed supplement that is derived from an animal product. All of the described feed compositions commonly include at least one additional nutrient component.

[0008] The feed composition may include at least about 1 g/kg of the leucine source and/or threonine source. In some embodiments, the feed composition includes at least about 2 g/kg of the leucine source and/or threonine source. The feed composition may include up to about 10 g/kg of the leucine source and/or threonine source. [0009] As used herein, L-Leu includes leucine as a free amino acid and leucine salts {e.g., Leu(HCl)). Where amounts of L-Leu are recited herein, the amounts relate to leucine on a free amino acid basis. As used herein, L-Thr includes threonine as a free amino acid and threonine salts {e.g., Thr(HCl)). Where amounts of L-Thr are recited herein, the amounts relate to threonine on a free amino acid basis. [0010] As used herein, "a leucine-producing microorganism, a threonine-producing microorganism, and/or a microorganism that produces both leucine and threonine" may include a single microorganism that produces both leucine and threonine and mixtures of microorganisms that produce leucine and/or threonine. "A leucine- producing microorganism, a threonine-producing microorganism, and/or a microorganism that produces both leucine and threonine" may include a microorganism that produces amino acids in addition to leucine and/or threonine (e.g., histidine, lysine, methionine, and/or cysteine).

[0011] As used herein, "a leucine-rich protein and/or threonine-rich protein" may include a single protein that is leucine-rich and threonine rich and mixtures of proteins that are leucine-rich and/or threonine rich. A "leucine-rich protein and/or threonine- rich protein" may include a single protein that is rich in amino acids in addition to leucine and/or threonine, (e.g., histidine, lysine, methionine, and/or cysteine). [0012] The feed composition may include fermentation constituents formed during fermentation of a leucine-producing microorganism, a threonine-producing microorganism, and/or a microorganism that produces both leucine and threonine. As used herein, "fermentation constituents" may include any suitable constituent(s) from a fermentation broth. For example, fermentation constituents may include dissolved and/or suspended constituents from a fermentation broth. The suspended constituents may include undissolved soluble constituents (e.g., where the solution is supersaturated with one or more components) and/or insoluble materials present in the fermentation broth. The fermentation constituents may also include at least a portion of the biomass formed during a fermentation. The fermentation constituents may include substantially all of the dry solids present at the end of a fermentation (e.g., by spray drying a fermentation broth and the biomass produced by the fermentation) or may include a portion thereof. For example, the crude fermentation product from fermentation of a leucine-producing microorganism, a threonine-producing microorganism, and/or a microorganism that produces both leucine and threonine may be fractionated and/or partially purified to increase the leucine and/or threonine content of the material which may still contain fermentation constituents in addition to the leucine and/or threonine.

[0013] The feed composition may include a crude protein fraction having a leucine content of at least about 10.3 wt.% and/or a threonine content of at least about 6.2 wt.%. In suitable embodiments, the crude protein fraction may have a leucine content of at least about 11%, at least about 13%, at least about 15%, at least about 17%, and suitably at least about 19%; and/or the crude protein fraction may have a threonine content of at least about about 6%, at least about 8%, at least about 10%, at least about 12%, and suitably at least about 14%. The feed composition may include a crude protein fraction having a leucine content of about 10.3 - 14.0 %, and more commonly 10.3 - 12.0 wt.%. The feed composition may include a crude protein fraction having a threonine content of about 6.2 - 9.3 wt.%, and more commonly about 6.2 - 8.4 wt.%.

[0014] The feed composition may include a leucine and/or threonine source having a leucine and/or threonine content on a free amino acids basis of at least about 400 grams per kilogram dry solids {i.e., 40 wt.% (dsb)). In suitable embodiments, the leucine and/or threonine source has a leucine and/or threonine content on a free amino acids basis of at least about 500 grams per kilogram dry solids, at least about 600 grams per kilogram dry solids, at least about 700 grams per kilogram dry solids, and/or at least about 800 grams per kilogram dry solids. [0015] The feed composition may include a rumen-protected leucine and/or threonine source which may include rumen-protected L-Leu and/or L-Thr. The rumen-protected leucine and/or threonine source may include a rumen-protected leucine-rich protein and/or threonine-rich protein, which may be of non-animal origin. The L-Leu and/or L-Thr may be rumen-protected by reacting the amino acid with at least one reducing carbohydrate (e.g., a reducing sugar). A leucine-rich protein and/or threonine-rich protein may be rumen-protected by reacting the protein with at least one reducing carbohydrate (e.g., a reducing sugar). Suitable reducing carbohydrates may include xylose, lactose, fructose, and/or glucose. The selected amino acid and/or selected protein may be rumen-protected by coating the amino acid and/or the protein with at least one fatty acid. Suitable fatty acids may include at least partially hydrogenated vegetable oils, such as soy bean oil. The coated product may be further coated with a surfactant.

[0016] The rumen-protected leucine source and/or threonine source may be capable of delivering at least about 40% of rumen-protected leucine and/or threonine post- ruminally. More commonly, the rumen-protected leucine and/or threonine source may be capably of delivering at least about 50%, 60%, 70%, 80%, and suitably about 90% of rumen-protected leucine and/or threonine post-ruminally. [0017] The feed composition may include additional rumen-protected amino acid sources. For example, the composition may include rumen-protected limiting amino acids such as rumen-protected histidine, lysine, methionine, and/or phenylalanine. Feed compositions comprising rumen-protected amino acid sources are described in U.S. published application no. 2006-039955, the disclosure of which is incorporated by reference herein in its entirety.

[0018] The composition may be used in several forms including, but not limited to, complete feed form, concentrate form, blender form and base mix form. Feed forms for increasing milk production in diary cattle by balancing the essential amino acids via a particular complete feed, concentrate, blender or base mix form of the composition are described in U.S. Patent No. 5,145,695 and U.S. Patent No. 5,219,596, the disclosures of which are incorporated by reference herein in their entireties.

[0019] If the composition is in the form of a complete feed, the percent protein level (crude protein content) may be about 10 to about 25 percent, more suitably about 14 to about 24 percent (or about 14 to about 19 percent); whereas, if the composition is in the form of a concentrate, the protein 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 protein level in the composition may be about 20 to about 30 percent, more suitably about 24 to about 26 percent; and if the composition is in the form of a base mix, the protein level in the composition may be about 55 to about 65 percent. Unless otherwise stated herein, percentages are stated on a weight percent basis. [0020] The complete feed form composition may contain wheat middlings, corn, soybean meal, corn gluten meal, distillers grains or distillers grains with solubles, salt, macro-minerals, trace minerals and/or vitamins. Other ingredients may commonly include, but not be restricted to sunflower meal, canola meal, cotton seed meal, whole cotton seed, brewers grain, linseed meal, malt sprouts and soybean hulls. [0021] The concentrate form composition generally contains wheat middlings, corn, soybean meal, corn gluten meal, distillers grains or distillers grains with solubles, salt, macro-minerals, trace minerals and vitamins. Alternative ingredients would commonly include, but not be restricted to sunflower meal, canola meal, cotton seed meal, whole cotton seed, brewers grains, linseed meal, and malt sprouts. The blender form composition generally contains wheat middlings, corn gluten meal, distillers grains or distillers grains with solubles, salt, macro-minerals, trace minerals and/or vitamins. Alternative ingredients would commonly include, but not be restricted to, corn, soybean meal, sunflower meal, cotton seed meal, whole cotton seed, brewers grains, linseed meal, malt sprouts and soybean hulls.

[0022] The base form composition generally contains wheat middlings, corn gluten meal, and/or distillers grains or distillers grains with solubles. Additional ingredients would commonly include, but are not restricted to soybean meal, sunflower meal, cotton seed meal, whole cotton seed, brewers grains, linseed meal, malt sprouts, macro-minerals, trace minerals and/or vitamins.

[0023] The complete feed form composition, concentrate form composition, blender form composition, and base form composition may also include a product that has an enhanced amino acid content with regard to one or more selected amino acids. In particular, the product may have an enhanced amino acid content with regard to one or more limiting amino acids for milk production. The product may have an enhanced amino acid content because of the presence of free amino acids in the product and/or the presence of proteins or peptides that include the amino acid in the product. For example, the product may have an enhanced content of leucine and/or threonine present as free amino acids and/or present in leucine-rich protein and/or threonine rich proteins. Typically, the product is derived from a non-animal source such as microorganisms {e.g., bacteria and yeast) and/or plants. The product may include non-animal and/or animal proteins {e.g., a leucine-rich animal protein and/or a threonine-rich animal protein produced in recombinant bacteria, yeast, and/or plants). [0024] The product may have an enhanced content of one or more amino acids, in particular, one or more essential amino acids determined to be limiting for milk production. Limiting amino acids may include leucine, threonine, histidine, lysine, methionine, phenylalanine, isoleucine, and/or tryptophan, which may be present in the product as a free amino acid or as a protein(a) or peptide(s) that is rich in the selected amino acid. For example, the product may include at least one leucine-rich protein and/or threonine-rich protein.

[0025] As defined herein, a leucine-rich protein will typically have at least about 15% leucine residues per total amino acid residues in the protein, and more typically, at least about 20% and/or 25% leucine residues per total amino acid residues in the protein. In suitable embodiments, a leucine-rich protein may have at least about 30% leucine residues and/or at least about 35% leucine residues per total amino acid residues in the protein.

[0026] As defined herein, a threonine-rich protein will typically have at least about 10% threonine residues per total amino acid residues in the protein, and more typically, at least about 15% and/or 20% threonine residues per total amino acid residues in the protein. In suitable embodiments, a threonine-rich protein may have at least about 25% threonine residues and/or at least about 30% threonine residues per total amino acid residues in the protein.

[0027] As disclosed herein, a product may have an enhanced content of leucine and/or threonine. A product with an enhanced content of leucine typically has a leucine content (including free leucine and leucine present in a protein or peptide) of at least about 10 wt.%, 11 wt.%, 13 wt.%, 15 wt.%, and suitably at least about 17 wt. % relative to the weight of the total amino acid content of the product, (as determined by the crude protein content of the product), and suitably at least about 19 wt. % relative to the weight of the total amino acid content of the product. A product with an enhanced content of threonine typically has a threonine content (including free threonine and threonine present in a protein or peptide) of at least about 5 wt.%, 6 wt.%, 8 wt.%, 10 wt.%, and suitabley at least about 12 wt. % relative to the weight of the total amino acid content of the product, (as determined by the crude protein content of the product), and suitably at least about 14 wt. % relative to the weight of the total amino acid content of the product.

[0028] A product with an enhanced content of leucine and/or threonine may be produced in a microbial fermentation process. In one example, a bacteria or yeast that overproduces leucine and/or threonine is grown in a fermentation system and the fermentation broth and/or fermentation biomass are further processed to produce a product that has an enhanced content of leucine and/or threonine. The fermentation broth and/or biomass may be dried (e.g., spray-dried), to produce the product with an enhanced content of leucine and/or threonine.

[0029] Leucine and/or threonine or a product having an enhanced content of leucine and/or threonine may be at least partially purified from the fermentation broth or lysed biomass. For example, leucine and/or leucine-rich proteins may be isolated based on characteristics such as isoelectric point. Similarly, threonine and/or threonine-rich proteins may be isolated based on characteristics such as isoelectric point. In one embodiment, the desired isoelectric point for a leucine-rich protein and/or threonine-rich protein may be varied by using recombinant technology to alter the amino acid composition of the protein (e.g., to create a protein having a selected leucine and/or threonine content and a desired isoelectric point). [0030] Chemical characteristics of leucine and/or threonine (e.g., isoelectric points (pi), molecular weight and/or hydrophobicity/hydrophilicity) may be used to selective precipitate leucine and/or threonine, preferentially extract leucine and/or threonine (e.g., into organic solvents), or preferentially bind leucine and/or threonine to various ion exchange resin or metal chelation matrices. Similarly, chemical characteristics of leucine and/or threonine may be used to purifty leucine-rich proteins and/or threonine-rich proteins for subsequent use in feed or food. Leucine-rich proteins and/or threonine-rich proteins may display unique binding properties that may facilitate isolation of the proteins.

[0031] Leucine-rich proteins and/or threonine-rich proteins may be selected from proteins described in the literature. A leucine-rich protein and/or threonine-rich protein may also comprise specific fragments of known proteins that have an increased leucine and/or threonine content compared to the full-length native protein. For example, a portion of a known protein (e.g., N-terminal portion, internal portion, and/or C-terminal portion) may be selected as a leucine-rich protein and/or threonine- rich protein. A leucine-rich protein and/or threonine-rich protein does not need to retain its native function to be suitable for the compositions or methods described herein. [0032] A leucine-rich protein and/or threonine-rich protein may be in the form of recombinantly-engineered proteins. For example, a poly-leucine motif and/or poly- threonine motif present and/or engineered in a recombinant protein. The recombinantly-engineered proteins may have an enhanced content of other amino acids in addition to leucine and/or threonine. In particular, the proteins may have an enhanced content of one or more of the essential amino acids, or the proteins may have an enhanced content of one or more of the other limiting amino acids for milk production, which may include histidine, lysine, methionine, phenylalanine, isoleucine, and tryptophan. As such, the recombinantly-engineered proteins may be designed to include a selected profile of amino acids. In addition to limiting amino acids for milk production, the proteins may be engineered to contain cysteine residues to enable the formation of intramolecular and/or intermolecular di-sulfide bonds. The ratios of the amino acids in the recombinantly-engineered proteins may be varied or designed to match the ratios that are predicted to be optimal for dairy cattle based on feeding studies or predictions. In one embodiment, the selected profile of amino acids, (e.g., in a recombinantly produced protein), is similar to the profile of a feed supplement that is derived from an animal product (e.g., blood meal). After a protein has been designed and its gene has been cloned into an expression vector, the protein may be expressed (or over-expressed) in a recombinant system using a microbial host (such as E.coli., Corynebacterium, Brevibacterium, Bacillus, Yeast), plants, and the like.

[0033] In order to optimize the expression of the protein in the host, the gene that encodes the protein may be designed to utilize specific tRNAs that are prevalent in the host. Alternatively, selected tRNAs may be co-expressed in the host to facilitate expression of the protein.

[0034] The recombinantly-engineered proteins may include specific sequences to facilitate purification of the proteins. For example, the proteins may include histidine tags. The proteins may also include "leader sequences" that target the protein to specific locations in the host cell such as the periplasm, or that target the protein for secretion. For example, the host cell may be a bacteria, and protein may include a bacterial secretion signal sequence such as the pectate lyase secretion signal sequence. [0035] The recombinantly-engineered proteins may also include protease cleavage sites to facilitate cleavage of the proteins in the abomasum and enhance delivery of amino acids in the protein to the small intestine. For example, one such protease is pepsin, one of the protein-digesting enzymes of the abomasum in cattle. Pepsin demonstrates a preferential cleavage of peptides at hydrophobic, preferentially aromatic, residues in the Pl and Pl' positions. In particular, pepsin cleaves proteins on the carboxy side of phenylalanine, tryptophan, tyrosine, and leucine residues. As such, the protein may include one or more pepsin cleavage sites. [0036] In another example, the product may include leucine-rich proteins and/or threonine-rich proteins augmented with peptides or proteins that have an enhanced content of other amino acids, in particular limiting amino acids. For example, a product may include one or more proteins that have an enhanced content of one or more of the same or different amino acids. As such, the product may include multiple proteins, peptides, and/or amino acids.

[0037] The leucine-rich proteins and/or threonine-rich proteins (or peptides) may be over-expressed in a microbial host (such as a species of Eschrichia, Corynebacterium, Brevibacterium, Bacillus, Yeast), plants and the like. An entire microbial biomass may be spray-dried and used in the animal feed or the leucine-rich proteins and/or threonine-rich proteins and related proteins or peptides may be at least partially purified from the biomass. Alternatively, where the microbial host excretes leucine and/or a leucine-rich protein (and/or threonine and/or a threonine-rich protein) to form an enriched broth, the amino acid enriched broth may be separated from the biomass produced by the fermentation and the clarified broth may be used as an animal feed ingredient, e.g., either in liquid form or in spray dried form. In one embodiment, leucine-rich proteins and/or threonine-rich protein may be purified by binding histidine tags in the proteins to a matrix that includes nickel metal. [0038] It may be desirable to use microbial hosts that do not contain lipopolysaccharides ("LPS") that have endotoxic effects, for example a Gram-positive bacteria, such as Corynebacteria and Brevibacterium. Gram-negative bacteria, such as E. coli, often include LPS that have an endotoxic effect. Selection of a bacteria that does not include endotoxic LPS may be particularly important when a biomass is to be prepared and used as a leucine and/or threonine source, because the majority of LPS remain associated with bacteria and are not released substantially into the fermentation broth unless the bacteria are lysed. As such, endotoxic LPS would be expected to be localized within the biomass after fermentation. [0039] The product may include ingredients that have been treated to facilitate rumen bypass. For example, the product may include treated leucine and/or treated leucine-rich proteins (and/or threonine and/or threonine-rich proteins). The selected amino acid and/or selected protein may be reacted with one or more reducing carbohydrates {e.g., xylose, lactose, glucose, fructose, and the like). In various embodiments, the selected amino acid {e.g., leucine and/or threonine) and/or the selected protein {e.g., a leucine-rich protein and/or a threonine-rich protein) may be coated with polymeric compounds, formalized protein, fat, mixtures of fat and calcium, mixtures of fat and protein, or with metal salts of long chain fatty acids. The selected amino acid and/or selected protein may be coated with vegetable oils (such as soy bean oil), which may be modified. For example, the selected amino acid and/or the selected protein may be coated with at least partially hydrogenated vegetable oils. In particular, the selected amino acid and/or the selected protein may be coated with a mixture of a metal salt of a fatty acid {e.g., zinc stearate) and a fatty acid {e.g., stearic acid). The selected amino acid and/or the selected protein may be coated with pH- sensitive polymers. A pH-sensitive polymer is stable at ruminal pH, but breaks down when it is exposed to abomasal pH, releasing the selected amino acid and/or the selected protein for digestion in the abomasums and absorption in the small intestine. [0040] In one aspect, the disclosed method includes several steps. First, an amino acid or a protein that is rich in one or more amino acids is synthesized. As noted above, a suitable amino acid may be leucine and/or threonine and a suitable protein may be a leucine-rich protein and/or a threonine-rich protein. The selected amino acid and/or the selected amino acid-rich protein may be synthesized using a microbial fermentation system to produce a fermentation biomass, which may be dried {e.g., spray-dried) to provide a dried fermentation biomass. Alternatively, the amino acid and/or protein may be present in the fermentation broth, which may be separated from the fermentation biomass {e.g., via filtration) and spray-dried to produce a dried fermentation broth that has an enhanced content of the amino acid and/or protein. Further, the amino acid and/or amino acid-rich protein may be isolated or at least partially purified from either the biomass and/or broth prior to preparing a dried product. The dried fermentation biomass, dried fermentation broth, and/or dried product may be coated with a coating to provide a coated product and/or treated (e.g., by reacting the dried fermentation biomass, dried fermentation broth, and/or dried product with a reducing carbohydrate such as lactose and/or xylose). The coating may be hydrophobic. The coating and/or treatment may protect the product and enable it to pass through the rumen with reduced degradation and to deliver at least a portion of the product to the abomasum and/or small intestine. As such, the coating and/or treatment allows the coated and/or treated products to bypass the rumen, (i.e., allows rumen bypass). The coated and/or treated product may be fed to a ruminant to improve milk production as well as to improve milk protein composition.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1 is a schematic representation of a model for microbial growth. NDF - "neutral detergent fiber"; NFC - "non-fiber carbohydrates"; VFA - "volatile fatty acids"; RDP - "rumen degradable protein"; rH - "pH of the rumen". [0042] FIG. 2 is a schematic representation of a typical spin disk process for encapsulating products.

DETAILED DESCRIPTION

[0043] Rate limiting amino acids in ruminant feed (e.g., leucine and/or threonine) and their concentration in feed is directly correlated to milk production in dairy cows. Blood meal is currently used in animal feed and is a rich source of rate limiting amino acids. Further, amino acide present in blood meal may not be significantly degraded in the rumen. Replacements for blood meal do not have a similar amino acid profile and may be lacking in one or more rate limiting amino acids. As such, a feed lacking blood meal would need to be supplemented with one or more rate limiting amino acids to fulfill amino acid requirements. In addition, as milk yields increase there is a corresponding increase in amino acid requirements. This increase in other amino acid requirements may need to be met as well.

[0044] Protein must escape ruminal degradation and pass to the small intestine to supply sufficient amounts of amino acids. The primary methods developed to prevent fermentative digestion of amino acids include (1) coating a product that has an enhanced amino acid content with a composition that protects the product from degradation in the rumen and (2) structural manipulation of the amino acid to produce amino-acid analogs that demonstrate reduced degradation in the rumen {e.g., by reacting the amino acid with a reducing sugar in a Maillard reaction). Single amino acid residues are more readily degraded in the rumen than amino acids present in proteins or peptides, and as such, leucine-rich proteins and/or threonine-rich protein may provide an advantage over leucine and/or threonine, respectively. Further, proteins with significant secondary or tertiary structure {e.g., relating to di-sulfide bond formation) may display better rumen protection.

[0045] In addition to providing a source of leucine and/or threonine for ruminant feed, leucine-rich proteins and/or threonine-rich proteins may closely resemble the "leucine-rich proteins and/or threonine-rich proteins" that are present in feed supplements {e.g., blood meal). For example, blood meal may include the bovine hemoglobin alpha chain, SwissProt. Accession No. P01966, which has a leucine content of more than 14% (leucine residues/total residues).

[0046] Other leucine-rich proteins and/or threonine-rich proteins are known from the literature.

[0047] As noted above, fragments of proteins may be suitable as leucine-rich proteins (or peptides) and/or threonine-rich proteins (or peptides). For example, proteins may be truncated at the N-terminus or at the C-terminus to create a leucine- rich protein and/or threonine-rich protein, where the protein includes a leucine-rich internal amino acid sequence and/or threonine-rich amino acid sequence, respectively. Fragments may be of any length, however, particularly suitable fragments may include at least about 20 amino acids.

[0048] Amino Acid Demand. Limiting amino acids may be supplied to an animal to increase production of a chosen animal product {e.g., milk) by supplementing the animal's feed with the limiting amino acid. Limiting amino acids may be identified by analyzing the amino acid profile of the chosen animal product (i.e., output profile) and comparing this profile to the profile of amino acids supplied to the animal (i.e., input profile). Methods for determining amino acid requirements are known in the art and are described in U.S. Patent No. 5,145,695 and U.S. Patent No. 5,219,596, which are incorporated by reference herein in their entireties.

[0049] Supply of Amino Acids. Ruminants derive amino acids from two sources: (1) microbial protein as determined by microbial growth; and (2) protein that remains undegraded in the rumen (i.e., "rumen undegraded protein" or "RUP"). Microbial growth may be predicted based on the carbohydrates available for fermentation in the rumen (e.g., starch, sugar, neutral detergent fiber, pectin, and beta-glucan), the supply of rumen degradable protein, and pH of the rumen. Because microbial proteins are not fully digestible, the supply of microbial amino acids supplied by the microbial protein must be adjusted based on the digestibility of the protein to provide a digestible microbial amino acid value.

[0050] The second source of amino acids is feed ingredients that remain undegraded after passing from the rumen to the abomasum (i.e., the bypass protein fraction). Amino acids within a feed ingredient are processed and utilized (i.e., degraded) by microbes in the rumen at different rates. As such, different amino acids will have different undegradable essential amino acid ("UEAA") values. In addition, a UEAA value may be adjusted based on the digestibility of an amino acid in the small intestine to provide a digestible UEAA value. The amount of essential amino acids that pass from the rumen can be estimated using the techniques described in Craig et el., "Amino Acids Released During Protein Degradation by Rumen Microbes," (1984) Journal of Animal Science, 58:436 - 443. The sum of digestible microbial amino acids and digestible UEAA' s is the digestible amino acid contribution that will be provided to the small intestine. For dairy cows, this is sometimes referred to as dairy digestible amino acid ("ddAA") for the amino acid in question, e.g., dairy digestible leucine ("ddAA LEU"), threonine ("ddAA THR"), and/or histidine ("ddAA HIS"). [0051] In diet formulation, the predicted digestible microbial amino acid contribution from rumen fermentation is subtracted from the animal's amino acid requirements, as determined by the animal's profile. The amounts of amino acids that need to be supplied as UEAA' s from feed are the difference between the animal's amino acid requirements and the amino acids supplied from digestible microbial amino acids.

[0052] The amino acid profile of milk can be compared to the profile of amino acids produced by microbes within the digestive tract of the animal (i.e., microbial amino acid profile). Differences between the microbial and milk amino acid profiles indicate where amino acids may be in excess or limiting. However, this amino acid profile comparison provides only part of the needed information in order to increase production of a chosen animal product. The efficiency with which the body incorporates amino acids in the small intestine into a chosen animal product must also be considered. By determining the output/input amino acid profile ratio and by determining the efficiency of incorporation, dairy digestible amino acid requirements may be determined. It has been established that histidine, lysine, methionine, phenylalanine, leucine, and/or threonine are likely to be limiting amino acids for milk production in dairy cows. A similar determination may be performed for the amino acid profile of muscle.

[0053] Synthesis of leucine-rich products and/or threonine-rich products. Leucine- rich products (and/or threonine-rich products) may include products that have an enhanced content of leucine (and/or threonine) as a free amino acid and/or products that include leucine-rich proteins (and/or threonine-rich proteins). Leucine-rich products (and/or threonine-rich products) may be produced by methods known in the art. For example, a leucine-rich fermentation broth (and/or threonine-rich fermentation broth) may be used as a source of leucine (and/or threonine). The leucine-rich fermentation broth (and/or threonine-rich fermentation broth) may be produced by single-cell organisms (e.g., microorganisms such as bacteria or yeast) and/or plants that are selected or engineered to overproduce leucine (and/or threonine). Suitable microorganisms may include microorganisms belonging to the genus Eschrichia, Bacillus, Microbacterium, Arthrobacter, Serratia, and Corynebacterium. Gram-negative bacteria are known to produce lipopolysaccharides ("LPS"), which are endotoxins. As such, it may be desirable to select a Gram- positive bacteria (and/or Gram-variable bacteria) as the host-cell, {e.g., Corynebacteria and Brevibacteria), particularly when a biomass is to be prepared. Because the majority of LPS remain associated with the host-cell and are not released into the fermentation broth until the host-cell is lysed, Gram-negative bacteria such as E. coli. may be suitable for producing a leucine broth (and/or threonine broth). [0054] The leucine-rich fermentation broth (and/or threonine-rich fermentation broth) may be spray-dried and used directly as a leucine source (and/or threonine source) or the broth may be concentrated. In another embodiment, leucine (and/or threonine) may be at least partially purified from the fermentation medium and biomass. The microbial produced leucine (and/or threonine) may then be prepared based on rumen bypass technology and added to feed at the required level. [0055] Alternatively, microbes may be engineered to accumulate and retain leucine (and/or threonine) and the microbes may be prepared as a spray-dried biomass product. Optionally, the biomass may be separated by known methods, such as separation, decanting, a combination of separation and decanting, ultrafiltration or microfiltration. The biomass product may be further treated to facilitate rumen bypass. In one embodiment, the biomass product may be separated from the fermentation medium, spray-dried, and optionally coated to facilitate rumen bypass, and added to feed as a leucine source (and/or threonine source). [0056] In a further embodiment, microbes may be engineered to produce leucine- rich proteins (and/or threonine-rich proteins). Leucine-rich proteins (and/or threonine-rich proteins) may include known and characterized proteins. Alternatively, a recombinantly engineered protein that has a chosen amino acid profile may be cloned into an expression vector and introduced into a suitable host cell {e.g., microbe).

[0057] The leucine-rich proteins (and/or threonine-rich proteins) may be secreted into the fermentation media, or alternatively, the leucine-rich proteins (and/or threonine-rich proteins) may accumulate in the microbes. The microbes may be prepared as a spray-dried biomass product, or the leucine-rich proteins (or peptides) and/or threonine-rich proteins (or peptides) may be isolated from the microbial biomass to provide a leucine-rich product and/or threonine-rich product. In any case, the leucine-rich product and/or threonine-rich product may be further treated to enhance rumen bypass. The treated product then may be added to feed as a leucine source and/or threonine source.

[0058] Construction and expression of a leucine-rich protein or peptide ("LRP") and/or a threonine-rich protein or peptide ("TRP") in a microbial host, Escherichia coli. Construction of a LRP and/or TRP expression construct AArcpET30(Xa/LIC), may be performed as follows. Primers are designed with compatible overhangs for the pET30(Xa/LIC) vector (Novagen, Madison, WI) for cloning a gene that encodes a LRP and/or TRP. The pET vector has a 12 base single stranded overhang on the 5' side of the Xa/LIC site and a 15-base single stranded overhang on the 3' side of the Xa/LIC site. The plasmid is designed for ligation-independent cloning, with N- terminal His and S-tags and an optional C-terminal His-tag, The Xa protease recognition site (IEGR) is positioned in front of the start codon of the gene of interest, such that the fusion protein tags can be removed.

[0059] Suitable primers for amplifying a gene encoding a selected LRP and/or TRP can be purchased for pET30 Xa/LIC cloning. It is possible to design primers that are internal to the gene such that the peptide that is generated has a higher percentage of leucine and/or threonine residues per total amino acids than the native protein sequence.

[0060] It is reported that alterations of tRNA concentrations and aminoacyl-tRNA synthetases influence amino acid biosynthesis. In addition, tRNA can have large effects on the expression and over-expression of heterologous genes in microbial expression systems through reduced translation and errors in amino acid sequences of protein products. {See, e.g., O'Neill etal, J. Bacteriol. 1990 Nov;172(ll):6363~71); Smith et al, Biotechnol Prog. 1996 Jul-Aug;12(4):417-22); Dieci et al, Protein Expr Purif. 2000 Apr;18(3):346-54). Thus, to increase the expression of the LRP and/or TRP for example, it would be beneficial to simultaneously express the corresponding leucyl-tRNA gene and/or threonyl-tRNA gene as well. It is also possible to design primers to introduce a selected LRP and/or TRP gene into an operon so that both the LRP (and/or TRP) and the leucyl-tRNA synthetase (and/or threonyl-tRNA synthetase) are co-expressed. [0061] Depending on the source of the specific leucine-rich protein (and/or threonine-rich protein), the codon bias of the respective gene could be changed to match the host microbe codon usage in order to achieve higher expression of heterologous proteins. {See, e.g., Baca et al., Int'l J. of Parasitology. 30: 113-118). Codon usage tables are available from many sources.

[0062] The following is one version of a PCR protocol which can be used to amplify a selected gene that encodes a leucine-rich protein and/or threonine-rich protein. In a 50 μL reaction, 0.1-0.5 μg template, 1.5 μM of each primer, 0.4 mM each dNTP, 3.5 U Expand High Fidelity™ Polymerase, and Ix Expand™ buffer with Mg²⁺ were added (Roche, Indianapolis, IN). The selected thermocycler program includes a hot start at 96⁰C for 5 minutes, followed by 29 cycles including the following steps: 94°C for 30 seconds, 40-65°C for 1 minute (gradient thermocycler) and 72°C for 2 minutes. After the 29 cycles, the sample is maintained at 72°C for 10 minutes and then stored at 4°C.

[0063] The PCR product is gel purified from 0.8 or 1% TAE-agarose gels using the Qiagen gel extraction kit (Valencia, CA). The PCR product is quantified by comparison to standards on the agarose gel, and then treated with T4 DNA polymerase following the manufacturer's recommended protocols for Ligation Independent Cloning (Novagen, Madison, WI).

[0064] Briefly, about 0.2 pmol of purified PCR product is treated with 1 U T4 DNA polymerase in the presence of dGTP for 30 minutes at 22°C. The polymerase removes successive bases from the 3' ends of the PCR product. When the polymerase encounters a guanine residue, the 5' to 3' polymerase activity of the enzyme counteracts the exonuclease activity to prevent effectively further excision. This creates single stranded overhangs that are compatible with the pET Xa/LIC vector. The polymerase is inactivated by incubating at 75°C for 20 minutes. [0065] The vector and treated insert are annealed as recommended by Novagen. About 0.02 pmol of treated insert and 0.01 pmol vector are incubated for 5 minutes at 22⁰C; 6.25 mM EDTA (final concentration) is added; and the incubation at 22°C is repeated. The annealing reaction (1 μL) is added to NovaBlue™ Singles competent cells (Novagen, Madison, WI), and incubated on ice for 5 minutes. After mixing, the cells are transformed by heat shock for 30 seconds at 42°C. The cells are placed on ice for 2 minutes, and allowed to recover in 250 μL of room temperature SOC for 30 minutes at 37°C with shaking at 225 rpm. Cells are plated on LB plates containing kanamycin (25-50 μg/mL).

[0066] Plasmid DNA from cultures that grow on the LB plates with kanamycin is purified using the Qiagen spin miniprep kit (Valencia, CA) and screened for the correct inserts. The sequences of plasmids that appeared to have the correct insert are verified by dideoxy chain termination DNA sequencing (SeqWright, Houston, TX) with S-tag and T7 terminator primers (Novagen), and internal primers. The sequence verified plasmid is transformed into the expression host BL21(DE3) according to Novagen protocols.

[0067] Expression of a LRP and/or TRP in transformed E. coli cells may be performed as follows. Fresh plates of transformed E. coli cells are prepared on LB medium containing 50 μg/mL kanamycin. Overnight cultures (5 niL) are inoculated from a single colony and grown at 30°C in LB medium with kanamycin. Typically, a 1 to 5 ml inoculum is used for induction in 100 ml - 500 ml LB medium containing 50 μg/mL kanamycin. Cells are grown at 37°C and sampled every hour until an OD₆₀₀ of 0.35-0.8 is obtained. Cells are then induced with 0.1 mM IPTG. The entire culture volume is centrifuged after approximately 4-10 hours growth (post-induction), for 20 minutes at 4⁰C and 3500 rpm. The supernatant is decanted and both the broth and the cells (washed once with sterile distilled water) are separately frozen at -8O⁰C, if immediate analysis is not anticipated. Cell extracts are prepared for protein analysis using Novagen BugBuster™ reagent with benzonase nuclease and Calbiochem protease inhibitor cocktail III according to the Novagen protocol. The level of protein expression in the cell extracts is analyzed by SDS-PAGE using 4-15% gradient gel (Bio-Rad, Hercules, CA).

[0068] Once the appropriate induction conditions (e.g., time and temperature) that results in maximum LRP and/or TRP expression is determined, cells are cultured under those conditions and the cell pellet is resuspended in an appropriate amount of a suitable isotonic buffer, for example, physiological saline (0.85% NaCl pH 7.0). This cell suspension is then lysed using methods known to those skilled in the art, such as treatment in French Pressure cells. The lysed cells are centrifuged at 10,000 - 15,000 rpm for 20 -30 min at 4^eC to separate the biomass and cell debris and generate a cell- free extract that contains the LRP and/or TRP. The extract, which contains the LRP and/or TRP, is spray dried to generate a product of LRP and/or TRP that can be added to animal feed as is, or after being subjected to suitable encapsulation to ensure survival through the rumen. Purification and/or concentration of LRP and/or TRP from transformed E. coli cells may be performed using techniques described in the literature.

[0069] The codon usage of the microbial host is taken into consideration in designing the synthetic gene that will be translated into the desired LRP and/or TRP, such that rare codons are not used. Codon usage in E. coli is expected to be different from that of Corynebacterium for example. Codon usage tables are known and available in the art.

[0070] Based on a protocol described in Stemmer et al, Gene 1995 Oct 16; 164(l):49-53, it is possible (1) to determine the best codons to use to design the nucleic acid sequence that will encode the desired peptide, (2) to design the required number of overlapping oligonucleotides spanning the length of the synthetic nucleic acid, and (3) to assemble the synthetic gene using PCR that relies not on DNA ligase but uses the properties of DNA polymerase to build longer DNA fragments during the PCR assembly reaction. The synthetic nucleic acid encoding a LRP and/or TRP can then be cloned into the desired vector containing the appropriate antibiotic/selection marker to ensure expression of the synthetic LRP and/or TRP in the host of choice for example plants E.coli, Corynebacterium, Brevibacterium, Bacillus, Yeast and/or plants.

[0071] As noted above, it is reported that alterations of tRNA concentrations and aminoacyl-tRNA synthetases influence amino acid biosynthesis. In addition, tRNA can have large effects on the expression and over expression of heterologous genes in microbial expression systems through reduced translation and errors in amino acid sequences of protein products. {See, e.g., O'Neill et al, J. Bacteriol. 1990 Nov;172(ll):6363-71; Smith et al, Biotechnol Prog. 1996 Jul-Aug;12(4):417-22); Dieci et al, Protein Expr Purif. 2000 Apr;18(3):346-54). Thus, to increase the expression of the synthetic or recombinant LRP and/or TRP, for example, it may be beneficial to simultaneously express the corresponding leucyl-tRNA and/or threonyl- tRNA, respectively. It may be beneficial to simultaneously express the amino acyl- tRNA synthetase gene as well {i.e., leucyl-tRNA synthetase and/or threonyl-tRNA synthetase, respectively).

[0072] Expression of a synthetic or recombinant LRP and/or TRP in transformed E. coli cells may be performed as follows. Fresh plates of transformed E. coli cells are prepared on LB medium containing 50 μg/mL kanamycin. Overnight cultures (5 mL) are inoculated from a single colony and grown at 30⁰C in LB medium with kanamycin. Typically, a 1 to 5 ml inoculum is used for induction in 100 ml - 500 ml LB medium containing 50 μg/mL kanamycin. Cells are grown at 37°C and sampled every hour until an OD₆₀₀ of 0.35-0.8 was obtained. Cells are then induced with 0.1 mM IPTG. The entire culture volume is centrifuged after approximately 4-10 hours growth (post-induction), for 20 minutes at 4⁰C and 3500 rpm. The supernatant is decanted and both the broth and the cells (washed once with sterile distilled water) are separately frozen at -8O⁰C if immediate analysis is not anticipated. Cell extracts are prepared for protein analysis using Novagen BugBuster™ reagent with benzonase nuclease and Calbiochem protease inhibitor cocktail HI according to the Novagen protocol. The level of protein expression in the cell extracts is analyzed by SDS- PAGE using 4-15% gradient gel (Bio-Rad, Hercules, CA).

[0073] Once the appropriate induction time that results in maximum LRP and/or TRP expression is determined, cells are cultured under those conditions and the cell pellet is resuspended in an appropriate amount of a suitable isotonic buffer, for example physiological saline (0.85% NaCl pH 7.0). This cell suspension is then lysed using methods known to those skilled in the art, such as treatment in French Pressure cells. The lysed cells are centrifuged at 10,000 - 15,000 rpm for 20 -30 min at 4⁰C to separate the biomass and cell debris and generate a cell-free extract that contains the LRP and/or TRP. This extract, which contains the LRP and/or TRP, can be spray dried to generate a product of LRP and/or TRP that can be added to animal feed as is, or after being subjected to suitable treatment and/or encapsulation to ensure survival through the rumen. [0074] Purification or concentration of synthetic or recombinant LRP and/or TRP from E. coli cells may be performed if necessary. The LRP and/or TRP produced can be subjected to further concentration and purification using techniques described in the literature.

[0075] Purification of LRP and/or TRP after a fermentation experiment may be performed as follows. Cells expressing the LRP and/or TRP are first disrupted using techniques known in the literature for example, using multiple passes through a French press cell at 960 psi on gauge (-19,000 psi in cell). The cell debris are separated from the LRP and/or TRP by centrifugation at 15,000 rpm at 4⁰C. The cell free extract or supernatant contains the LRP and/or TRP and is subjected to further methods to specifically bind the LRP and/or TRP and separate them from the other proteins in the cell free extract.

[0076] One method to purify LRP and/or TRP is based on the ability of a histidine- tag sequence to bind to a histidine binding resin. The LRP and/or TRP may be engineered to include a histidine-tag sequence ("his-tagged"). The his-tagged LRP and/or TRP may be purified by binding to the resin and performing metal chelation chromatography techniques. A "His Bind Kit" is commercially available from Novagen. The his-tag of the LRP and/or TRP bind to Ni²⁺ cations which are immobilized on the histidine-binding resin. The unbound proteins are washed away and the his-tagged LRP and/or TRP can be recovered by elution with imidazole. The his-tagged LRP and/or TRP can be dialyzed to remove the imidazole and then concentrated or spray dried for addition to a feed composition as is, or subjected to appropriate treatment to minimize degradation in the rumen.

[0077] In addition to producing leucine-rich products and/or threonine-rich products in fermentation systems, leucine-rich products and/or threonine-rich products also may be produced in transgenic plant systems. Methods for producing transgenic plant systems are known in the art.

[0078] Rumen protection of leucine-rich products and/or threonine-rich products. Leucine and/or leucine-rich products (and/or threonine and/or threonine-rich products) {i.e., ingredients) may be treated and/or coated or encapsulated to decrease degradation in the rumen (i.e., to facilitate rumen bypass). A suitable coating may have a relatively high melting temperature as described below. [0079] Suitable coatings may include a mixture of a hydrophobic, high melting point compound and a lipid. The combination of one or more, hydrophobic, high melting point compounds (e.g., mineral salts of fatty acids such as commercial grade zinc stearate) with one or more type of lipid, forms a coating material that can protect the content and functionality of the coated ingredient(s). These coatings can be formulated to meet the needs of high temperature and pressure processing conditions as well as protection of the amino acid payload from the microbial environment of the rumen. Suitable coatings are described in U.S. Patent Publication No. 2003/0148013, which is incorporated herein by reference in its entirety.

[0080] Hydrophobic, high melting point compounds typically have a melting point of at least about 7O⁰C, and more desirably, greater than 100°C. In particular, zinc salts of fatty acids, which have a melting point between about 115°C and 130°C, are suitable hydrophobic, high melting point compounds.

[0081] The lipid component typically has a melting point of at least about 0°C and more suitably no less than about 40°C. The lipid component may include vegetable oil, such as soybean oil. In other embodiments, the lipid component may be a triacylglycerol with a melting point of about 45-75°C. Commercial grade stearic acid may be selected as a representative lipid from a group including but not limited to: stearic acid, hydrogenated animal fat, animal fat (e.g., animal tallow), vegetable oil, (such as crude vegetable oil and/or hydrogenated vegetable oil, either partially or fully hydrogenated), lecithin, palmitic acid, animal oils, wax, fatty acid esters (C₈ to C₂₄), fatty acids (C₈ to C₂₄).

[0082] The coating may be present in the coated product in an amount from 1-2000 wt.%, relative to the weight of the coated ingredient (e.g., as leucine, threonine, a leucine-rich protein, and/or a threonine-rich protein). Commonly, the coating represents about 15 to 85 wt.%, relative to the weight of the coated ingredient. More commonly, the coating represents about 20 to 60 wt.% and/or 30 to 40 wt.%, relative to the weight of the coated ingredient. The coating may prepared from a hydrophobic mixture. The coating may include a surfactant. [0083] The coating uses one or more, hydrophobic, insoluble compounds combined with a lipid. For example, commercial grade zinc stearate is extremely hydrophobic and completely insoluble in water. The addition of commercial grade zinc stearate to the coating formula may improve the protection level of the ingredient and its functionality, significantly as compared to a lipid only coating. For example, by combining zinc stearate with a somewhat insoluble lipid such as commercial grade stearic acid, the coating compound may provide better protection from leaching {i.e., loss of the active ingredient from the coated product), when the coated product is in an aqueous medium. As such, the benefit of the present coating composition may be utilized in feeds designed for ruminants to bypass the rumen and deliver the active ingredient to the small intestine.

[0084] In addition to facilitating rumen bypass, the coating may also be useful for protecting the coated ingredients against heat and pressure experienced during the manufacturing process (pelleting and extrusion). The coating composition may be useful in all types of production processes where heat is applied and heat susceptible ingredients are used. Ingredients which may benefit from this form of protection are ingredients that are subject to heat damage or degradation, such as amino acids, proteins, enzymes, vitamins, pigments, and attractants.

[0085] In addition to protecting ingredients from heat related damage or loss there is also the need to protect ingredients to damage or loss attributable to association or chemical reaction with other ingredients. The method of encapsulation may prevent harmful association with other ingredients. As such, the method of encapsulation provides the ability to prepackage or combine ingredients in a formulation, where the ingredients would be usually packaged individually.

[0086] The coating composition may be prepared in a number of ways. Preferably, the preparation process includes making a solid solution of the zinc organic salt component and the lipid component. In one embodiment, the zinc organic salt and the lipid component may be melted until they both dissolve and form a solution. The solution may then be allowed to solidify to form a solid solution. [0087] In addition to the zinc organic acid component and the lipid component, the coating may include other ingredients. For example, the coating may include an one or more emulsifying agents such as glycerin, polysaccharides, lecithin, gelling agents and soaps, which may improve the speed and effectiveness of the encapsulation process. Additionally, the coating may include an anti-oxidant to provide improved protection against oxidation effects. Further, the coating composition may include other components that may or may not dissolve in the process of forming the solid solution. For example, the coating composition may include small amounts of zinc oxide and other elements or compounds.

[0088] A suitable coating may be prepared from a partially hydrogenated vegetable oil such as soybean oil. Other suitable vegetable oils, which be at least partially hydrogenated, include palm oil, cottonseed oil, corn oil, peanut oil, palm kernel oil, babassu oil, sunflower oil, safflower oil, and mixtures thereof. [0089] A suitable coating may be prepared from a mixture that includes a partially hydrogenated vegetable oil and additional constituents, such as a wax. Suitable waxes include beeswax, petroleum wax, rice bran wax, castor wax, microcrystalline wax, and mixtures thereof. In some embodiments, a suitable coating is prepared from a mixture that includes about 85-95% partially hydrogenated vegetable oil (preferably about 90%) and about 5-15% wax (preferably about 10%). [0090] The coating may include an agent for modifying the density of the coated substrate, for example, a surfactant, such as polysorbate 60, polysorbate 80, propylene glycol, sodium dioctylsulfocsuccinate, sodium lauryl sulfate, lactylic esters of fatty acids, polyglycerol esters of fatty acids, and mixtures thereof. The surfactant may be applied to a substrate that has been pre-coated with a mixture that include a partially hydrogenated vegetable oil and a wax.

[0091] A coated substrate (or pre-coated substrate) may be prepared by spraying a hydrophobic mixture that includes a partially hydrogenated vegetable oil (85%-95%) and a wax (5%-15%) on a substrate that includes L-Leu and/or a leucine-rich protein (and/or L-Thr and/or a threonine-rich protein). Optionally, a pre-coated substrate may be further coated by spraying the surface of the pre-coated substrate with a surfactant to form the coated substrate. The coated substrate may have the following composition: substrate (40-80%); hydrophobic mixture (20-60%); surfactant (0-40%) (optional). The coated substrate may have a specific gravity of about 0.3 - 2.0 (more commonly about 1.3 - 1.5). In one embodiment, the coated substrate includes: about 50% substrate; about 35% hydrophobic mixture; and about 15% surfactant. The coated substrate may be prepared by pre-coating the substrate with a hydrophobic mixture, and subsequently coating the pre-coated substrate with a surfactant. [0092] After the coating composition is prepared, it can then be used to prepare the protected ingredient. One suitable procedure for preparing the protected ingredient uses encapsulation technology, preferably microencapsulation technology. Microencapsulation is a process by which tiny amounts of gas, liquid, or solid ingredients are enclosed or surrounded by a second material, in this case a coating composition, to shield the ingredient from the surrounding environment. A number of microencapsulation processes could be used to prepare the protected ingredient such as spinning disk, spraying, co-extrusion, and other chemical methods such as complex coacervation, phase separation, and gelation. One suitable method of microencapsulation is the spinning disk method. In the spinning disk method, an emulsion and/or suspension of the active ingredient and the coating composition is prepare and gravity-fed to the surface of a heated rotating disk. As the disk rotates, the emulsion/suspension spreads across the surface of the disk to form a thin layer because of centrifugal forces. At the edge of the disk, the emulsion/suspension is sheared into discrete droplets in which the active ingredient is surrounded by the coating. As the droplets fall from the disk to a collection hopper, the droplets cool to form a microencapsulated ingredient (i.e., a coated product). (See, e.g., the schematic representation of a suitable spinning disk coating system shown in FIG. 2). Because the emulsion or suspension is not extruded through orifices, this technique permits use of a higher viscosity coating and allows higher loading of the ingredient in the coating.

[0093] The encapsulation of ingredients for use in animal feeds are described in U.S. Patent Publication No. 2003/0148013, which is incorporated herein by reference in its entirety.

[0094] Amino acids (such as leucine and/or threonine) and/or proteins (such as leucine-rich proteins and/or threonine-rich proteins) may also be chemically altered to protect the amino acid in the rumen and to increase the supply of specific amino acids provided to the abomasums and small intestine. For example, methionine hydroxyl analog (MHA®) has been used as an amino acid supplement. In addition, amino acids may be provided as amino acid/mineral chelates. Zinc-methionine and zinc- lysine complexes have been used as amino acid supplements. [0095] A leucine source, which may include L-Leu and/or a leucine-rich protein, (and/or a threonine source, which may include L-Thr and/or a threonine-rich protein) may be reacted with a reducing carbohydrate to protect leucine (and/or threonine) from rumen-degradation (e.g., by performing a Maillard reaction). For example, L- Leu and/or a leucine-rich protein (and/or a L-Thr and/or a threonine-rich protein) may be reacted with reducing sugars such as, but not limited to, xylose, glucose, fructose, lactose, mannose, ribose, and mixtures thereof. Sugar sources may include corn products and hydrolysates of corn products (e.g., at least partially hydrolyzed corn starch or modified corn starch), molasses and hydrolysates of molasses, hemicelluloses and hydrolysates of hemicelluloses, sugars contained in spent sulfite liquors, and mixtures thereof.

[0096] A leucine source, which includes L-Leu and/or a leucine-rich protein, (and/or a threonine source, which includes L-Thr and/or a threonine-rich protein) may be reacted with a reducing sugar in a reaction mixture to form a treated leucine source (and/or threonine source). The treated source then may be added to a feed composition. Alternatively, a leucine source, which includes L-Leu and/or a leucine- rich protein, (and/or a threonine source, which includes L-Thr and/or a threonine-rich protein) may be added to a feed composition to form a supplemented feed composition. The supplemented feed composition may be reacted with a reducing sugar in a reaction mixture to protect amino acids present in the supplemented feed composition, including amino acids present in the leucine source (and/or threonine source). The leucine source (and/or threonine source) may include one or more constituents of a fermentation broth formed by fermenting a leucine-producing microorganism (and/or a threonine-producing microorganism) in a nutrient broth. [0097] The Maillard reaction mixture typically includes at least about 1 mole of reducing sugar per 1 mole of free amino acids. Typically, the reaction mixture includes at least about 3-5 moles of reducing sugar per 1 mole of free amino acids. The reaction mixture typically has a pH of about 4.0 - 10.5, (suitably about 6.0 - 8.5). The reaction mixture typically has a moisture content of about 6 - 40%, (suitably about 15 - 25%). The reaction mixture typically is heated to a temperature of about 20 - 150^QC, (suitably about 80 - 110^δC and/or about 90 - 100^eC) for a time period of about 0.5 - 72 hours, (suitably about 1 -4 hours). The reaction mixture may be subjected to pressure (e.g., pressures of about 2000 - 3500 KPa (about 300 - 500 p.s.i.)). The reaction mixture may be subjected to pressure before, during, or after the reaction mixture is heated. The reaction mixture may be extruded and/or pelleted. [0098] From a standpoint of providing a protected product, yeast may be a particularly suitable host for expressing leucine-rich proteins (and/or threonine-rich proteins) and/or L-Leu (and/or L-Thr). A lysine-accumulating yeast has been shown to accumulate from 4 to 15% of its dry weight as lysine. The majority of the lysine is contained in vacuoles that are stable when incubated with rumen fluid, but immediately released when exposed to pepsin, one of the protein-digesting enzymes of the abomasum. Thus, this organism may be a useful host for expressing proteins and/or amino acids as described herein and providing a protected feed supplement that may increase the amount of proteins and/or amino acids available for intestinal absorption.

[0099] Feeding formulations that have an enhanced content of one or more essential amino acids. Initially, an empirical approach was taken to generate essential amino acid requirements for lactating cows. The essential amino acid composition of rumen microbial protein was compared to the essential amino acid composition of milk protein (Table 1). (The same may be done for muscle protein as an indicator of amino acid requirements for growth, maintenance and reproduction.)

[0100] Amino acids predicted to be limiting were then candidates for further study. Once amino acid requirements were determined, a method was developed to satisfy those amino acid requirements. The first step was to account for microbial amino acid production in the rumen. A microbial model for amino acid production is provided in Figure 1. Microbial amino acid production is determined by microbial growth, which in turn is determined by carbohydrate concentrations that are fermented in the rumen including starch, neutral detergent fiber ("NDF"), sugars, and residual non-fiber carbohydrates ("RNFC") such as pectin and beta-glucan. [0101] To determine the amino acid contribution of rumen microbial protein to an animal's diet, the total rumen microbial protein is multiplied by the percent of each specific amino acid present in the protein. Many researchers have found that the amino acid composition of rumen microbial protein to remain fairly constant. Digestibility of bacterial amino acids is assumed to be 80% for each amino acid. The resulting amounts of amino acids provided by rumen microbial protein were then subtracted from the amino acid requirements. The deficits, (i.e., the differences between the requirements and the amino acids supplied from rumen microbial protein), indicated the amounts of amino acids that should advantageously be supplied as undegradable essential amino acids (UEAAs) in feed. [0102] Feed ingredients high in UEAAs (or "bypass" amino acids) were evaluated to determine potent sources of UEAAs. Blood meal has been used as a common source of UEAAs in the past. Blood meal is also a good source of leucine (Table 2).

[0103] Animal amino acid requirements. Amino acids required in feeds for dairy cows are called Dairy Digestible Amino Acids ("ddAA"). The sum of the digestible microbial amino acid plus the digestible rumen undegraded essential amino acid (UEAA) concentration of that same amino acid is the ddAA. Dairy Digestible Amino Acids represent the supply of total digestible AA to the small intestine. The total amino acid requirements of a dairy animal may be determined as follows. The total amount of an amino acid required ("TAAR") is equal to the amount required for maintenance ("Maintenance Amino Acid" or "MAA") plus the amount of the amino acid required for milk production ("Milk Amino Acid Output" or "MAAO") plus the amount of the amino acid required for growth ("Growth Amino Acid" or "GAA") (i.e., TAAR = MAA + MAAO +GA A). [0104] Encapsulation. The process displayed in Figure 2 represents microencapsulation by spin disk technology. Other microencapsulation processes include spraying, centrifugal co-extrusion, and chemical means. The numerical denotations in Figure 2 refer to the following: 1 - SWECO; 2 - K-TRON Twin Screw Volumetric Feeder; 3 - Boiler; 4 - Fat Heating Tank; 5 - VIKING Fat Pump; 6 - Slurry Mixing Vessel; 7 - Disk; 8 - Heat Ring; 9 - Collection Tank; 10 - Transfer Conveyor; and 11 - Storage Tank.

[0105] The process begins by preparing the coating, for example; a water-soluble nutrient may be protected from water solubility by using a fat coating. The coating is melted by heating the coating to its melting point in the fat holding tank until the coating is liquefied. The nutrient is typically a dry powder of an amino acid, biomass, peptide or protein is prepared. (In some cases, if the nutrient particle size is too large, the nutrient can be passed through a screen (e.g., a SWECO screener)). The nutrient is placed in a volumetric feeder, which delivers a known, accurate concentration of the nutrient {e.g., as a dry powder) at a constant rate.

[0106] The liquid fat is added to the slurry vessel at a controlled rate using a metering pump. The rate of addition is selected such that the liquid fat combines with the nutrient in a chosen ratio. For example, if a coated product has 35% of a nutrient and the product is produced at a rate of 100 lbs/hour, the melted fat must be added at a rate of 65 lbs/hour and the volumetric feeder must deliver the nutrient at a rate of 35 lbs/hour.

[0107] The melted fat and nutrient are mixed together in the slurry vessel to create an emulsion or suspension. The emulsion/suspension is discharged from the bottom of the vessel and is applied as a layer to a rotating disk underneath the vessel. The emulsion/suspension spreads across the disk because of centrifugal forces. As the layer approaches the edge of the disk, the layer is sheared into discrete particles (i.e., droplets or microcapsules) that contain the nutrient surrounded by the coating. As the particles falls from the disk, the coating cools and solidifies. The coated particle falls into the collection hopper and from the collection hopper onto the transfer conveyor. The conveyor moves the bulk the high melting point coating cools and solidifies. The capsules fall into the collection hopper, down the sides of the collection hopper walls and down onto the transfer conveyor. The conveyor moves the bulk particles to bulk storage for further packaging.

[0108] Feed Formulations. Products having an enhanced content of leucine and/or threonine may be included in feed formulation. Tables 3 - 10 provide examples of feed formulations having an enhanced leucine content and/or threonine content. [0109] For example, Table 3 shows one example of a complete feed having an enhanced leucine content and/or threonine content. Table 3 lists the relative amounts of feed ingredients that can be used to make up this exemplary complete feed having an enhanced leucine content and/or threonine content. The complete feed composition includes a leucine-rich protein (and/or threonine-rich protein) which has a leucine content of about 10% (and/or a threonine content of about 10%). Table 4 lists the amounts of a number of common nutrients that are present in the complete feed composition set forth in Table 3.

[0110] Table 5 shows one example of a feed concentrate having an enhanced protein content. Table 5 lists the relative amounts of feed ingredients that can be used to make up this exemplary feed concentrate having an enhanced leucine content (and/or threonine content). The feed concentrate includes leucine-rich protein (and/or a threonine-rich protein) which has a leucine content of about 10% (and/or a threonine content of about 10%). Table 6 lists the amounts of a number of common nutrients that are present in the feed concentrate set forth in Table 5.

[0111] Table 7 shows one example of a supplement having an enhanced content of rumen-protected leucine (and/or rumen-protected threonine). Table 7 lists the relative amounts of feed ingredients that can be used to make up this exemplary supplement. The supplement includes a rumen-protected leucine source (and/or rumen-protected threonine source), such as rumen protected leucine and/or a rumen protected leucine- rich protein which has a leucine content of about 10% (and/or rumen protected threonine and/or a rumen protected threonine-rich protein which has a threonine content of about 10%). Table 8 lists the amounts of a number of common nutrients that are present in the supplement set forth in Table 7.

[0112] Table 9 shows one example of a complete feed composition having an enhanced content of rumen-protected leucine. Table 9 lists the relative amounts of feed ingredients that can be used to make up this exemplary feed composition. The feed composition includes a rumen-protected leucine source (and/or threonine source such as rumen protected leucine and/or a rumen protected leucine-rich protein which has a leucine content of about 10% (and/or rumen protected threonine and/or a rumen protected threonine-rich protein which has a threonine content of about 10%). Table 10 lists the amounts of a number of common nutrients that are present in the feed composition set forth in Table 9.

ILLUSTRATIVE EMBODIMENTS

[0113] In one embodiment, a feed composition is provided. The feed composition includes a leucine source and at least one additional nutrient component. The leucine source includes L-Leu and fermentation constituents from fermentation of a leucine- producing microorganism. The feed composition has a crude protein fraction having a leucine content of at least about 10.3 wt.%. Commonly, the feed composition has a crude protein fraction having a leucine content of about 10.3 - 14.0 wt.%, 10.5 - 13.0 wt.%, and suitably 10.5 - 12.0 wt.%.

[0114] In some embodiments, the crude protein fraction may represent at least about 10 wt.% of the feed composition. Commonly, the crude protein fraction represents at least about 14-19 wt.% of the feed composition.

[0115] At least a portion of the leucine source may be protected against rumen degradation. For example, the L-Leu present in the leucine source may be reacted with a reducing carbohydrate and/or coated with a coating mixture. The coating mixture may include at least one fatty acid. The coating mixture may include partially hydrogenated vegetable oil (e.g., soybean oil) and/or a surfactant. [0116] The fermentation constituents may include soluble and/or insoluble constituents from the fermentation broth formed during fermentation of the leucine- producing microorganism. The fermentation constituents may include dissolved and/or undissolved constituents from the fermentation broth formed during fermentation of the leucine-producing microorganism. The fermentation constituents may include biomass formed during fermentation of the leucine-producing microorganism.

[0117] In some embodiments, the leucine-producing microorganism is a Corynebacteήum. In other embodiments, the leucine-producing microorganism is a Brevibacterium.

[0118] In some embodiments, the leucine source is rumen-protected and the feed composition provides, post-ruminally, a desirable amount of the leucine present in the rumen-protected leucine source. For example, in some embodiments, the feed composition may provide at least about 50% of the rumen-protected leucine post- ruminally. For example, about 1 g of leucine present in the rumen-protected leucine source may result in about 500 mg of the leucine present in the rumen-protected leucine source being delivered post-ruminally. In other embodiments, at least about 60%, 70%, and suitably 80% of leucine present in the rumen-protected leucine source is capable of being delivered post-ruminally.

[0119] In another embodiment, a feed composition is provided. The feed composition includes a leucine source and at least one additional nutrient compound. The leucine source includes L-Leu and fermentation constituents from fermentation of a leucine-producing microorganism. The leucine source has a leucine content on a free amino acids basis of at least about 400 grams per kilogram dry solids. [0120] At least a portion of the leucine source may be protected against rumen degradation. In some embodiments, at least about 50%, 60%, 70% and suitably 80% of the leucine present in the rumen-protected leucine source is capable of being delivered post-ruminally.

[0121] In another embodiment, a feed composition is provided. The feed composition includes a rumen-protected leucine source and at least one additional nutrient component. The rumen-protected leucine source includes rumen-protected L- Leu and/or a rumen-protected leucine-rich protein of non-animal origin. In some embodiments, at least about 50%, 60%, 70% and suitably 80% of the leucine present in the rumen-protected leucine source is capable of being delivered post-ruminally. [0122] In some embodiments, the leucine source has a leucine content on a free amino acids basis of at least about 400 grams per kilogram dry solids. The leucine- rich protein, which may be present in the rumen-protected leucine source, may have a leucine content of at least about 15% relative to total number of amino acids in the protein.

[0123] In some embodiments, the rumen-protected L-Leu and/or the rumen- protected leucine rich protein of non-animal origin has been reacted with at least one reducing sugar (e.g., lactose and/or xylose). In some embodiments, the rumen- protected L-Leu and/or the rumen-protected leucine rich protein of non-animal origin has been coated with a coating mixture that includes at least one fatty acid. The coating mixture may include partially hydrogenated vegetable oil (e.g., soy bean oil), and/or a surfactant.

[0124] In other embodiments, a feed composition is provided. The feed composition includes a rumen-protected leucine source having at least about 40 wt.% (dry solids basis) L-Leu free amino acid. The feed composition may have a crude protein fraction which has a leucine content of at least about 10.3 wt.%. In one embodiment, the feed includes a crude protein fraction which has a leucine content of about 10.5 to 12.0 wt.%.

[0125] The rumen-protected leucine source may include fermentation constituents from fermentation of a leucine-producing microorganism.

[0126] In some embodiments, the leucine source is rumen-protected by reacting the leucine source with at least one reducing sugar to provide a rumen-protected leucine source. The reducing sugar may include lactose and/or xylose. [0127] The leucine source may be coated with a coating mixture that includes at least one fatty acid to provide a rumen-protected leucine source. For example, the leucine source may be coated with a hydrophobic mixture that includes a partially hydrogenated vegetable oil, such as soy bean oil. The hydrophobic mixture may include a wax, such as beeswax. The leucine source may be coated with surfactant. In some embodiments, the leucine source is coated with a hydrophobic mixture and then subsequently is coated with a surfactant.

[0128] In some embodiments, when the feed composition is fed to ruminant, at least about 50% of leucine present in the rumen-protected leucine source may be capable of being delivered post-ruminally. More commonly, when the feed composition is fed to ruminant, at least about 60%, 70%, and suitably 80% of leucine present in the rumen- protected leucine source may be capable of being delivered post-ruminally. [0129] In one embodiment, a feed composition is provided. The feed composition includes a threonine source and at least one additional nutrient component. The threonine source includes L-Thr and fermentation constituents from fermentation of a threonine-producing microorganism. The feed composition has a crude protein fraction having a threonine content of at least about 6.2 wt.%. Commonly, the feed composition has a crude protein fraction having a threonine content of about 6.4 - 9.3 wt.%, 6.4 - 8.8 wt.%, and suitably 6.4 - 8.6 wt.%.

[0130] In some embodiments, the crude protein fraction may represent at least about 10 wt.% of the feed composition. Commonly, the crude protein fraction represents at least about 14-19 wt.% of the feed composition.

[0131] At least a portion of the threonine source may be protected against rumen degradation. For example, the L-Thr present in the threonine source may be reacted with a reducing carbohydrate and/or coated with a coating mixture. The coating mixture may include at least one fatty acid. The coating mixture may include partially hydrogenated vegetable oil (e.g., soybean oil) and/or a surfactant. [0132] The fermentation constituents may include soluble and/or insoluble constituents from the fermentation broth formed during fermentation of the threonine- producing microorganism. The fermentation constituents may include dissolved and/or undissolved constituents from the fermentation broth formed during fermentation of the threonine-producing microorganism. The fermentation constituents may include biomass formed during fermentation of the threonine- producing microorganism.

[0133] In some embodiments, the threonine-producing microorganism is a Corynebacterium. In other embodiments, the threonine-producing microorganism is a Brevibacterium.

[0134] In some embodiments, the threonine source is rumen-protected and the feed composition provides, post-ruminally, a desirable amount of the threonine present in the rumen-protected threonine source. For example, in some embodiments, the feed composition may provide at least about 50% of the rumen-protected threonine post- ruminally. For example, about 1 g of threonine present in the rumen-protected threonine source may result in about 500 mg of the threonine present in the rumen- protected threonine source being delivered post-ruminally. In other embodiments, at least about 60%, 70%, and suitably 80% of threonine present in the rumen-protected threonine source is capable of being delivered post-ruminally. [0135] In another embodiment, a feed composition is provided. The feed composition includes a threonine source and at least one additional nutrient compound. The threonine source includes L-Thr and fermentation constituents from fermentation of a threonine-producing microorganism. The threonine source has a threonine content on a free amino acids basis of at least about 400 grams per kilogram dry solids.

[0136] At least a portion of the threonine source may be protected against rumen degradation. In some embodiments, at least about 50%, 60%, 70% and suitably 80% of the threonine present in the rumen-protected threonine source is capable of being delivered post-ruminally.

[0137] In another embodiment, a feed composition is provided. The feed composition includes a rumen-protected threonine source and at least one additional nutrient component. The rumen-protected threonine source includes rumen-protected L-Thr and/or a rumen-protected threonine-rich protein of non-animal origin. In some embodiments, at least about 50%, 60%, 70% and suitably 80% of the threonine present in the rumen-protected threonine source is capable of being delivered post- ruminally.

[0138] In some embodiments, the threonine source has a threonine content on a free amino acids basis of at least about 400 grams per kilogram dry solids. The threonine- rich protein, which may be present in the rumen-protected threonine source, may have a threonine content of at least about 10% relative to total number of amino acids in the protein.

[0139] In some embodiments, the rumen-protected L-Thr and/or the rumen- protected threonine rich protein of non-animal origin has been reacted with at least one reducing sugar (e.g., lactose and/or xylose). In some embodiments, the rumen- protected L-Thr and/or the rumen-protected threonine rich protein of non-animal origin has been coated with a coating mixture that includes at least one fatty acid. The coating mixture may include partially hydrogenated vegetable oil (e.g., soy bean oil), and/or a surfactant.

[0140] In other embodiments, a feed composition is provided. The feed composition includes a rumen-protected threonine source having at least about 40 wt. % (dry solids basis) L-Thr free amino acid. The feed composition may have a crude protein fraction which has a threonine content of at least about 6.2 wt.%. In one embodiment, the feed includes a crude protein fraction which has a threonine content of about 6.4 to 9.3 wt.%.

[0141] The rumen-protected threonine source may include fermentation constituents from fermentation of a threonine-producing microorganism.

[0142] In some embodiments, the threonine source is rumen-protected by reacting the threonine source with at least one reducing sugar to provide a rumen-protected threonine source. The reducing sugar may include lactose and/or xylose.

[0143] The threonine source may be coated with a coating mixture that includes at least one fatty acid to provide a rumen-protected threonine source. For example, the threonine source may be coated with a hydrophobic mixture that includes a partially hydrogenated vegetable oil, such as soy bean oil. The hydrophobic mixture may include a wax, such as beeswax. The threonine source may be coated with surfactant.

In some embodiments, the threonine source is coated with a hydrophobic mixture and then subsequently is coated with a surfactant.

[0144] In some embodiments, when the feed composition is fed to ruminant, at least about 50% of threonine present in the rumen-protected threonine source may be capable of being delivered post-ruminally. More commonly, when the feed composition is fed to ruminant, at least about 60%, 70%, and suitably 80% of threonine present in the rumen-protected threonine source may be capable of being delivered post-ruminally.

LIST OF ILLUSTRATIVE EMBODIMENTS

[0145] Embodiment 1. A feed composition comprising: (a) a leucine source which includes L-Leu and fermentation constituents from fermentation of a leucine- producing microorganism; and (b) at least one additional nutrient component; wherein the feed composition has a crude protein fraction having a leucine content of at least about 10.3 wt.%.

[0146] Embodiment 2. The feed composition of embodiment 1, comprising at least about 10 wt.% of the crude protein fraction. [0147] Embodiment 3. The feed composition of embodiment 1, wherein at least a portion of the leucine source is protected against rumen degradation. [0148] Embodiment 4. The feed composition of embodiment 1, wherein the fermentation constituents include at least one of soluble and insoluble constituents from a fermentation broth formed during fermentation of the leucine-producing microorganism.

[0149] Embodiment 5. The feed composition of embodiment 1, wherein the fermentation constituents include at least one of dissolved and undissolved constituents from a fermentation broth formed during fermentation of the leucine- producing microorganism.

[0150] Embodiment 6. The feed composition of embodiment 1, wherein the fermentation constituents include biomass formed during fermentation of the leucine- producing microorganism.

[0151] Embodiment 7. The feed composition of embodiment 1, wherein the leucine-producing microorganism is a Corynebacterium.

[0152] Embodiment 8. The feed composition of embodiment 1, wherein the leucine producing microorganism is a Brevibacterium.

[0153] Embodiment 9. The feed composition of embodiment 1, wherein at least about 50% of leucine present in the rumen-protected leucine source is capable of being delivered post-ruminally.

[0154] Embodiment 10. A feed composition comprising: (a) a leucine source which includes L-Leu and fermentation constituents from fermentation of a leucine- producing microorganism; and (b) at least one additional nutrient component; [0155] wherein the leucine source has a leucine content on a free amino acids basis of at least about 400 grams per kilogram dry solids.

[0156] Embodiment 11. The feed composition of embodiment 10, wherein at least a portion of the leucine source is protected against rumen degradation. [0157] Embodiment 12. The feed composition of embodiment 10, wherein at least about 50% of leucine present in the rumen-protected leucine source is capable of being delivered post-ruminally. [0158] Embodiment 13. A feed composition comprising a rumen-protected leucine source which includes at least about 40 wt.% (dsb) L-Leu.

[0159] Embodiment 14. The feed composition of embodiment 13, wherein the feed composition has a crude protein fraction which has a leucine content of at least about 10.3 wt.%.

[0160] Embodiment 15. The feed composition of embodiment 13, having a crude protein fraction which has a leucine content of about 10.5 - 12.0 wt.%. [0161] Embodiment 16. The feed composition of embodiment 13, wherein the rumen-protected leucine source further comprises fermentation constituents from fermentation of a leucine-producing microorganism.

[0162] Embodiment 17. The feed composition of embodiment 13, wherein at least about 50% of leucine present in the rumen-protected leucine source is capable of being delivered post-ruminally.

[0163] Embodiment 18. The feed composition of embodiment 13, wherein the rumen-protected leucine source includes leucine which has been reacted with at least one reducing sugar.

[0164] Embodiment 19. The feed composition of embodiment 18, wherein the at least one reducing sugar includes lactose.

[0165] Embodiment 20. The feed composition of embodiment 13, wherein the leucine source has been coated with a coating mixture that includes at least one fatty acid.

[0166] Embodiment 21. The feed composition of embodiment 20, wherein the coating mixture includes partially hydrogenated vegetable oil. [0167] Embodiment 22. The feed composition of embodiment 13, comprising at least about lg/kg of the rumen-protected leucine source.

[0168] Embodiment 23. A feed composition comprising: (a) a rumen-protected leucine source which includes at least one of (i) rumen-protected L-Leu, (ii) a rumen- protected leucine rich protein of non-animal origin; and (iii) a mixture thereof; and (b) at least one additional nutrient component. [0169] Embodiment 24. A feed composition comprising: (a) a threonine source which includes L-Thr and fermentation constituents from fermentation of a threonine- producing microorganism; and (b) at least one additional nutrient component; [0170] wherein the feed composition has a crude protein fraction having a threonine content of at least about 6.2 wt.%.

[0171] Embodiment 25. The feed composition of embodiment 24, comprising at least about 10 wt.% of the crude protein fraction.

[0172] Embodiment 26. The feed composition of embodiment 24, wherein at least.a portion of the threonine source is protected against rumen degradation. [0173] Embodiment 27. The feed composition of embodiment 24, wherein the fermentation constituents include at least one of soluble and insoluble constituents from a fermentation broth formed during fermentation of the threonine-producing microorganism.

[0174] Embodiment 28. The feed composition of embodiment 24, wherein the fermentation constituents include at least one of dissolved and undissolved constituents from a fermentation broth formed during fermentation of the threonine- producing microorganism.

[0175] Embodiment 29. The feed composition of embodiment 24, wherein the fermentation constituents include biomass formed during fermentation of the threonine-producing microorganism.

[0176] Embodiment 30. The feed composition of embodiment 24, wherein the threonine-producing microorganism is a Corynebacterium. [0177] Embodiment 31. The feed composition of embodiment 24, wherein the threonine-producing microorganism is a Brevibacterium.

[0178] Embodiment 32. The feed composition of embodiment 24, , wherein at least about 50% of threonine present in the rumen-protected threonine source is capable of being delivered post-ruminally.

[0179] Embodiment 33. A feed composition comprising: (a) a threonine source which includes L-Thr and fermentation constituents from fermentation of a threonine- producing microorganism; and (b) at least one additional nutrient component; [0180] wherein the threonine source has a threonine content on a free amino acids basis of at least about 400 grams per kilogram dry solids.

[0181] Embodiment 34. The feed composition of embodiment 33, wherein at least a portion of the threonine source is protected against rumen degradation.

[0182] Embodiment 35. The feed composition of embodiment 33, , wherein at least about 50% of threonine present in the rumen-protected threonine source is capable of being delivered post-ruminally.

[0183] Embodiment 36. A feed composition comprising a rumen-protected threonine source which includes at least about 40 wt.% (dsb) L-Thr.

[0184] Embodiment 37. The feed composition of embodiment 36, wherein the feed composition has a crude protein fraction which has a threonine content of at least about 6.2 wt.%.

[0185] Embodiment 38. The feed composition of embodiment 36, having a crude protein fraction which has a threonine content of about 6.4 to 9.3 wt.%.

[0186] Embodiment 39. The feed composition of embodiment 36, wherein the rumen-protected threonine source further comprises fermentation constituents from fermentation of a threonine-producing microorganism.

[0187] Embodiment 40. The feed composition of embodiment 36, wherein at least about 50% of threonine present in the rumen-protected threonine source is capable of being delivered post-ruminally.

[0188] Embodiment 41. The feed composition of embodiment 36, wherein the rumen-protected threonine source includes threonine which has been reacted with at least one reducing sugar.

[0189] Embodiment 42. The feed composition of embodiment 41, wherein the at least one reducing sugar includes lactose.

[0190] Embodiment 43. The feed composition of embodiment 36, wherein the threonine source has been coated with a coating mixture that includes at least one fatty acid.

[0191] Embodiment 44. The feed composition of embodiment 43, wherein the coating mixture includes partially hydrogenated vegetable oil. [0192] Embodiment 45. The feed composition of embodiment 36, comprising at least about lg/kg of the rumen-protected threonine source. [0193] Embodiment 46. A feed composition comprising: (a) a rumen-protected threonine source which includes at least one of (i) rumen-protected L-Thr, (ii) a rumen-protected threonine-rich protein of non-animal origin; and (iii) a mixture thereof; and (b) at least one additional nutrient component.

EXAMPLES

Maillard Reaction Protocols

[0194] 1. Equal amounts of sugar (1.5 g xylose, fructose, lactose, or glucose) and amino acid (1.5 g leucine or threonine) are placed in a 50 ml centrifuge tube. Water is added (0.9 ml) and the tube is capped. The tubes are incubated in a 80⁰C water bath for up to 2 hours. Samples are freeze dried, then redissolved in 40 ml H₂O. Maillard Reaction Products are detected by measuring the absorbance at 420nm. Samples are diluted in water, if necessary, to obtain an absorbance of less than 2.0 absorbance units.

[0195] 2. Leucine or threonine (0.5g) and dialdehyde starch (0.5g) are dissolved in 10 ml 0.05M sodium phosphate buffer (pH 8.0) in a 50 ml centrifuge tube. The tube is capped and incubated at 65°C or 100⁰C for up to 4 hours. Solubilized Maillard products are stored at 4°C. Maillard Reaction Products are detected by measuring the absorbance at 420nm. Samples are diluted in water, if necessary, to obtain an absorbance of less than 2.0 absorbance units.

Determination of Maillard-Protected Leucine and/or Threonine Degradation In Vivo

[0196] Free Amino Acid degradation in vivo may be determined relative to Co¹¹ in fistulated cows. Free leucine or leucine modified by the Maillard Reaction Protocol (and/or free threonine or threonine modified by the Maillard Reaction Protocol) are introduced into fistulated cows together with Cobalt II as a marker for digestion. After introducing the leucine or modified leucine (and/or threonine or modified threonine) into the cows, samples are periodically withdrawn and the amount of leucine (and/or threonine) is determined using standard protocols known in the art. The amount of leucine and/or threonine related to Co¹¹ is plotted versus time to calculate a degradation rate (k_d). The degradation rate for free leucine (and/or free threonine) is compared to the degradation rate for modified leucine (and/or modified threonine). Preparation of Coated Amino Acids

[0197] Coated leucine and/or Coated threonine is prepared by spraying commercial grade L-Leu and/or commercial grade L-Thr with a mixture of partially hydrogenated soy bean oil and wax to prepare a pre-coated L-Leu substrate and/or pre-coated L-Thr substrate. The pre-coated substrate may then be optionally subsequently coated with a surfactant using the methodology substantially as described in U.S. Patent Nos. 5,190,775; 6,013,286; and 6,106,871, the entire contents of which are incorporated herein by reference in their entireties.

Determination of Coated Amino Acid Degradation In Vitro

[0198] Coated amino acid degradation in vitro is determined by incubating various concentrations of free leucine (and/or free threonine) or coated leucine (and/or coated threonine) in a suitable degradation solution. Degradation rates are determined for various concentrations of amino acid, and plotted versus amino acid concentration.

[0199] AU references, patents, and/or applications cited in the specification are indicative of the level of skill of those skilled in the art to which the invention pertains, and are incorporated by reference in their entireties, including any tables and figures, to the same extent as if each reference had been incorporated by reference in its entirety individually.

[0200] It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. [0201] In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group. [0202] Also, unless indicated to the contrary, where various numerical values are provided for embodiments, additional embodiments are described by taking any 2 different values as the endpoints of a range. Such ranges are also within the scope of the described invention.

Claims

1. A feed composition having a crude protein content of at least about 14 wt. % and comprising at least one of:

(a) a rumen-protected leucine source, wherein leucine comprises at least about 10.3 wt.% of the crude protein content; and

(b) a rumen-protected threonine source, wherein threonine comprises at least about 6.2 wt.% of the crude protein content.

2. The feed composition of claim 1, comprising a rumen-protected leucine source, wherein leucine comprises at least about 10.3 wt.% of the crude protein content.

3. The feed composition of claim 2, wherein the rumen-protected leucine source has an L-Leu content of at least about 40 wt.% (dsb).

4. The feed composition of claim 2, wherein the composition is capable of delivering at least about 50% of the rumen-protected leucine post-ruminally for intestinal absorption.

5. The feed composition of claim 2, wherein the rumen-protected leucine source includes leucine which has been reacted with at least one reducing sugar.

6. The feed composition of claim 2, wherein the rumen-protected leucine source comprises a coating.

7. The feed composition of claim 1, comprising a rumen-protected threonine source, wherein threonine comprises at least about 6.2 wt.% of the crude protein content.

8. The feed composition of claim 7, wherein the rumen-protected threonine source has an L-Thr content of at least about 40 wt.% (dsb).

9. The feed composition of claim 7, wherein the composition is capable of delivering at least about 50% of the rumen-protected threonine post-ruminally for intestinal absorption.

10. The feed composition of claim 7, wherein the rumen-protected threonine source includes threonine which has been reacted with at least one reducing sugar.

11. The feed composition of claim 7, wherein the rumen-protected threonine source comprises a coating.

12. The feed composition of claim 6 or 11 , wherein the coating comprises a lipid component.

13. The feed composition of claim 12, wherein the lipid component comprises a fatty acid.

14. The feed composition of claim 12, wherein the coating further comprises a surfactant.

15. The feed composition of claim 6 or 11, wherein the coating comprises zinc stearate.

16. The feed composition of claim 1, wherein at least one of the leucine source and threonine source includes fermentation constituents from fermentation of a microorganism.

17. The feed composition of claim 1 comprising:

18. The feed composition of claim 1, further comprising at least one of:

(c) a rumen-protected histidine source;

(d) a rumen-protected lysine source;

(e) a rumen-protected methionine source; and

(f) a rumen-protected phenylalanine source.

19. The feed composition of claim 18, comprising a rumen-protected histidine source, wherein histidine comprises at least about 3-7 wt.% of the crude protein content.

20. The feed composition of claim 19, wherein the rumen-protected histidine source has an L-His content of at least about 40 wt.% (dsb).

21. The feed composition of claim 19, wherein the composition is capable of delivering at least about 50% of the rumen-protected histidine post-ruminally for intestinal absorption.

22. The feed composition of claim 19, wherein the rumen-protected histidine source includes histidine which has been reacted with at least one reducing sugar.

23. The feed composition of claim 19, wherein the rumen-protected histidine source comprises a coating.

24. The feed composition of claim 18, comprising a rumen-protected lysine source.

25. The feed composition of claim 24, wherein the rumen-protected lysine source has an L-Lys content of at least about 40 wt.% (dsb).

26. The feed composition of claim 24, wherein the composition is capable of delivering at least about 50% of the rumen-protected lysine post-ruminally for intestinal absorption.

27. The feed composition of claim 24, wherein the rumen-protected lysine source includes lysine which has been reacted with at least one reducing sugar.

28. The feed composition of claim 24, wherein the rumen-protected lysine source comprises a coating.

29. The feed composition of claim 23 or 28, wherein the coating comprises a lipid component.

30. A method for increasing milk production in a ruminant comprising feeding the ruminant the composition of claim 1.

31. The method of claim 30, wherein the rumen-protected leucine source comprises a coating and the rumen-protected threonine source comprises a coating.

32. The method of claim 31 , wherein the coating comprises a lipid component.

33. The method of claim 32, wherein the lipid component comprises a fatty acid.

34. The method of claim 32, wherein the coating further comprises a surfactant.