Title of Invention	"High Tryptophan Soybean Meals and Methods of Preparing The Same"
Abstract	Abstract "HIGH TRYPTOPHAN SOYBEAN MEAL" The present invention is directed to a soybean meal with high tryptophan content and its method of manufacture. The high tryptophan content soybean meal is to be used as an ingredient in animal feeding operations. Also provided are products from the further processing of the soybean meal.

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The present invention involves die fields of genetic engineering, plant 5 breeding, grain processing, and animal nutrition. The present invention relates to a novel high tryptophan soybean meal to be used as an ingredient in animal feeding operations. Animal species raised for meat Jack the ability to manufacture a number of •• amino acids and tlierefore are required to obtain these amino acids from their d;^et, 10 The amino acids which must be obtained from the diet are referred to as essential amino acids. Plants are able to synthesize all twenty of the essential amino acids and therefore serve as the primaiy source of these amino acids for animals. Tryptophan is one of these essential amino acids, and at the same time, is unden-epresented in the amino acid profile of many feed ingredients. 15 Economical sources of protein, such as, by-products from the com milling and animal rendering plants, are commonly used in animal feeds. Examples of these types "of by-products Include corn gluten meal, distiller's grains with solubles, meat and bone meal, feather meal, and poultry meal. Unfortunately, the tryptophan content in these by-products is deficient for various animal requirements, and therefore limits the 20 amounts that may be used in certain feed formulations. Soybean meal is one of the major ingredients of animal feed that provides protein and essential amino acids. Vv'hen soybean meal is formulated in feed rations, the inclusion rate is typically calculated based on satisfying the most limiting essential amino acid. This limiting essential amino acid is typically tryptophan, resulting in the 25 remaining essential amino acids being formulated in excess of dietary requirements- The excess amino acids end up as waste. The need therefore exists to provide soybean meals with higher concentrations of tryptophan. SUMMARY OF THE INVENTION The present invention described herein relates to a high tryptophan content 30 soybean meal derived from the processing of one or more soybeans having a high total tryptophan content. The present invention includes the use of a high tryptophan content soybean jneal in the animal feed industry. 2 Thus, m a first aspect, the present invention is directed to a soybean meal having a total tryptophan content greater than about 0.78 weight percentage on a dry matter basis (wt.%), wherein no exogenous tryptophan has been added. In one embodiment of the present invention, the soybean mea! has at least about O.IO wt.% 51. free tryptophan. In another embodiment the soybean meal has at least about 0.43 wt.% free tryptophan. In a further embodiment, the soybean meal has a protein content of at least about 44 wt.% or higher. In addition the soybean meal may further have a protein bound tryptophan content comprising transgenlcally modified protein, wherein the transgenlcally modified protein contains at least 8 wt.% tryptophan 10 residues. The present invention relates to a method of making a soybean meal having at least about 0,78 wt.% total tryptophan comprising; introducing into regenerable cells « '• of a soybean plant a trahsgene comprising an isolated nucleic acid molecule encoding an enzyme in the tryptophan biosynthetic pathway, wherein the isolated nucleic acid 15 molecule is operably linked to a promoter functional in a plant cell, to yield transformed plant cells; and regenerating a plant from said transformed plant cells wherein the cells of the plant express the enzyme encoded by the isolated nucleic acid molecule in an amount effective to increase the tryptophan content in the soybean grain of the plant relative to the tryptophan content in the grain of an untransformed 20 soybean plant of the same genetic background; and producing a soybean meal from the grain of the transformed plant, In one aspect of the present invention the method includes a transgene which encodes a monomeric anthranjiate synthase comprising an anthraniiate synthase alpha domain and an anthraniiate synthase beta domain. The method further includes a 25 transgene that encodes a feedback insensitive maize anthraniiate synthase alphasubunit. The method frirther includes any of the transgenes that encode phosphoribosylanthranilate transferase, phosphoribosylanthranilate isomcrase, indolet 3-phosphate synthase, or tryptophan synthase. In another aspect, the present invention is directed to a method of producing a 30 high tryptophan content soybean meal comprising: a) selecting soybean grain having a total tiyptopJian content of greater than about 0,65 wt.%; and b) extracting an oil from said grain to produce a soybean meal. In one embodiment of the present 3 invention, the method of producing a high tryptophan content soybean meal may also use a soybean grain having a free tryptophan of greater than about 0,15 •wt.%. In another aspect, the present invention is directed to incorporating the soybean meal into animal feed, including feed for animal producer, feed for 5 companion animals, and feed for aquacuiture. The soybean meal of the present invention is also useftil as a fermentation feed source. I. In another aspect, the present invention is directed to a high tryptophan content, fiill fat soybean meal for use in animal feeds, The high tryptophan content, fill! fat soybean meal may optionally be extruded. 10 In another aspect, the present invention is directed to a high tryptophan content soybean isolate or soybean protein concentrate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention describes a new feed ingredient, a high tryptophan ^ content soybean meal. The meal of the present invention is useful in animal feeding 15 operations, as an aquacuiture feed source and as a component of a fermentation media. The following de/mitions are used herein; Exogenous Tryptophan: Tryptophan that is not an intrinsic part of the soybean from which the soybean meal has been produced, Exogenous tryptophan may be 20 '• added to the meal or to the feed, in order to increase the concentration. Free Tryptophan: Tryptophan in the free acid form and not part of an oligopeptide, polypeptide, or protein, i Full Fat Soybean Meal; A soybean product, produced similar to .soybean meal, except omitting the oil extraction step. 25 Protein Content: Weight percentage of protein contained in soybean seeds or soybean meal. Soybean Meal: A feed ingredient that is a product of processing soybean "grain. The phrase "soybean meal", as used herein, refers to a'defatted, desolventized, toasted, and ground soybean materia!, 30 Soybean Protein Isolate: A preparation from soybean grain made by removing the majority .of non-protein components and containing not less than 90% protein on a moisture-fi^ee basis. ' 4 Soybean Protein Concentrate: A preparation from soybean grain made by '• removing most of the oil and water soluble non-protein constituents and containing not less than about 65% protein on a moisture-free basis. Transgenc: A nucleic acid molecule, including at least a pronnoter sequence, a 5 coding region, and a transcription termination sequence, inserted into the genome of a cell via gene splicing techniques. Total Tiyptophan Content: The sum of free tryptophan and protein bound tryptophan contents. Free Tryptophan Content: The weight percentage of free tryptophan of the to ' soybean grain or soybean meal. Protein Bound Tryptophan Content: The weight percentage of tryptophan that Is Incorporated into proteins or peptides in the soybean seed or soybean meal. The phrases "protein bound tryptophan" and "peptide bound tr>'ptophan" arc herein used interchangeably. 15 High Tryptophan Soybean Varieties The high tryptophan content soybean meal of the present invention involves the use of a high tryptophan soybean variety or varieties. There are various methods of producing a high tryptophan soybean variety. Tryptophan in soybean grain e.xists in two different forms: protein bound and. 20 free, Technical approaches for increasing the concentration of free tryptophan in grain include: 1) increasing synthesis, 2) decreasing degradation, or 3) increasing transport from the site of synthesis to the site of storage. Additionally, tlie combination of any or all of above approaches can be used to achieve optimal results. Increased synthesis of tryptophan in soybean plants can be achieved by 1) over 25 expressing a key enzyme or enzymes in the biosyrithetic pathway, or 2) expressing at least one key enzyme in the biosyntlietic pathway that is less sensitive or insensitive to the feedback inhibition as compared to the corresponding endogenous enzyme. Examples of these methods are described in US, Patent Publication Nos. 2003/0097677 and 2003/0213010, iiereln incorporated by reference. 30 Decreased degradation of tryptophan can be achieved by 1) reducing the amount of the enzyme(s) responsible for degradation, or 2) reducing the effectiveness of the degradation enzymes by expressing aninhibitor of that enzyme; or 3) by expressing a mutant form of the degradation enzyme that would competitively inhibit 5 ^ I, the activity of tiie native enzyme. The amount of tiie enzyme may be reduced by gene suppression techniques such as antisense suppression, sense co-suppression, RNA interference, or other techniques well known in the art. Plants possess multiple forms of amino acid transporters characterized 5 according to their specificity for, or affinity lo. individual amino acids. Over expression of a tryptophan transporter or expression of a more effective tryptophan transporter would facilitate transport of tryptophan from the plastJds to other compartments such as cytosohc space, extra cellular space, or vacuoles. See, for example, U.S. Patent Publication No. 2003/0188332. 10 Protein bound tryptophan can be increased by over expressing a storage protein that contains a high level of tryptophan. The high tryptophan protein can be a native protein or a modified form of a native protein. Examples of these methods are disclosed in PCT Applications WO 98/45458, WO 98/20133, and WO 99/29882. Additionally, protein bound tryptophan can be increased on a weight 1S percentage basis by increasing the overall concentration of protein in soybean grain, relative to other components such as carbohydrate and lipid. High protein soybeans can be obtained fay screening the natural germplasm of* soybeans or mutant populations of soybeans. Another method of increasing protein bound tryptophan is to suppress the 20 expression of native storage proteins that are inherently low in'tryptophan. In this method, the amino acid composition of the grain changes in favor of higher levels of tryptophan as compared to a non-suppressed parental line. An example of this method, as specifically applied to corn, but applicable to soybeans, is disclosed in U.S. Patent 6,326,527, 25 A further method of increasing protein bound tryptophan in soybeans is to engineer the nucleic acid sequences encoding a major storage protein by substituting tryptophan codons in place of those coding for other amino acids. The resulting expressed protein, tlios, has higher levels of tryptophan, thereby increasing the total tryptophan level in the plant. An example of this method is disclosed in U.S. Patent 30 Publication No. 2003/0200558. In yet another method, free tryptophan levels can be increased in a target tissue, and at the same time, a complementary protein sink can be created, which 6 results in an increase in protein bound tryptophan. An example of this method is described in U.S. Patent 6,080,913, One of ordinary skill in tlie art will recognize that other methods of producing • a soybean grain having high tryptophan content exist, and may be used to generate the 5 high tryptophan content soybean meal of the present invention. . Soybean Processing and Products In one aspect of the present invention, high tryptophan soybeans arc processed into high tryptophan content soybean meal. IMany methods are Icnqwn for the processing of raw soybeans into soybean meal. The high tryptophan content soybean 10 meal of the present invention may be prepared using these methods to process high tryptophan soybean grain. Illustrative processes for soybean meal preparation include those taught in U.S. Patents 4,992,294; 5.225,230; 5,773,051; and 5,866,192. Typically, commercial soybean processes start with the step of receiving the soybeans from the field by any 15 conventional transport means. The soybeans are typically received in a dirty and often wet condition and may be cleaned with a vibrating screen. In this step the soybeans are separated from non-soybean material, for example, rocks, sticks, leaves, stems, dirt, weed seeds, and unwanted fragments of soybeans. The cleaned soybeans, in combination witli the loose hulls that are not removed by the vibrating screen, are 20 transferred to an aspirator in which most of the remaining loose hulls are removed by air, The soybeans are then transferred to storage, and the removed loose hulls are collected as a by-product for further processing. At this point in the processing, the soybeans typically contain about 12% water, but the actual water content of the soybeans may vary based on a host of 25 different factors. If tlie water content of the soybeans is in excess of about 12%, then the soybeans may be subjected to a drj'ing step to reduce the water content below about 12% prior to placing in storage. The control of the water content is essential to prevent mold and microbial contamination during storage. The processing procedures from this point forward may vary depending upon 30 the desired end products. For example, the soybeans may be first dehulled using such conventional equipment as cracking roils or hammer mills in combination with a conventional aspiration system. Alternatively, the hulls may not be removed prior to furtlier processing. See, for example, U.S. Patent 5,225,230. In order to deactivate 7 antinutritional factors, such as trypsin inhibitors, the soybeans may be subjected to For cracking processes, clean, dry, whole soybeans are fed to coarsely corrugated roller mills or "crackers." These crackers can have one or more sets of 5 rolls. Soybean pieces, called "cracks," are formed. The goal of the cracking step is to maximize the pieces that are 1/4"* to l/S"* the size of the starting soybean, and to minimize the formation of fines, which are pieces less than 1 mm in diameter. From the cracking milts, particles ot"whole soybeans (cracks) are conveyed to multistage aspiration dehulling systems, which typically employ 1 to 3 stages. Bach 10 stage consists of an aspirator and a size screening system. At each stage, the fiberrich hulls are first removed by means of a coimtercurrent air stream and a cyclone. The heavier, fiber-lean, meats fraction is conveyed to a screening system that removes at least one additional fraction by size, and yields one stream for fiirther aspiration. Alternatively, screening can be employed prior to aspiration. The "hulls" stream is 15 typically combined with other soybean byproducts and used as an animal feed ingredient. The once dehuUed meats are then dehulled a second time to bring them to less than about 3% crude fiber (4.28% crude fiber on a defatted, dry basis) using a 2 stage commercial pre-extraction process. However, the single stage systems can also be employed to yield meats. 20 The resulting meats are then heat conditioned, such as in a rotary or stack cooker. The residence times of the cracks are typically between about 20 and about 40 minutes. Discharge temperatures typically are in the range of about 120 to 180°F. Lower conditioning temperatures may be employed if a greater fines production in tlie flaker is tolerable. 25 The conditioned meats are then fed to smooth roller mills called flakers. A force of greater than about 500 IcPa-gaugc (72.5 psig) is typically applied to the rolls. Flake thicltnesses of less than about 0,75 nun (0.030") are preferably produced' in order to obtain maximum oil recovery in the subsequent oil extraction,step.- Optionaily, the cracking and dehulling steps are eliminated, or done subsequent to the 30 conditioning step. An additional option would be to expand a percentage of the flaked soybeans to form "collets" prior to oil extraction. Other process variations include conditioning prior to the cracking step, and eliminating the dehulling step prior to oil extraction. A soybean meal of the present invention produced In a process 8 '•• having the variation of eliminating the dehulling step would be considered a high tryptophan and high fiber soybean nicaJ, This product could be a specialty feed ingredient in a swine production operation. The next step iii the process of generating soybean meal is the extraction of 5 oil. This extraction step is typically done using a lipophilic solvent, but may also be done by mechanical extraction. In tiiis process, the soybean meal is contacted with a suitable solvent (e.g., hexane) to remove tlie oil to a content of typically less than about 1% by weight. One example of a conventional solvent extraction procedure is . ' described in U.S. Patent 3.721.569. 10 However, if a "full fat" soybean meal is desired.then the oil bearing meal is not subjected, to oil (also known as fat or lipid) extraction. In this embodiment of the present invention, the resulting product would be a high tryptophan content, full fat soybean meal. ' At this stage, the solvent extracted, defatted soybean meal tj^pically contains 15 about 30% solvent by weight. Prior to being used as an animal feed, the meal is typically processed through a desolventizer-toaster 03T) to remove the residua! solvent and to heat the protein fraction sufficient to inactivate trypsin inhibitors and other naturall)' occurring toxicants (antifeedants). Typically, steam contacts the soybean meal and the Itcat of vaporization released fi'om the condensing steam 20 vaporizes the solvent, which is subsequently recovered and recycled. As an alternative to solvent extraction, the soybean meal is defatted mechanically using, for example, a screw piess. This mechanioally extracted or "expeller" soybean meal typically contains between about4 ind about 8% residual oil. If the intended use of the meal is as a feed supplement for ruminants, then the meal 25 may first be heated and dried in a specified hianner, such as that taught in U.S; Patent 5,225,230, before oil is extracted mechanically. The defatted soybean meal is then dried and typicaUy ground or pelletized, and then milled into a physical .state suitable for use as a food supplement (jr as an animal feed. Further processing of the soybean or the meal may optionally be done to make 30 the resulting feed more palatable, available, and/or digestible in animals. These processes include addition of enzymes or nutrients, and heat treatment of the meal. Additionally, further processing may be done to the nseal, such as pelleting, to make it more compact and dense in distribution. 9 Further processing of the soybean mea! can produce soybean floxir, soybean protein concentrates, and soybean protein isolates that have food, feed, and industrial uses. The high tryptophan content soybean mea! of the present invention can be further processed into any of the products described below. 5 Soybean flours are produced simply by grinding and screening the defatted soybean meol. Soybean protein concentrates, having at least about 65 wt.% protein, are made by removing soluble carbohydrate material from defatted soybean meal. Aqueous alcohol extraction (60-80% ethanoi in water) or acid leaching at the '•• isoelectric pH 4.5 of the protein are ll)e most common methods bf removing the 3 0 soluble carbohydrate fraction. A myriad of applications ha,ve been developed for soybean protein concentrates and texturized concentrates in processed foods, meat, poultry, fish, cereal, and dairy systems, any of which can be employed with the high tryptophan content soybean mea! of the present invention. Soybean protein isolates are preferaljly produced through standard chemical 15 isolation, drawing the protein out of the defatted soybean flake tlirough solubilization (alkaft extraction at pH 7-10) and separation followed by isoelectric precipitation. As ' a result, isolates are at least about 90 wt.% protein. They are sometimes high in sodium and minerals (ash content), a property that can limit their application. Their major applications have been in dairy substitution, as in infant formulas and milk 20 replacers. Soybean flours are often used in the man\ifacturt«g of meat extenders and analogs, pet foods, baking ingredients, and other food products. Food products made fi^om soybean flour and isolate include baby food, candy products, cereals, foocl drinks, noodles, yeast, beer, ale, and the like. 25 • One of skill in the art v/ili recognize that variations in the above described procedures may be made without departing from the spirit of the present invention. The hightryptophan content soybean meal of the present invention can be fiirther processed into any of the products described above. Feed Formulations 30 The higii tryptophan content soybean meal of the present invention is used in various feed formulations, in a preferred embodiment, the hightryptophan conbnt soybean meal of tiie present invention is used in feed formulations for simple stomach animals, such as swine and pdultry. Due to the higher tryptophan content of the 10 soybean meal of the present invention, inclusion rates arc commonly reduced as compared to commodity soybean meal. Use of the soybean meal of the present invention in feed fonnuktions will reduce or eliminate the need to add exogenous sources of tryptophan. These characteristics of the soybean meal of the present 5 invention provide the benefit to the animal {jroducer and formulator of having more options in feed formulation. Tlie high tryptophan content soybean meal of the present invention allows a formulatpr to use less expensive ingredients in animal feeds which lowers the feed cost for animal producers. Shown in the table below is a comparison of broiler 10 grower diets using the high tryptophan content soybean meal of the present invention (C), a formulation with no animal by-products (A), and a formulation with animal byproducts (B), As can be seen, by being able to use meat and bope meal (MBM) and corn gluten meal with the high tryptophan content soybean meal (HT) of the present '. invention, the cost per ton of feed is reduced 4-6 dollars. 15 ; ^___ ' Ingredients j (A) ' 1 ~~(B) j (Q ' No animal by- With animal by- HT plus animal prodiicts \ products by-products Com 63 64 69 MBM ~ ' ^~~T'~ ""~ 4 ^ • Corn gluten meal 4 - 4 ! I •„ Cost,$/2.000LB I 146 144 140 Listed in the table below are selected feed ingredients and feed formulations, and their crude protein (CP), lysine (Lys), and tryptophan (Trp) contents. It can be seen that certain ingredients containing low tryptophan content yet high protein can be 20 used in formulations with the high tryptophan content soybean meal of the prese/it invention. 25 • 11 Total Amino Adds (%) ' ' Ingredient Name CP Lys I Trp | Trp/Lys (*100) I Trp/CP{*l00l Com gluten feed- 21.5 0,63 ' 0.07 11 0.326 Com gluten meal 60,0 1.02 0.31 30 _ .„___9::ill„_ MBM 51.S 2,61 0.28 ' ' 11 "" "'o.544"" Feather meal 84i5 2,08 0,54 26 0.639 DDGS^ 27.2 0.75 0,19 25 0.700 i Com 8.3 • 0.26 0.06 23 0,723 . Poultry byproduct 64.1 3.32 0.48 14 0.749 Distiller grain 24.8 0.74 . 0.2 27 0,606 Bakefy byproduct 10.8 0.27 0.1 37 0.926 Peanut meal . 49.1 1.66 i 0.48 29 0.978 Sunflower meal 42.2 1.2 0.44 37 1,043 Fish meal, Menhaden 62.9 4.81 0.66 14 1.049 Rice Bran HF 13.3 0.57 0.14 | ' 25 1.053 Mllo 9.2^ 0.22 0.1 I 45 1.087 Cotton meal 41.4 1.72 0.48 26 1.159 Barley 10.5 0.36 0.13 36 1.238 MIdds 15.9 0.57 0,2 35 1.258 Canola meal 35.6 2.08 0.4S 22 1.264 Rice 7.9 0.3 0-1 33 1.266 Soytieanmeal 47.S 3.02 0.6 S ^ 1.368 Blood meal, conventional 77,1 7.04 1.08 15 1.401 Wheat 11.5 0.38 0.26 68 2.261 Feed Formulations ^ Broiler grower 20 1 0.18 18__ 0.900 Layer 15 0.69 0.16 23 1'.067 Turkey Starter B 26 1.S 0.24 16 0.823 Turkey Grower B 19 1 0J8 18 0.947 Turkey Finisher B 14 0.65 0.13 20 0.929 Swine grower 20- SOkg _18 0;9S 0.17 18 0.844 Swine finisher 80- 120 kg ^ J 13.2 . 0.8 I i 0,11 • I 18 1 0.833 'DDGS denotes distiller's dried grains with solubles. Data extracted from NRC pou!tr>' (1994) and NRC swine (1998) The present invention is further detailed in the foiJowing Examples, which are 5 offered by way of illustration and are not intended to limit the present invention in any manner. EXAMPLE 1 This example describes the generation of transgenic high tryptophan soybeans used to prepare tlie high tryptophan content soybean meal of the present invention. 12 The high tryptophan soybeans designated GM_Ai5238:0015, were generated as described by Weaver et al (jU.S. Patent PublicBtion No, 2003/0213010, already incorporated by I'efercnce), Briefly, soybean plants were rransformed with the vector pMON39325, containing the coding sequence for a feedback insensitive maize 5 antliranilate synthase (AS) a-subunit driven by a 7S a' promoter. An event containing a high tryptophan level was selected and numbered GM_A15238, Rl seeds from this event were grown under greenhouse conditions to generate Rl plants. Using the Invader® Assay, (Third Wave Technologies, Inc., Madison, WI) identifications of homozygous and 10 heterozygous plants were made. One gene positive homozygous plant (Gly[_A15238:0015) and one gene negative homozygous piaat (GM_A15238:0017) were selected and advanced to further generations. The generation of soybean grain for high tryptophan content soybean meal preparation was executed under the guidance of USDA regulation for regulated transgenic material {see, for example 7 15 CFR§340). EXAMPLE 2 This example sets forth methods of analysis for free and total tryptophan, and total protein in soybean seeds and meal. Free Tryptophan 20 Amino acids in the soybean meal are detected using a prc-column primary amine dcrivatization with o-phthalaldehyde (OPA). The resulting ammo acidadduct, an isoindole, is hydrophobic and possesses excellent fluorescence characteristics, which can then be detected on a tluorescence detector, chromatography, separation is achieved through the hydrophobicity of the R-groups 25 located on each amino acid. To help stabilize the fluorophor, a thiol is added such as • 2-mercaptoethaiiol or 3-mercaptopropionic acid. Seed and meal samples are ground to 1 mm screen fineness or finer. Ground , samples are stored at 5°C prior to analysis. For analysis the samples are brought to room temperature and then weighed directly into conical centrifuge tubes (2.0 ml 30 capacity). Tlie sample to extraction solvent ratio is equal to or less than 30 mg/ml, A 5% ti-ichloroacetic acid (TCA) solution, (part no.VW3372-l, VWR Scientific, West 13 Chester, PA) is added to each sample and then mixed by vortex for about 30 minutes. The samples are allowed to sit overnight (16 hours) to ensure extraction completion. The samples are then mixed by vortex for about 30 minutes, centrifuged for 30 minutes at 3000 rpm, and the supernatant is 5 saved and stored at -80°C prior to analysis. The amino acids are analyzed by HPLC (model 1100, Agilent Technologies, Inc., Palo Alto, CA) with flourescence detection (FLD) and a Zorbax Eclipse-AAA, XDB C-18 coiiimn, Zorbax Eclipse-AAA guard column, and the following 10 parameters: Analytical time to run method: 14.0 minutes Total elapsed time per nm: Approximately 17 minutes Typical and minimum sample size: Typical: 50 mg Minimum:. 30 mg 15 Typical analytical range: 7.8-800 pmol/nL. ' The mobile phases are (A) 40mM Na^HPO/i Buffer at pH=7.8 with 0.001% sodium azide and (B) acetonitriic: MeOH; H2O (45:45:10 v/v). All reagents are HPLC grade and all solvents are High Purity grade from Honeywell, Burdick and Jackson (Muskegon, Michigan), Below is a chart showing the gradient of tlie mobile 20 phase used and the HPLC settings. Time (min.) %B 0,00 5.0 1.00' 5.0 . 9.80 35.0 12.00 100.0 12,50 5.0 14.00 5.0 Temperature: 40'*C Column Flow: 2.00mL/min FLD Settings: Excitation: 340 nm 25 Emission: 450 nm Peakwidth; > 0.2 min. PMTGnim'lO 14 Fluorescence Scan: Excitation Range: 220-380 nm, Step 5 nm Emission Range: 300-500 nm, Step 5 nm Crude protein analysis follovved AOAC® Official Method 990.03, (2000), (AOAC® International, Gaithersburg, MD); and amino acid profiles followed AOAC® 5 Official Method 982.30 E(a,b,c),CHP. 45.3,05, (2000). EXAMPLE 3 This example sets forth the production of soybean meal k the pilot plant scale. Ijhe soybean meal of the present invention, used in the feeding trials described herein, was prepared at a pilot plant scale, by a solvent extraction process. 10 The high tryptophan soybeans, GM_A15238:0015 (descHbed in Example 1), as well as the parental line A4922 (Asgrow peed Company, Des Moines, lA), and the negative transgcnlo "tsoline, GM_A15238:0017, were cleaned and then dried in a Behlsn Wicks drier (Behlen Manufacturing Company, Columbus, NE) to between 10 and 10.5% moisture. The cleaned and dried soybeans were then stored in covered, ] 5 portable bins for 1 -3 days to allow the meats to loosen from the hulls. The beans were then fed into a single strand Ferreli-Ross (A. T. Ferrell Company Inc., Bluffton, IN) cracking mill. TTie cracking rolls operated at ambient temperature at a gap setting of 8, corresponding to 1.9 mm. TUe rolls operated at a differential speed ratio of 1.5:1 with the slower roll running at 700 ppm. 20 The cracks produced from the cracking mills were conveyed to a multistage aeromechanica! dehulling system (Kice Zigzag Aspirator, Kice Industries, Wichita, KS) to remove the hulls from the meats. The aspirator was operated at an absolute pressure of 1-2.4 inches of water. The resulting hulls were collected and fed into a hammer mill, The product from (lie hammer mill was sent to a gravity table where 25 the meat rich fraction was separated from the hulls and collected. The meats cbllccted this "way were blended with the aspirated cracks fraction (blended meats fraction) prior to flaking. The blended meats fraction was then conveyed at 66-IB8 kg/hour to a Scott Tenderblend conditioner (model number SJC2, Scott Equipment Company, New 30 Prague, MN) and heated to obtain an exit temperature of 55-67°C and moisture content of 9,5%. The conditioned blended meats fiaction was fed into aRoskamp flaking roll model 2862 (28" diameter X 62'.; wide, CPM Roslcamp Champion,^ 15 « Waterloo, lA) where they were flaked to a thickness of 0,23-0.36 mm, at 6(fC^ using a gap setting of 0.010 inch. , • The flakes were then fed to a Crown Iron Works model 2 percolation extractor (Crown Iron Works Co., Roseviile, MN) for oil extraction. The extractor was 5 operated using a residence time of approximately 37 minutes, a hexane to meal weight ratio of 1:1, and a throughput of approximately 140 kg/hour. The solvent extracted meal was then conveyed via a Crown Schnecken pre-desolventizer to a two-deck Crown desolventlzer toaster (DT), Tlie pre-desolventizer was operated under a pressure'of 0,2 inches of water to provide a discharge temperature of 50°C. Tk^e, DT 10 was operated under the following conditions: the top deck temperature of 91-104'C; bottom deck temperature of lOMOS^C; and DT vapor temperature of 75 ± 5°C. The resulting meal had an exit moisture level of 16-19% and a urease level corresponding toapHriseof0.15±0.5, The desolventized meal was then dripd to a moisture level of 8.5-9.5% and 15 then hm-nmermilled to a particle size small enough to pass through a 12/64 inch screen. Tlie resulting soybean meals were used in stability tests and in broiler feed trials, described herein below. EXAMPLE 4 20 This example describes and compares protein and tryptophan contents of commercial and high tryptophan content soybean meals, and the corresponding soybean grain used to produce the meals. Shown below in Table 2 are the results from analysis of high tryptophan content soybean meal (HT SBM) of the present invention, commodity soybean meal, commodity soybeans, and a control meal. The 25 • control and the high tryptophan soybean meals were processed at the pilot scale as described in Example 3. Also included in Table 2 arc values for a soy isolate and a soy concentrate, included for comparison, I 30 16 •t t "^ Table 2. Comparative analysis of soybeans and soybean meals. I Moisture Protein % FreeTrp Total Trp/Protein , % (ppm) Tr'p % ratio (xlOO) Commodity " 40 0.54 1.35 ' soybean Commodity SBM 12 47?7 oM Oo Control soybean 4^77 40l " 211 0^65 L62 Control SBM ' 1037 326 0.54 I f f soybean 4J7 40l "1307 "091 l27 HTSBM 8^1 4341 L20 Soybean isolate SSJ LOS 126 Soybean 64 OS MO ' concentrate i Analysis methods used to generate Table 2 are described in Example 2, EXAMPLES 5 This example describes the stability determination of free tryptophan in the high tryptophan content soybean meal of the present invention, during processing and • storage. The high tryptophan content soybean meal, described in Examples 3 and 4 above, was used in the stability determinations described herein. Process samples 10 were taken at various stages and analyzed for free tiyptophan, as described in' Exaraple'2. The analytical results from tliese samples are summarized below in Table 3. The results demonstrate that there is no significant loss of free tryptophan concentration during the production of high tryptophan content soybean meal. The finished soybean meal retained about 98% of the initial free tryptophan contained in 15 the soybean grain, when normalized to a defatted, dehuiled, and moisture free basis. As a comparison, the soybean meal that wa^ subjected to an additional heating time in the DT step of 90 minutes (overcooked soybean meal), had a significantly lower concentration of tiyptophan, indicating tliat degradation was possible under more severe heating conditions, • 20 17 t Tabie3. Stability and retention office tryptophan during processing. gam pie Free trp* (ppm) % retention • HT soybean (whole) 5032 100% Soybean meal after hexane ext. 4755 94% 5 Finished soybean meal 4927 9S% Overcooked soybean meal** 4284 85% *ciata normalized to defatted, dehulled, and moisture free basis for comparison purposes ** overcooked meal was generated by increasing the time in the DT by 90 10 minutes. Stability testing was conducted to determine the stability of the free and total tryptophan during storage of the meal. Samples of the high tryptophan content soybean meal, described in Ejcamples 3 and 4 above, were stored at 4*C, 22'C, and 15 38°C, for 6 months in environmental chambers (Enconair Model GC8-2JH1, Enconair Ecological Chambers Inc., Winnipeg, Mannitoba, Canada). The samples that were stored at 38°C were also controlled at 60% humidity, A sample of approximately 600 grams high tryptophan content soybean meal was contained in Nalgene jars. Subsamples were analyzed at the time points specified below in Tables 4 and 5, with 20 each time point analysis being run in duplicate. The results are shown in Tables 4 and 5, The results indicate that both free and protein bound tryptopbaii are stable in the high tryptophan content soybean, meal of the present invention, over 6 months, even at elevated temperatures (SS^C). 25 Table 4. Stability of free tryptophan in high tryptophan soybean meal during storage. ! ;. I 4'C I 22'C I ' 3 8°C/gO% humidity Time 0 100.0 1 Month 2M 2 Month.-i 10^4 105.0 99,8 3MontKs 99^2 4 Months 96.8 | 97.0 9U 5 Months 94.2 6 Months I 98.0 | 96.7 | 95.9 18 ft s Table 5. Stability of total tryptophan in high tryptophan soybean meal during storage. I 4'C ' 22°C I 38°C/60% humidity Time 0 J j 100.0 ~ ' 1 Month j "" [ 87.9 2 Months I 92.5 91.7 90.9 3 Months ^ M;0 4 Months 89J 90:5 91.0 5 Months I 90;7 6 Months I 91.7 | 91.1 j 89.9 In Tables 4 and 5, each data point represents! the average of 2 replicates. 5 EXAMPLE 6 This example describes a broiler feeding study using a high tryptophan content • soybean meal produced as described in Example 3. A feeding study was performed using a randomized block design comprising 7 dietary treatments and 10 replicates per treatment. The treatments, analysis of the two 10 soybean meals, and the feed formulations used in the study are described in Tables 6 throBgh 8. Seventy PeterSime (Zulte, Belgium) cages in 3 batteries were divided into 10 blocks (replicates). The blocks were distributed in such a way that the position and level of the cages withiii each battery was a blocking factor. A total of 3fiO male broilers (birds) of the strain Ross 308 (Welp Hatchery, Bancroft, lA) were used in this 15 21 day trial. When the birds were 7 days of age, they were weighed, randomly assigned to pens, and-the test was initiated. The birds had ad libitum access to water and feed throughout the growing period. Mash diets were used across all age periods. Table 6. Description of treatments for feeding trials; Treatment Description Number .. 1 Basal diet, 57% of Trp, 100% of other amino acids 2 As Treatment 1, plus lov/er level of parental soybean meal 3 As Treatment 1, plus higher level of parentaj soybean meal 4 As Treatincnt 2, add free tryptophan to equal level of Treatment 6 5 As Treatment 3, add &ee tryptophan to equal level in Treatment 7 19 4 6 As Treatment 1, lower level of positive isbline soybetui meal (HT) ^^^^^^^^ 7 As Treatment 1, higher level of positive isoiine soybean meal QYT) ' Tablo 7, Measured nutrient concentration of test soybean meal {%) II 11 ' II I, • II riiiiiff Nutrients Parental Control (Parent of HT) Positive Trp Isollne (HT) Ash, % 6.060 6.150 Moisture, % 11.850 • 9.450. Fat,% 0,850 . 1.200 Protein, % 49.940 50.710 ADF, % 3.000 3,250 NDF,% 5.500 5.750 • Threonine 2.015 -2,030 Cysteine 0.640 0.656 Valine 2.410 2.425 Methionine 0.750 0.780 Isoieuclne 2.240 2.245 Leucine 3,665 3.880 Phenylaianlne ' 2505 2,505 Hislidine 1.325 1.340 Lysine 3.205 3.220 Arglnine 3.616 3.600 Tryptophan 0^5 1.226 Body weight and feed intake measurements were recorded at approximately 5 day 7,14,21, and 28 of the trial to allow for calculation of average daily gain, feed intake, and feed to gain ratio during the 7-14,14-21,21-28, and overall periods. Mortality was also recorded throughout the trial. ,, The room temperature was controlled at 90 ± 2"? at day 1 and then decreased 1°F each day until the end of the trial, with daily highs and lows recorded. There was 10 a 23 hour lighting used for the entii-e experiment with I hour dark ptriod-from midniglit to 1:00 am. Each pen housed 6 birds with a growing density of 0,58 square foot per bird at the start of the trial. Table 9 shows the factorial analysis of broiler performance data using treatments 2 to 7. For comparison purposes, the average performance of control is 15 also listed. Average trends are very similar among testing periods. Results of? to 28 . days of age indicate that there were significant differences for feed:gain ratio between the main effects of soybean meal level (P value 20 * I formulated to have tryptophan as the first limiting nutrient, the positive response of performance due to increasing SBM levels could be attributed to the increased tryptophan content, Performance averages among the main effect of tryptophan source confirms this conclusion. Treatments of parental SBM plus free tryptophan 5 (P+T, Treatments 4 and 5) and positive tryptophan isoline SBM (HT, Treatments 6 and 7) had the same amount of dietary tryptophan, and their performances were very shnilar (0-28d feedrgain ratios of 1.611 and 1.624 for P+T and HT, respectively). However, birds fed parental SBM diets (Treatments 2 and 3), which contained a lower level of tryptophan, gave a significantly poorer performance (0-28d feed;gain ratio of 10 1.801). The results of the trial further indicate that the tryptophan in the transgenic high tryptophan meal (HT) is identical lo the synthetic tryptophan supplied exogenously (P+T), with respect to bird performance. Table 8. Ingredient and nutrient compositions of ' btoavailafaiiity trials. Values given are on a weight % basis. Btml.tm tower levd HIgftw level Lowor level Higher level Lower level Higher level of SBM of petenlor of psremil ofpeiental ofpatentnl ofpoiidve pojitive (jotine Ingredients «ndnu«ienu SBM SBM SBM + I>ee SBM + ftee IsoSltwsSBM SBM vp Up TRMTI tRMT2 TRMT3 TRMT4 • TRMT5 TRMT6 7RMT7 Cm-RneGtowiJ 46,689 41689 4W89 46.689 46.689 46.689 4^.689 CornOlulenMeolfiOKProiein 2S.568 28.568 28.568 28.568 28.568 28.568 2«.568 Oil-Com • J.768 3,768 3.768 3.768 3.768 3.76S 3.768 GoletiBlJed com starch 10.000 8J254 6.514 8.240 6.486 82OT 6.421 Solke Floe 200 Ff» 5.776 4.970 4.166 4,970 4.J66 5,016 4059 Parental SDM 2.590 5.171 2,590 5.17! HiyptOfdw. 98H • 0.0143 0.0285 Positive iJoHne SBM (HT) , 2.590 5.17! CALCIUM CARBONATE 1.591 1,583 1.575 1.583 1.575 1.583 1.576 Phojphale-MonoDicilcium 1.526 !,506 5,486 1.506 1.4B6 1,506 1,487 Broiler Vitamin Premix 0.125 0,125 0.125 • 0.125 0.!25 0.125 0.12$ Poultiy Trace Mineral I're'mix 0.050 0.050 0.050 0.050 0.650 0,050 0.050 SALT 0,428 0.431 0,434 0,431 0.434 0.431 0.434 CMOUNECHLORlDE-60 0.191 0,178 0.164 0,178 0.164 0,178 0.184 L-LYSINEHCL 0,807 0.807 0.807 0.807 0.807 0.807 0,807 THREONINE 0.013 0.013 0.013. 0.013 0,0)3 0,013 0.013 l,_AtgWne, r«e base 0,4!9 0,419 Q.4I9 0.419 0.4!9 0,419 0,419 Blue late color 0,050 0.050 0.05O 0,050 0,050 0.050 0,050 Total 100,000 100.000 iOO.OOO 100.000 100.000 100.000 lOO.OOO I 20 21 « Table 9. Main effects and interactions of tryptophan source and SBM level on broiler performance. Tfeatment 7'1id li-Zli 21-28d 0-28d Basal 2.58 2A0 .Zi9 ZAi Trpiottra PareiiM 1.758a 1.784a 1,773a 1.801a P Po$.{$olim 1.623b 1,626b LWOb 1.624b SBM level low l,7S0a 1.818» 1,7924 1,809« High 1.461b 1.533b ]5S3b lJ20b Parattat ' Low 1.932 1,957 1.914 1.979 Wgh 1.618 1.628 1.632 1.623 Paraiti\!*trp Low 1.675 1.739 1,682 1,728 Wgh 1,392 1.481 1.528 1,481 Poa.isolmt JLoiu 1.673 1.762 1,804 1,755 High 1,373 1,490 1,501 1,461 Trp source p-vatue <.oooi> SEW 0,017 0,015 0,029 0.0J5 CriHcoJKsM^ei 0,053 0.046 0.092 0,04« SBM level P-vnlm «,0001 <.0m> SBM 0.014 0.0U 0,023 0.012 CrtfiMlRmi/e 0.043 0.036 0.072 0.037 Sovrtx X Level P-vatue 0.3331 0.0310 0.1377' 0.0S43 SEM ' 0,024 0,02t 0.041 0,021 5 EXAMPLE? • This example describes the generation of high tryptophan, high protein soybean varieties useful in preparing the high tryptophan content soybean meal of the present invention. ,, Soybeans, at the R3 generation, that are homozygous for the maize 10 anthraniiate synthase a gene (described in U.S. Patent Publication No. 2003/0213010) were cro.ssed to a high protein soybean variety EXP3103REN (described in PCT Application PCT/US05/002503) to produce Fl seed. Th^ resulting Fl seed was planted and grown to maturity to produce F2 seed. The resulting F2 seed was,planted and resulting plants were genotyped with respect to glyphosate resistance and 22 tryptophan content, Plants identified as heterozygous for the glyphosate resistance and high tryptophan genes were cuiled. 'The resulting F2:3 seed was collected in a single plant harvest and analyzed tor free tryptophan (described in Example 2 above), total protein and total oil content using raethodologies well known in the art. The 5 results of the F2:3 selections indicate that the high tryptophan phenotype is expressed in a high protein germplasm, at approximately the expected frequency, while maintaining the acceptable oil level (Table 10), Table 10. Effects of high protein levels on tryptophan content in transgenic soybeans, Number of Ave; Protein Event ID "Plants/event (wt%) Ave011(vyt%) AveTrp(ppm) 28893 [ 5 46.5 19.8 2511 _ _ _ _ -__ j__ ___ _ _ _ . _ -_ 19,9 ' 2427 _ _ - _-_ --- __,_ 30^5*-; 6 4636 [ I9J 2866 _____ 3 46l isfj lis? 10 A single line from each of the events described in Table 10 were advanced to field trials to evaluate seed composition, tolerance to glyphosate herbicide and general agronomics. The field trials utilized a randomized spilt plot design with duplicates of the following 3 glyphosate treatments; no glyphosate, 1.5 lbs glyphosate acid 15 equivalent (ae) /A at V3 and R], and 1,5 lbs glyphosate ae/A at V3 and 3,0 lbs glyphosate ae/A at HI stage. All plots are harvastedat maturity and subsamples are analyzed for tryptophan, oil protein, chlorosis, necrosis, plant height, maturity, and yield. The results of the F2;4 trials confirm the earlier result that the high tryptophan trait is expressed in the high protein germplasm. Additionally, the results indicate that 20 that tiie presence of the high tryptophan trait does not affect the glyphosate tolerance. This example provides an adtiitional soybean source for use in generating theJiigh tryptophan content soybean meat of the present invention. These soybeans are processed into high tryptophan content soybean meal as described above in Example 3, 25 While the present invention has been disclosed in this patent application by reference to the details of preferred embodiments of the present invention, it is to be 23 understood that the disclosure is intended in an illustrative rather than a limiting . sense, as it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the present invention and the scope of appended claims. V. CLEANCOW CLAIMS .J We claim: 1. A soybean meal, comprising greater than about 1.3 wt.% total tryptophan, wherein no exogenous tryptophan has been added and wherein the soybean has a high tryptophan content as a result of over expressing at least one gene encoding an enzyme in the tryptophan biosynthetic pathway. 2. The soybean meal as claimed in claim 1, wherein the soybean is not transgenic. 3. The soybean meal as claimed in claim 1, wherein the soybean is transgenic. 4. The soybean meal as claimed in claim 1, wherein the total tryptophan content further comprises a free tryptophan concentration of at least 0.10 wt.%. 5. The soybean meal as claimed in claim 1, wherein the total tryptophan content further comprises a protein bound tryptophan content. 6. The soybean meal as claimed in claim 5, wherein the protein bound tryptophan content comprises transgenic protein. 7. The soybean meal as claimed in claim 6, wherein the transgenic protein contains at least about 20% tryptophan residues. 8. The soybean meal as claimed in claim 1, wherein the enzyme is selected from the group consisting of anthranilate synthase, phosphoribosylanthrarulate transferase, phosphoribosylanthranilate isomerase, indole-3 -phosphate synthase, and tryptophan synthase. 9. The soybean meal as claimed in claim 8, wherein the enzyme is anthranilate synthase. 10. The soybean meal as claimed in claim 1, wherein the gene is a transgene. i 11. The soybean meal as claimed in claim 10, wherein the transgene encodes a feedback insensitive anthranilate synthase. 12. The soybean meal as claimed in claim 10, wherein the transgene encodes a monomeric anthranilate synthase. 13. The soybean meal as claimed in claim 1, wherein the gene is over expressed as result of a transgenic transcription factor. 14. The soybean meal as claimed in claim 1, wherein the soybean expresses a transgene that encodes an enzyme cataly2ing a reaction in the tryptophan biosynthetic pathway, and wherein the enzyme is less sensitive or insensitive to feedback inhibition as compared to an endogenous enzyme catalyzing the same reaction. 15. The soybean meal as claimed in claim 14, wherein the transgene encodes an anthranilate synthase. 16. The soybean meal as claimed in claim 15, wherein the anthranilate synthase is a maize C28 variant. 17. The soybean meal as claimed in claim 1, wherein the activities of one or more of the enzymes in the degradation pathway for tryptophan are reduced. 18. The soybean meal as claimed in claim 17, wherein the enzymes in the degradation pathway include chorismate mutase, tryptophanase, and tryptophan oxidase. 19. The soybean meal as claimed in claim 17, wherein the activities are reduced due to gene suppression of a gene encoding at least one of the enzymes in the degradation pathway for tryptophan. 20. The soybean meal as claimed in claim 19, wherein the suppression is accomplished using antisense or sense co-suppression technology. 21. The soybean meal as claimed in claim 1 as and when used in an animal feed, a fermentation feed, a soybean protein or an aquaculture feed. 22. A method of making a soybean meal as claimed in claim 1, comprising: a. introducing into regenerable cells of a soybean plant a transgene comprising an isolated nucleic acid molecule encoding an enzyme in the tr3rptophan biosynthetic pathway; wherein the isolated nucleic acid molecule is operably linked to a promoter functional in a soybean cell, to yield transformed soybean plant cells; b. regenerating a soybean plant from said transformed soybean plant cells wherein the cells of the plant express the enzyme encoded by the isolated nucleic acid molecule in an amount effective to increase the tryptophan content in a grain of the plant relative to the tryptophan content in a grain of an untransformed soybean plant of the same genetic background; and c. producing a soybean meal from the grain of the transformed plant. 23. The method as claimed in claim 22, wherein the transgene encodes a monomelic anthranilate synthase comprising an anthranilate synthase a-domain and an anthranilate synthase ^- domain. 24. The method as claimed in claim 22, wherein the transgene encodes a feedback insensitive maize anthranilate synthase a-subunit. 25. A method of producing soybean meal as claimed in claim 1, comprising: a. selecting soybean grain having a total tryptophan content of between 0.65 wt.% and 1.2wt.%;and i i b. extracting an oil from said grain to produce a soybean meal. 26. The method as claimed in claim 25, wherein said grain has a free tryptophan of greater than 0.

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