Classifications

• • A—HUMAN NECESSITIES
  • A21—BAKING; EDIBLE DOUGHS
  • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
  • A21D6/00—Other treatment of flour or dough before baking, e.g. cooling, irradiating, heating
• • A—HUMAN NECESSITIES
  • A21—BAKING; EDIBLE DOUGHS
  • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
  • A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
  • A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
  • A21D2/14—Organic oxygen compounds
  • A21D2/22—Ascorbic acid
• • A—HUMAN NECESSITIES
  • A21—BAKING; EDIBLE DOUGHS
  • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
  • A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
  • A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
  • A21D2/24—Organic nitrogen compounds
  • A21D2/245—Amino acids, nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis

Definitions

• the present invention relates generally to polymeric structures wherein one or more polymers (either biopolymers such as proteins or synthetic polymers) are crosslinked by tyrosine bonds formed through peptides respectively associated with each polymer or polymer region, as well as isolated peptides useful in deriving such polymeric structures.
• the invention has particular applicability in the context of grains such as wheat, wherein the crosslinking property of grain protein can be altered by genetically altering a gene which expresses the plant protein in order to cause the altered gene to express a greater or lesser number of tyrosine bond- forming subunits.
• the invention also is concerned with monitoring and assessing wheat or flour samples for dough forming potential, monitoring subsequent dough formation and modifying the physical properties of the dough during the course of dough mixing.
• the levels of tyrosine, dityrosine, phosphotyrosine, and other tyrosine bonded compounds are measured in growing wheat and in flour to predict dough forming properties, based on the potential level of tyrosine bonds that may be produced during mixing of the flour with water to produce a dough.
• manipulation of dityrosine or phosphotyrosine content can occur during wheat growth, especially in response to environmental conditions to ensure consistent growth of wheat which provides high quality flour for optimum dough products.
• the actual levels of tyrosine bonds formed in dough during mixing may also be monitored and manipulated as needed by the addition of oxidizing/reducing agents, free radical scavengers or tyrosine analogs to consistently produce high quality doughs.
• bonds incorporating tyrosine can be analyzed at different stages of end-product formation (e.g. baking).
• dough is produced by mixing wheat flour and water.
• Dough made from wheat flour has a viscoelastic property not exhibited by doughs made from other cereals. This viscoelastic property is believed to be derived from gluten protein.
• the glutenin subunits one of the two classes of storage proteins which are part of the gluten complex in wheat, are known to directly affect dough formation and bread making quality.
• Present theories regarding dough formation were developed with the idea that only disulfide crosslinks are involved in the mechanism of gluten structure formation. It was believed that these disulfide bonds were formed and/or broken and reformed during the mixing process and were ultimately responsible for the characteristics exhibited by a particular sample of dough.
• a dough intended to be used for bread may have different desirable properties than a dough made for breakfast cereal processing.
• similar flours used in dough processing may exhibit different characteristics during mixing due to environmental conditions present when the grain used to make the flour was growing or genetic differences.
• some varieties of wheat are less effected by specific environmental conditions than other varieties. Dough manufacture is affected by many different variables and it was heretofore impossible to predict with reasonable accuracy the qualities that any dough will exhibit during mixing based on an a priori analysis of the flour or wheat used or knowledge of the conditions under which the wheat was grown.
• oxidizing/reducing agents can affect the properties and consistency of the dough as desired.
• a common modifier and improver of doughs potassium bromate (KBrO 3 ) is known to have positive effects on dough quality. Due to KBrO 3 's dough improving effects, it was a common ingredient in most dough formulas. Unfortunately, KBr0 3 has been determined to be potentially carcinogenic at certain levels and its use in bread doughs has been banned in the United Kingdom, Japan and New Zealand. The United States has limited the use of KBrO 3 with maximum permitted levels of 50 or 75 ppm. However, following a request from the FDA in 1991, a majority of baking companies have voluntarily stopped using KBrO 3 .
• dough can become sticky and reduce operating efficiency causing expensive delays and product loss.
• the dough can be overdeveloped or overworked resulting in low quality products. There is a point in time during mixing of every dough where continued mixing beyond that point results in a dough of inferior quality. Stopping the mixing process prior to that point also results in unacceptable dough quality.
• Tyrosine refers to the tyrosine residues within a peptide or protein chain.
• Tyrosine bonds in the context of plant proteins refers to bonds between a tyrosine residue within a peptide or protein chain and another chemical moiety, free or within a polypeptide, and embraces dityrosine species as well as multiple bonds between respective tyrosine residues and a common bridging moiety.
• tyrosine bonds refers to radical bonds other than peptide bonds formed by two peptides, each peptide including therein at least one tyrosine residue and often including a tyrosine pair made up of two peptide-bonded tyrosine residues. These bond forming peptides may be a part of a protein or coupled to a protein, another biopolymer, or a synthetic polymer.
• “Dityrosine species” embrace dityrosine, isodityrosine, trityrosine, di-isodityrosine, and analogs thereof.
• Free tyrosine refers to the amino acid tyrosine when not within a peptide or protein chain.
• Tyrosine pair refers to two peptide bonded tyrosine residues (YY), either alone or in a larger amino acid sequence.
• Dityrosine refers to two tyrosine residues linked together by biphenyl or ether linkages.
• Densine analytical reference standard refers to two tyrosine moieties linked through a biphenyl linkage and having the following structure.
• Optimum with respect to a dough's viscoelastic properties refers to when a dough exhibits desired physical characteristics based on the dough's eventual end-use taking into account the fact that doughs having different eventual end-uses may have different desired viscoelastic characteristics.
• "Analysis” with respect to tyrosine, dityrosine or phosphotyrosine content refers to any technique for determining tyrosine, dityrosine, phosphotyrosine and/or tyrosine bond content such as amino acid analysis of protein or protein hydrolysates, elucidation and analysis of appropriate nucleic acid sequences, and any other physical analytical methods (e.g. NMR).
• isolated means altered “by the hand of man” from its natural state., i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
• a polynucleotide or polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
• the present invention is predicated upon the discovery that a class of tyrosine- containing peptides form tyrosine bonds (as defined above) in the protein fraction of wheat, wheat flour, wheat doughs and final products derived from such doughs, and that such tyrosine bonds have a profound and heretofore unrecognized effect upon final product quality.
• This discovery makes it possible to test wheat during growth thereof to determine the tyrosine bond level therein and to alter if necessary the growth conditions of the wheat so as to change the tyrosine bond level in the final harvested wheat protein.
• tyrosine bond levels can be measured in flour so as to permit a baker to adjust formulation or baking conditions for optimum results.
• wheat may be genetically altered using known techniques such as site directed mutagenesis in order to increase or decrease the level of tyrosine bond formation.
• isolated peptide crosslinkers can be provided which can crosslink with polymers (either intrapolymer or interpolymer) to yield new non- naturally occuring polymers and composite polymeric structures.
• isolated peptides can be used as such crosslinkers wherein the peptides are selected from the group consisting of: ( 1 ) peptides having the sequence X a YYX b ; (2) peptides having the sequence X a QXGXYPTSX b ; (3) peptides having the sequence X a QXGYXPTSX b ; (4) peptides having the sequence X a GQGQXGXYPTSXQQX b ; (5) peptides having the sequence X a GQGQXGYXPTSXQQX b , and (6) reversals of all of the foregoing, wherein each X independently represents any amino acid residue, and the sum of a + b ranges from 0-14.
• Particularly preferred isolated peptides include YY (i.e., a tyrosine pair), and QQGYYPTS or QPGYYPTS.
• YY i.e., a tyrosine pair
• QQGYYPTS QPGYYPTS
• a "reversal" of a peptide refers to a reverse-order amino acid sequence between the amino and carboxyl ends of a given peptide.
• QQGYYPTS its reversal would be STPYYGQQ.
• the invention thus includes non-naturally occurring polymers made up of a polymer chain with one or more of the foregoing peptides within or attached to the polymer chain.
• the class of polymers susceptible to modification is extremely broad, and embraces biopolymers ( e -g-j proteins, polysaccharides, starches, nucleic acids, lipids) as well as the synthetic polymers described herein.
• any naturally occurring wheat or other plant protein made by being modified to include therein the peptide(s), either within the normal protein sequence or as a side chain or end cap to the protein.
• synthetic polymers can be modified with the peptide(s) as internal, side chain, or end cap substituents.
• Such non-naturally occurring polymers can be reacted and crosslinked to form composite polymers.
• the reaction conditions are dependent upon the specific polymers in question, but the level of tyrosine bond formation in the composites can be altered by the presence of a number of reagents. For example, if it is desired to foster an increase in tyrosine bonds in the composite polymers, the crosslinking reaction is aided by the presence of oxidizing agents and/or free radical generators.
• Typical oxidizing agents are group consisting of KBr0 3 , ascorbic acid and azo dicarbon amide (ADA), while common free radical generators are selected from the group consisting of the peroxides, peroxidases, and catalases.
• reducing agents and/or free radical scavengers may be employed in the crosslinking reaction.
• the reducing agents include cysteine, glutathione, betamercaptoethanol and DTT, whereas the scavengers are typically selected from the group consisting of BHT, cysteine, glutathione, and t-butyl catechol.
• One class of polymeric structures in accordance with the invention includes a pair of discrete naturally occurring biopolymers (e.g., proteins including enzymes, plant proteins, plant storage proteins) coupled together through one or more tyrosine bonds, typically forming dityrosine.
• the biopolymers in the composite polymeric structure may be the same or different. However, in all cases the tyrosine bond(s) are formed by two peptides respectively associated with each of the discrete biopolymers, and with at least one of the peptides being non-naturally occurring with respect to the corresponding biopolymer.
• each of the bond- forming peptides individually has from 2 to about 28 amino acid residues therein and including a tyrosine residue; in many cases, the peptides include a tyrosine pair, the latter being made up of two peptide-bonded tyrosine residues.
• the peptides are associated with each biopolymer. In the case of protein biopolymers, the peptide may be within and form a part of the sequence of the protein, or alternately may be attached as a side chain or terminal group on the protein. If necessary or desirable, the bond-forming peptides may be coupled to the corresponding biopolymers through conventional coupling agents.
• leucocarpus Amaranthus caudatus, Amaranthus cruentus
• barley Hydeum vulgare
• malting barley buckwheat (kasha) (Fagpyrum esculentum), canary seed (Phalaris canariensis), false melic grass (Schizachne purpurascens), maize (Zea mays), millet, common millet, (Panicum miliaceum), red millet, (Eleusine coracana), bulrush millet (Pennisetum typhoideum), foxtail millet (Setaria italic ⁇ ), proso millet (Panicum miliaceum), finger millet (Eleusine coracana), pearl millet, bulrush millet, cattail millet (Pennisetumglaucum), fonio millet (Digitariaexilis), oats (Avena sativa), quinoa
• biopolymers include flax (genus Linum), cotton (genus Gossypium), industrial hemp (Cannabis sativa), wool (sheep fiber), wood (tree fiber), silk (produced by silkworm (moth) larvaeBombyx mori), mohair (Merino sheep, angora goats), cashmere (goats), jute (Corchorus c ⁇ psul ⁇ ris or C. olitorius), kanaf (Hibiscus c ⁇ nn ⁇ binus), sugarcane (S ⁇ cch ⁇ rum officin ⁇ rum), and sorghum (Sorghum vulgare).
• Another polymeric structure in accordance with the invention can be made up of only a single naturally occurring biopolymer or synthetic polymer having respective portions thereof coupled together through one or more tyrosine bonds.
• the tyrosine bonds are also formed by two peptides respectively associated with the biopolymer or synthetic polymer portions, and with at least one of said peptides being non-naturally occurring with respect to the biopolymer or synthetic polymer.
• This type of polymeric structure would be possible with relatively large proteins or the like having a conformation permitting portions thereof to come into close adjacency for tyrosine bond formation.
• Such single biopolymers include enzymes, such as saccharifying enzymes, gluconase, carbohydrase, glucoamylase, protease, pectinase, mannase, urease, cellulase, pentosanase, xylanase, lysozyme, catalase, invertase, isomerase, lipase, hydolase, deaminase, phosphatase, dehydrogenase, oxidase, esterase, lyase, aminoacylase, amyloglucosidase, peroxidase, aspartase, galactosidase, catalase, lactase, debranching enzyme, alcalase, nuclease, polyphenoloxidase, carboxyl peptidase, cellulase, crosslinking enzymes, dehydrogenase, dextranase
• the invention also includes formation of polymers and composite polymeric structures made up of synthetic polymers having the peptides hereof within the polymeric chain or attached thereto, or in the case of composites plural polymers coupled together through one or more tyrosine bonds.
• the tyrosine bonds are formed by two peptides associated with the discrete synthetic polymers, those peptides being the same as the peptide sequences defined previously.
• Synthetic polymers useful in this aspect of this invention include polymers containing primary and secondary hydroxyl, primary and secondary amino, carboxyl and isocyanate groups; examples of suitable polymers would be the C 2 -C 4 polyalkylene glycols (e.g., the polyethylene glycols) and aminated and carboxy-capped derivatives thereof, polysaccharides and their carboxylated and aminated derivatives, the polyacrylates (e.g., polymethylmethacrylate) and derivatives thereof, and the polystyrene, polyethylene and polypropylene copolymers containing hydroxyl, amino or carboxyl functional groups.
• the peptides may be attached to the individual synthetic polymers in any fashion, such as a side chain or terminal group. These peptide crosslinkers would typically be attached to the synthetic polymers via known coupling agents.
• one important aspect of the invention involves the genetic manipulation of specific plant genes in order to introduce or alter the level of tyrosine bond- forming subunits in proteins expressed by those genes.
• wheat is a prime candidate for such genetic manipulation, inasmuch as wheats inherently have such bond-forming subunits in the protein fractions thereof.
• other plants such as those listed above and especially including wheat, soy, corn, rye, oats, triticale, sorghum, rice, and barley can be altered in this manner. Such altered plants can then be used to form wheat-like doughs having the stickiness, viscoelastic and structural properties similar to that of traditional wheat doughs.
• the invention provides a method of altering a crosslinking property of a protein by genetically altering a gene which expresses the protein in order to cause the altered gene to express a protein having a greater or lesser number of tyrosine bond- forming subunits therein, as compared with the naturally occurring gene.
• glutamine could be coded for using the codon sequence caa, cag; glycine could be coded for using the codon sequence ggt, ggc, gga, ggg; tyrosine could be coded for using the codon sequence tat or tac; proline could be coded for using the codon sequence cct, ccc, cca, or ccg; threonine could be coded for using the codon sequence act, ace, aca, or acg; and serine could be coded for using the codon sequence agt, age, tct, tec, tea, or teg. Other specific insertions or mutations would of course be subject to the same analysis.
• the invention also comprehends a method of growing a plant having genes in the genome thereof which express proteins including therein (1) peptides having the sequence X a YYX b ; (2) peptides having the sequence X a QXGXYPTSX b ; (3 ) peptides having the sequence
• each X independently represents any amino acid residue, and the sum of a + b ranges from 0-14.
• naturally occurring wheat or genetically altered grains described previously are embraced within the applicable plants.
• the method comprises the steps of periodically analyzing the plant or plant structure during growth thereof to determine the level of tyrosine bonds therein, and in response to such analysis applying a phosphate-containing nutrient to the plant or the soil adjacent the plant.
• a phosphate fertilizer is applied to the soil for this purpose.
• Such treatment at strategic time(s) during the plant growth has the effect of decreasing the rate of dityrosine and/or tyrosine bond formation in the plant.
• the invention has particular applicability in the context of wheat, wheat flour, wheat doughs and final wheat-based products, and provides teclmiques for optimizing the foregoing.
• wheat kernels can be grown which form high quality flours and consistently result in optimum flour dough product preparation despite differences in initial flour quality, environmental stresses which occurred during wheat kernel development, genetic differences or mixing times
• the preferred dough monitoring method includes preparing a dough in the normal fashion and monitoring tyrosine, dityrosine and phosphotyrosine levels as well as tyrosine bond formation.
• Tyrosine residues can bond and/or form crosslinks between and among other chemical residues or moieties, e.g., tyrosine residues, quinones, hydroquinone, dihydroxyphenylalanine (DOPA), dopaquinone, semiquinones, glutathione (GSH), cysteine, catechols and various carbohydrates. Some of these compounds may also act as a bridge between tyrosine residues in proteins.
• DOPA dihydroxyphenylalanine
• GSH glutathione
• Structures including tyrosine residues include dityrosine, isodityrosine, trityrosine and other potential structures involving covalent bonds between and among tyrosine residues as well as crosslinks between tyrosine residues and other compounds.
• Typical tyrosine-bonded chemical moieties found in flours or doughs may include other tyrosine residues, quinones, hydroquinone, dihydroxyphenylalanine (DOPA), dopaquinone, semiquinones, glutathione (GSH), cysteine, catechols and various carbohydrates as well as other structures which could form tyrosine bonds.
• DOPA dihydroxyphenylalanine
• GSH glutathione
• cysteine catechols and various carbohydrates as well as other structures which could form tyrosine bonds.
• the most significant tyrosine- bonded moieties consist of tyrosine residues bound to other tyrosine residues through a bonding mechanism other than peptide bonding. NMR analysis has confirmed that the principal tyrosine bonding in wheat, wheat flours and wheat doughs is in the form of dityrosine.
• oxidizing/reducing agents metal chelating agents, free radical scavengers, free tyrosine or adjusting the dough pH during processing can affect the properties and consistency of the dough as desired.
• Phosphorylation blocks the formation of tyrosine bonds and therefore interferes with the ability of a flour to form a dough.
• This phosphorylation can occur during processing by the addition of phosphorous or phosphorous-containing compounds as a dough ingredient or additive, or alternatively, during wheat kernel growth by the application of phosphorous or phosphorous-containing fertilizers to the ground adjacent growing wheat plants. This phosphorous is then taken up by the plant wherein it interferes with the formation of tyrosine bonds.
• predetermined standards for an optimum range of tyrosine bonds will govern the monitoring and any subsequent modification of tyrosine formation in the dough.
• the monitoring provides continuous feedback indicating the approximate range or levels of tyrosine bonds at individual stages in the process. If there are too many tyrosine bonds, this information is used for example to direct the addition of a specific amount of the amino acid tyrosine, a tyrosine analog, free radical scavengers or metal chelating agents to the dough to prevent over-formation of tyrosine bonds. If this factor is not monitored or tyrosine is not added, continued mixing will cause the dough to become too sticky resulting in reduced processing efficiency.
• the present invention also allows for mixing to progress past the point in time at which, the dough has an optimum number of tyrosine bonds and the dough exhibits desired viscoelastic properties. If free tyrosine is added to the dough once an optimum range of tyrosine bonds is reached, mixing may continue without a significant subsequent increase in the number of tyrosine bonds and corresponding loss of desired viscoelastic properties. This occurs due to an inhibition of tyrosine residues within the protein or peptide chains binding with other tyrosine residues within the protein or peptide chains brought about by the added free tyrosine occupying the binding sites of the tyrosine residues within the protein or peptide chains. Thus, mixing may continue without a significant corresponding increase in tyrosine bonds and loss of desired viscoelastic properties.
• mixing may continue for up to about 10 minutes after reaching the optimum range of tyrosine bonds while retaining +/- 10% of the desired viscoelastic properties. More preferably, mixing may continue for up to about 20 minutes after reaching the optimum range of tyrosine bonds while retaining +/- 10% of the desired viscoelastic properties. Still more preferably, mixing may continue for up to about 10 minutes after reaching the optimum range of tyrosine bonds while retaining +/- 20% of the desired viscoelastic properties. Even more preferably, mixing may continue for up to about 20 minutes while maintaining +/- 20% of the desired viscoelastic properties.
• Increasing the pH of the dough will also result in a decrease in the rate of dough formation and may decrease the rate of tyrosine bond formation. Conversely, if there are not enough tyrosine bonds at a given stage of the mixing process, oxidizing agents may be added which may increase the rate of tyrosine bond formation. This will increase dough quality by causing development of necessary viscoelastic properties. Additionally, decreasing the pH of dough during processing will also affect dough characteristics and may promote tyrosine bond formation.
• the preferred method would also include using a computer program configured to achieve a predetermined range of tyrosine bonds in a dough by directing the manipulation and/or addition of additives to the dough during mixing. These steps would be carried out manually or automatically in response to the approximate number of tyrosine bonds found by analysis at any stage in the process. Any suitable analytical procedure could be followed, for example, by fluorescence detection. Following such analyses, the dough could be modified by the addition of the appropriate tyrosine bond formation modifier such as oxidizing agents, metal chelating agents, free radical scavengers, free tyrosine, and pH adjustment. Also, the physical mixing of the dough could be altered as necessary. This procedure could be carried out stepwise until the range of tyrosine bonds is within a predetermined range for a given dough application.
• a computer program configured to achieve a predetermined range of tyrosine bonds in a dough by directing the manipulation and/or addition of additives to the dough during mixing.
• dityrosine has been found to continue during the actual formation of the end-products. In other words, dityrosine levels continue to increase during baking. Thus, levels of dityrosine can be monitored at different stages of the baking process in order to further optimize knowledge of end-product quality.
• cultivars of wheat contain different levels of tyrosine and dityrosine as well as different rates of dityrosine formation, all of which affect later processing steps used to make end-products.
• cultivars generally considered to be of higher quality have or form little or no dityrosine in the developing or mature wheat kernels while cultivars of generally lower quality have elevated levels of dityrosine and greater rates of tyrosine bond formation in developing or mature wheat kernels.
• dityrosine levels can be monitored in wheat kernels as they develop and if dityrosine levels are increasing, phosphorous or fertilizer containing phosphorous can be applied to the ground adjacent the growing plants in order to limit or reduce the rate or limit further dityrosine formation. It has been found that premature dityrosine formation adversely affects dough formation and ultimately end-product formation.
• glutenin has a major role in contributing to the dough forming characteristics of wheat flour. This is because all glutenin proteins have tyrosine (Y) and tyrosine, tyrosine pair (YY) repeats throughout their structure.
• these repeats are found in a YYPTS motif or in the generalized peptide sequences, (1) peptides having the sequence X a YYX b ; (2) peptides having the sequence X a QXGXYPTSX b ; and (3) peptides having the sequence X a GQGQXGXYPTSXQQX b , wherein each X independently represents any amino acid residue, and the sum of a + b ranges from 0-14.
• Particularly preferred isolated peptides include YY (i.e., a tyrosine pair), and QQGYYPTS or QPGYYPTS).
• wheat "quality" in terms of any application for any end product may also be determined by the number and location of these repeat sequences within a particular variety of wheat.
• cultivars of wheat contain different levels of tyrosine, phosphotyrosine and dityrosine. Moreover, cultivars generally considered to be of higher quality have little or no dityrosine in developing or mature wheat kernels while cultivars of poorer bread-making quality have elevated levels of dityrosine in their developing or mature wheat kernels.
• tyrosine, phosphotyrosine and dityrosine levels can be monitored in wheat kernels as they develop and if dityrosine levels are increasing, fertilizer containing phosphorous can be applied to limit or reduce the rate of further dityrosine formation. Additionally, environmental conditions such as heat during wheat growth also impact the formation of tyrosine bonds thereby interfering with a flour's ability to form dough.
• tyrosine, phosphotyrosine and dityrosine levels in these flours can be able to account for any differences during subsequent processing.
• steps may be taken to reduce further dityrosine formation (e.g. by applying phosphorous).
• heat stress can be minimized tlirough monitoring and manipulation of dityrosine formation and wheat producers can consistently grow crops of high quality which produce optimum end- products.
• the starting flour used to make the dough may be screened to predict the dough forming potential for a particular use prior to initiating any mixing. This screening is done in much the same way as the monitoring of dough during mixing.
• the approximate levels of tyrosine, dityrosine, phosphotyrosine and/or tyrosine bonds in a flour sample are measured in order to assess and predict the dough forming potential based on the respective native, naturally occurring amounts of these compounds.
• the flour is analyzed to determine the amount of tyrosine therein because the amounts of tyrosine bonds therein is usually very small.
• the flour may be analyzed to determine the level of dityrosine.
• Flours may be grouped according to such levels found within the storage protein chains. It is believed that the glutenin subunits, and especially the YYPTS or QQGYYPTS motif repeats of the gluten protein chains occupy a more significant role with respect to a dough's viscoelastic properties. However, the gliadin subunits may still be of importance with respect to tyrosine bonds and their effect on a dough's physical characteristics.
• Flours having tyrosine, phosphotyrosine, dityrosine and/or tyrosine bond levels falling within a certain range would be grouped together and designated as having a certain grade.
• the numbers and locations of these motifs in the amino acid profiles of the wheat could also be determined and a grading scale developed. The grade would therefore indicate the approximate range of tyrosine and/or tyrosine bonds inherent in the flour or the number and locations of these motif repeats. This would allow users of flour for different applications to choose a flour that has a desired starting amount of tyrosine and/or tyrosine bonds or a particular number or location of these motif repeats which would contribute to the consistent production of high quality end-products.
• Figure 1 is a graph illustrating the increase of tyrosine bonds over time during mixing of a dough
• Fig. 2 is a graph illustrating the effect of ascorbic acid on tyrosine bond formation during mixing of a dough
• Fig. 3 is a graph illustrating a control mixograph during dough mixing
• Fig. 4 is a graph illustrating the effect on dough formation by adding free tyrosine during mixing
• Fig. 5 is a graph illustrating the effect of adding phosphotyrosine during mixing of a dough
• Fig.6 is a graph illustrating dityrosine (designated as DiY) used as a reference standard detected by fluorescence
• Fig. 7 is a graph illustrating the amount of tyrosine bonds present in a flour sample and dough samples mixed for five and ten minutes, respectively, detected by fluorescence;
• Fig. 8 is a graph illustrating dityrosine used as a reference standard detected by fluorescence
• Fig. 9 is a graph illustrating the amount of tyrosine bonds detected by fluorescence in a sample of flour
• Fig. 10 is a graph illustrating the amount of tyrosine bonds detected by fluorescence in a second sample of flour
• Fig. 11 is a graph illustrating the derivatized dityrosine used as a reference standard detected by fluorescence
• Fig. 12 is a graph illustrating the amount of tyrosine bonds detected by fluorescence in a sample of derivatized amino acids from a flour sample
• Fig. 13 is a graph illustrating the amount of tyrosine bonds detected by fluorescence in a sample of derivatized amino acids from a second flour sample;
• Fig. 14 is a graph illustrating the fluorescent compounds present in a sample of flour
• Fig. 15 is a graph illustrating the fluorescent compounds present in a dough mixed for ten minutes
• Fig. 16 is a graph illustrating the fluorescent compounds present in a flour sample
• Fig. 17 is a graph illustrating the fluorescent compounds including tyrosine present in a dough sample after one minute of mixing in the presence of an additional 1% free tyrosine
• Fig. 18 is a graph illustrating the fluorescent compounds including tyrosine present in a dough sample after five minutes of mixing in the presence of an additional 1% free tyrosine;
• Fig. 19 is a graph illustrating the fluorescent compounds including tyrosine present in a dough sample after ten minutes of mixing in the presence of an additional 1% free tyrosine;
• Fig. 20 is a graph comparing the peaks from a control dough mixed for ten minutes to a dough mixed for ten minutes with 1% free tyrosine added;
• Fig. 21 is a graph comparing the peaks from a sample of flour only and samples from a mixture of flour and water only after five minutes of mixing, after five minutes of mixing and 77 minutes of proofing, and after five minutes of mixing, 90 minutes of proofing and 27 minutes of baking;
• Fig. 22 is a graph comparing the peaks from a sample of flour only and samples from a full formula bake after five minutes of mixing, after five minutes of mixing and 27 minutes of proofing, after five minutes of mixing and 90 minutes of proofing, and after five minutes of mixing, 90 minutes of proofing and 27 minutes of baking; Fig.
• Fig. 23 is a graph comparing the peaks from a sample of flour only and samples from a dough comprising flour, water and lOOppm ascorbic acid taken after five minutes of mixing, after five minutes of mixing and 77 minutes of proofing, and after five minutes of mixing, 90 minutes of proofing and 27 minutes of baking;
• Fig. 24 is a graph comparing the peaks from a sample of flour only and samples from a dough comprising flour, water and 45ppm ADA taken after five minutes of mixing, after five minutes of mixing and 77 minutes of proofing, and after five minutes of mixing, 90 minutes of proofing and 27 minutes of baking;
• Fig. 25 is a graph comparing the peaks from a sample of flour only and samples from a dough comprising flour, water and 30ppm KBrO 3 taken after five minutes of mixing, after five minutes of mixing and 77 minutes of proofing, and after five minutes of mixing, 90 minutes of proofing and 27 minutes of baking;
• Fig.26 is a graph comparing the peaks from three different flour varieties grown under normal environmental conditions
• Fig. 27 is a graph comparing the peaks of KARL 92 wheat grown under different environmental conditions with NWS flour grown under normal environmental conditions
• Fig. 28 is a graph comparing the peaks of TAM 107 wheat grown under different environmental conditions with NWS flour grown under normal environmental conditions;
• Fig.29 is a graph comparing the peaks of flour and an extruded product made with that flour;
• Fig 30 is a graph illustrating the detection of dityrosine in maturing wheat kernels
• Fig. 31 is a graph comparing the tyrosine and phosphotyrosine levels from KARL 92 wheat grown under various temperature conditions;
• Fig. 32 is a graph comparing the tyrosine and phosphotyrosine levels from TAM 107 wheat grown under various temperature conditions;
• Fig. 33 is a graph comparing the dityrosine levels of doughs made with KARL 92 wheat flour which was grown under various temperature conditions;
• Fig. 34 is a graph comparing the dityrosine levels of doughs made with TAM 107 wheat flour which was grown under various temperature conditions;
• Fig. 35 is a graph comparing dityrosine levels of doughs formed from soft and hard wheats;
• Fig. 36 is a graph comparing the levels of dityrosine between Durum flour and doughs made with Durum flour;
• Fig. 37 is a graph comparing the levels of dityrosine in doughs having varying levels of cysteine.
• Fig. 38 is a graph comparing the levels of dityrosine doughs having varying levels of glutathione.
• Example 1 In this example, a standard wheat dough was measured during the dough mixing process in order to determine tyrosine bond levels therein.
• the dough in the experiment began as a regular mixture of wheat flour and deionized water, progressed during mixing to a good quality dough and proceeded to lose its elasticity and become too sticky, eventually proceeding to breakdown as mixing progressed.
• This data indicates that dough formation in the early stages of mixing and "breakdown" of dough during mixing after peak development are caused by the formation of tyrosine bonds. There does not appear to be any breaking of covalent bonds, but simply the accumulation of tyrosine bonds as mixing continues.
• Example 2 This example measured the effects of a common oxidizing agent, ascorbic acid, on tyrosine bond formation during dough mixing.
• Samples of wheat dough consisting of wheat flour and deionized water were taken during mixograph analysis at one, five, ten, fifteen, and twenty minutes after mixing began.
• Example 1 Prior to mixing, a solution of 5% ascorbic acid was added to the system. Protein was extracted from these samples with 70% ethanol for one hour and then dialyzed. The samples were vacuum dried and submitted for amino acid analysis. The amino acid analysis protocol utilized in Example 1 was utilized in this example. The dityrosine used as a reference standard for tyrosine bonds between tyrosine residues was obtained from the Department of Biochemistry and Biophysics, Oregon State University.
• tyrosine bond formation is enhanced with the addition of agents such as ascorbic acid and these may in turn be used to contribute to modifying the dough forming process in order to provide improved dough based products.
• Example 3 This experiment compared tyrosine formation consisting of crosslinks among tyrosine residues between control wheat flour, control wheat flour with l%(w/v) aqueous free tyrosine added and control wheat flour with l%(w/v) aqueous free phosphotyrosine added.
• a dough was prepared by mixing each of the respective flour samples with deionized water. For the samples containing the free tyrosine and free phosphotyrosine, these two latter ingredients were present in the water added to the control flour to produce the dough. Results of this example are given in Figs. 3, 4 and 5. Results
• the mixograph results from the control wheat flour dough are given in Fig.3.
• the line marked MP means midline mixing peak and is the point at which the dough is mixed to optimum for breadmaking properties.
• the lines marked ML and MR indicate points along the midline curve either 2 minutes to the left (ML) or 2 minutes to the right (MR) of the MP.
• the line marked TP is the envelope mixing peak. "Envelope” refers to the two lines seen, one outlining the top of the curve and the other outlining the bottom of the curve. The peak of the top line is the highest point on it.
• the lines marked TL and TR are just indicating points on the upper envelope curve either to the left (TL) or right (TR) of the envelope mixing point (TP).
• the line marked TX is an arbitrary time set for data collection that was set at 8 minutes in this case in order to determine the width of the curve two minutes prior to the end of the analysis.
• the line marked TT refers to curve tail and refers to the end of the analysis.
• Example 4 This example determined that tyrosine bonds in doughs were measurable by direct analysis with a fluorescence detector.
• Fig. 6 shows the dityrosine used as a reference standard eluting at about 18.9 minutes.
• Fig. 7 shows the amino acids present in a flour sample compared with dough samples that were mixed for five and ten minutes. Five minutes of mixing was near the optimum mixing point of this flour sample and therefore may represent the "ideal" number of these tyrosine bonds necessary for baking purposes. There is an easily detectable amount of tyrosine bonds formed at this point in mixing as is evidenced by the peaks at about 18.9 minutes and at about 22.5 minutes in the chromatogram.
• Fig. 7 also shows the amino acids present in the dough sample that was mixed for ten minutes. Again, the tyrosine bond peaks at about 18.9 and at about 22.5 minutes have increased. This level of tyrosine bond formation is probably indicative of an overformation of tyrosine bonds.
• the dough has now been mixed beyond its ideal for baking purposes. This sample was taken at a point in mixing that cereal chemists refer to as "breakdown" because the dough does not retain its resistance to extension and is not as elastic as it would be after only five minutes of mixing.
• Example 5 This example measured the approximate amounts of tyrosine bonds in different wheat flour samples.
• Samples of wheat flour were taken to determine the approximate level of tyrosine bonds therein prior to forming any dough. Knowledge of the amount of tyrosine bonds present in the flour provides an indication of the crosslink forming potential of a flour sample. It may also indicate the types or extent of modifications that may be necessary in order to produce an optimum dough product.
• the samples were hydrolyzed and amino acid analysis by HPLC was performed on the underivatized samples. The amino acids were then analyzed with fluorescence at a wavelength of 285 nm and compared to the dityrosine analytical standard which eluted at 19.579 minutes. The amino acid analysis protocol utilized in example 4 was utilized in this example. The dityrosine analytical reference standard was obtained from the Department of Biochemistry and Biophysics, Oregon State University.
• tyrosine bonds in flour samples were determined using fluorescence detection.
• Fig. 8 illustrates the tyrosine bond standard eluting at 19.579 minutes.
• Fig. 9 illustrates the approximate amount of tyrosine bonds present in a sample of flour as detected by fluorescence.
• the peak at 19.432 minutes represents approximately 83 pmol of tyrosine bonds/ ⁇ g of protein.
• the tyrosine bond level can then be used to predict the rate at which tyrosine bonds would be fo ⁇ ned during the mixing of a dough as well as predicting the amount and type of manipulation that may be needed to consistently produce an optimum dough product.
• the operator of the machinery used to produce a dough would know that a longer mixing time and/or the addition of oxidizing agents would be necessary to produce an optimum dough product. This would also allow any automated machinery to be precalibrated such that the initial tyrosine bond content of the flour would be taken into account when programming mixing times and ingredient additions.
• Fig. 10 illustrates the approximate amount of tyrosine bonds present in a second flour sample.
• the peak at 19.412 minutes represents approximately 105 pmol of tyrosine bonds/ ⁇ g of protein.
• Example 6 This example demonstrated a different and more sensitive method of measuring tyrosine bonds in wheat flour.
• Example 5 Samples of the flour used in Example 5 were hydrolyzed and subsequently derivatized and HPLC analysis was performed on these derivatized amino acids. The derivatiztion procedure of Cohen et al., Anal. Biochem. , 211 :279-287 (1993) was used. Results
• Fig. 11 shows the derivatized dityrosine standard eluting at
• Fig. 12 illustrates the derivatized tyrosine bonded species from the first sample eluting at 59.261 minutes. The area under the peak represents approximately 83 pmol of tyrosine bonds/ ⁇ g of protein.
• Fig. 13 illustrates the derivatized tyrosine bonded species from the second sample eluting at 59.266 minutes. The area under that peak represents approximately 105 pmol of tyrosine bonds/ ⁇ g of protein.
• flours may inherently have varying amounts of tyrosine bonds.
• the actual amount of tyrosine and tyrosine bonded species present in a given flour may correlate to the tyrosine bond forming potential of the flour during dough processing.
• measuring the initial content of tyrosine and tyrosine bonded species of the starting flour and thereafter measuring and controlling/manipulating the tyrosine and tyrosine bonded species in the dough during processing will result in higher quality end-products with decreased waste.
• Example 7 This experiment demonstrated that tyrosine residues link with several components in flour to form fluorescent compounds.
• a sample of flour was analyzed using HPLC followed by fluorescence detection. Results from this sample are given in Fig. 14.
• doughs made with this analyzed flour were formed by mixing the doughs for 10 minutes and then analyzing these doughs to determine whether other fluorescent compounds were formed.
• the sample was analyzed using HPLC followed by fluorescence detection. The results from this sample were compared to the flour sample.
• Fig. 14 shows the results of the flour sample and Fig. 15 shows the results of the dough sample.
• Fig. 15 shows other fluorescent compounds are evident by the peaks at 12.314 minutes, 17.076 minutes, 22.520 minutes and 36.424 minutes.
• the tyrosine bond peaks at 18.938 and 22.520 represent dityrosine. This shows that other fluorescent bonds that may be incorporating tyrosine are also being formed during the mixing process which may also affect dough forming characteristics. When combined with the knowledge of the starting tyrosine content, the rate and/or potential for forming bonds incorporating tyrosine could be predicted.
• Example 8 This experiment tested the tyrosine bond effects of adding a 1% (w/v) aqueous solution of free tyrosine to flour used to make a dough.
• the addition of free tyrosine may also prolong mixing times once a predetermined range of tyrosine residues within the gluten storage protein chains have formed bonds. This is due to the free tyrosine bonding with the tyrosine residues within the protein or peptide chains thereby preventing them from forming crosslinks with other tyrosine residues within the protein or peptide chains.
• a quantity of free tyrosine would be added once the range of tyrosine bonds reached a predetermined standard. This free tyrosine would inhibit further binding between tyrosine residues within protein or peptide chains allowing the mixing process to be extended without a change in the viscoelastic properties.
• the mixing time could be extended for about
• Example 9 This example compared HPLC peaks in a control dough and a 1% free tyrosine added dough in order to ascertain tyrosine bond levels therein.
• This example illustrates one method of dete ⁇ nining and setting an optimum range standard for tyrosine bonds in a given wheat flour-containing product. Finding a predetermined optimum standard for such products allows the producer of the product or machinery operator to compare the range or number of bonds present in a production run to that of an ideal product, thus permitting real-time modification of the product.
• This standard is found by producing the wheat flour-containing product under optimum processing conditions and taking samples at various stages of the production process. These samples are analyzed to determine the approximate range of tyrosine bonds present in the sample at each stage. This could be done with an infinitely large number of samples at an infinitely large number of stages in the process (e.g. every minute, every second, every 1/lOth of a second, etc.) in order to provide as narrow of a target or optimum range as possible. The range or number of tyrosine bonds found at each stage of the processing of ideal products is then used as a benchmark to control future processing of that particular product.
• the number of tyrosine bonds found at each stage of processing is compared to the optimum or ideal number for that stage and any necessary modifications for bringing the number of tyrosine bonds within the optimum range are made, thereby ensuring that an optimum product is made every time.
• this entire process is done through a computer program configured to direct the processing of any product utilizing tyrosine bonds.
• the preferred computer program is designed to direct the operator of the equipment to either manually or automatically:
• the ideal range may be based on bonds between and among tyrosine residues or between tyrosine residues and other compounds (which may bridge storage protein chains or be storage protein chain substituents) such as quinones, hydroquinone, dihydroxyphenylalanine, dopaquinone, semiquinones, glutathione, cysteine, catechols, various carbohydrates and analogs thereof (tyrosine bonds), all of which may be measured using the methods outlined in the examples above.
• bonds between and among tyrosine residues or between tyrosine residues and other compounds which may bridge storage protein chains or be storage protein chain substituents
• bonds such as quinones, hydroquinone, dihydroxyphenylalanine, dopaquinone, semiquinones, glutathione, cysteine, catechols, various carbohydrates and analogs thereof (tyrosine bonds), all of which may be measured using the methods outlined in the examples above.
• a dough comprising 99.8 g wheat flour and a 58% level of water absorption was formed. Fifty milligram (50 mg) dough samples were taken at various times during the mixing and immediately placed in 6 N HCl with 1% phenol and evacuated for amino acid hydrolysis. 100 gram pup loaves were prepared using a 90 minute fermentation according to AACC method 10-lOB. This method includes five minutes of mixing followed by 25 minutes of proofing (rising or fermenting) called the first punch stage, 52 more minutes of proofing termed the second punch stage, and finally, 13 more minutes of proofing (for a total proofing time of 90 minutes) termed the pan stage.
• Example 11 illustrates the HPLC profiles of the amino acid analyses of samples taken from this flour and water only bake.
• the line labeled A represents the amino acid content of the flour with no mixing or baking treatment.
• the line labeled B represents dough that has been mixed for 5 minutes.
• the line labeled C represents dough that has been proofing (rising or fermenting) for 77 minutes after being mixed for 5 minutes.
• the line labeled D represents a sample that has completed all stages of bread making and has been baked for 27 minutes. Two peaks of significant interest appear in the samples: the peak at about 18-18.5 minutes and the peak at
• Example 11 This example is similar to Example 11, and describes an additional test wherein tyrosine bond levels were measured during bread dough manufacture and baking.
• a full formula dough comprising 99.8 g flour, 67 g water, 2 g compressed yeast
• Example 11 100 g pup loaves were prepared using a 90 minute fermentation according to AACC method 10-10B. One hundred (100) mg dough samples were taken at the first punch stage pan stage, and after baking for 27 minutes. Dough and bread samples were immediately flash frozen and lyophilized. The freeze dried samples were ground with mortar and pestle and 50 mg of each sample was hydrolyzed for amino acid analysis. The 50 mg samples were hydrolyzed under vacuum with 6N HCl with 1% phenol at 115 °C for 24 hours.
• Example 11 As in Example 11 (Fig.21), dityrosine crosslinks were shown to be increasing during all stages of bread making and especially during the baking process. This is evident by the increases in the peaks at approximately 19 minutes and approximately 21-22 minutes which were found during each successive stage of the bread making process.
• This example differed from Example 11 in that it represents a full formula bake, meaning that it includes all of the necessary ingredients for bread making and a good quality loaf of bread resulted (in contrast to the loaf produced by the flour and water bake of Example 11, which was of low quality).
• dityrosine is apparent as the dough goes through the various mixing and baking stages and these increases appear to be greater than the increases seen in the flour and water only bake. This indicates that use of a full formulation dough enhances dityrosine crosslinking and contributes to end-product quality.
• Example 13 This example illustrates the effect of ascorbic acid on dityrosine bond formation during bread mixing and baking processes.
• Example 11 The same dough formula described in Example 11 (comprising flour and water only) was used for this example. However, 100 parts per million (ppm) of ascorbic acid was added to the water prior to mixing the water with the dough. Following formulation of the dough, the same analysis procedure used in Example 11 was followed. Again the samples were also compared to HPLC analysis of flour only.
• Fig. 23 illustrates the effect that ascorbic acid has on dityrosine during mixing and baking.
• ascorbic acid exerts its major influence on dityrosine during the baking process.
• the peak most influenced is the one at 21- 22 minutes while the other peaks are not significantly effected.
• the addition of ascorbic acid appears to have had little or no effect on the peak at approximately 19 minutes while greatly increasing (more than quadrupling) the dityrosine peak for all samples at approximately 21-22 minutes.
• the samples taken after 5 minutes of mixing and at the second punch stages do not show appreciable differences at any peak including the peak at 21-22 minutes.
• a significant difference is shown after baking the dough.
• ascorbic acid appears to increase the number of dityrosine bonds significantly, especially during the baking process.
• Example 14 This example illustrates the effects of ADA when added to the dough during mixing.
• Example 11 A dough was formed with the same levels of flour and water as in Example 11. Additionally, 45 ppm ADA was added to the water before the water was added to the flour to make the dough. The analysis procedure of Examples 11 and 12 was then followed to produce the results given in Fig. 24.
• Fig. 24 illustrates that ADA exerts an effect on dityrosine content when added to the mixing process of a dough. Significant effects are shown at the peak eluting at approximately 21-22 minutes while the other peaks are not significantly effected. Comparing Example 14 (Fig. 24) with Example 11 (Fig. 21), it is apparent that ADA greatly increases the dityrosine content of the dough during mixing and at the second punch stages of dough development. When compared with the results from Example 11, the addition of ascorbic acid appears to have had little or no effect on the peak at approximately 19 minutes while greatly increasing the dityrosine peak for all samples at approximately 21-22 minutes. Interestingly, the samples taken after 5 minutes of mixing and at the second punch stages do not show appreciable differences at any peak including the peak at 21 -22 minutes. However, a significant difference is shown after baking the dough. Thus, ascorbic acid appears to increase the number of dityrosine bonds significantly, especially during the baking process.
• Example 15 This example shows the influence KBr0 3 exerts when added to a flour and water only mixture.
• Example 14 This example was carried out using the same formula and procedures used in Example 14 with the exception that 30 ppm of KBrO 3 was added to the water before mixing it with the flour instead of the 45 ppm of ADA. Again, samples of the dough were taken after 5 minutes of mixing, at the second punch stage after 5 minutes of mixing and 77 minutes of rising and fermentation and at 27 minutes into the baking stage. These samples were analyzed as described in Example 11. Results of this example are given in Fig. 25.
• Example 16 This example compared three different flours, NWS control flour; KARL 92 control flour; and TAM 107 control flour.
• NWS flour and KARL 92 flour are generally regarded as flours which produce good quality bread while TAM 107 is generally regarded as being a flour which produces medium to poor quality bread.
• TAM 107 has an inherently higher level of dityrosine as shown by the peak at 21 -22 minutes than either NWS flour or KARL 92 flour. Because TAM 107 is regarded as being of lower quality than KARL 92 or NWS flour with respect to bread making quality, it is interesting to note that TAM 107 has a higher dityrosine level than either of the other two flours. Therefore, these results indicate that TAM 107 may form too many dityrosine bonds during kernel development. Consequently, these previously formed dityrosine bonds either prevent or do not allow the proper formation of a good quality dough.
• dityrosine bonds should form during the mixing and baking processes as the flour proteins interact with each other during mechanical processing (mixing) and as a result of ingredient interaction and not in the kernels during seed development.
• genetic differences contributing to dityrosine formation in kernels during seed development could be determined and manipulated (e.g. through phosphorylation) so as to produce flours which form certain levels of dityrosine at certain times during kernel development, dough mixing, dough development or baking.
• timing differences in dityrosine formation may be more or less desirable for different dough applications and end uses thereby permitting better flour selection and processing procedures based on these differences which will result in the consistent formation of more high quality end-products.
• Example 17 This example compared dityrosine levels from a good bread-making quality flour, KARL 92, when grown at various temperatures.
• KARL 92 flour and NWS flour were grown under normal field conditions. Additionally, one sample of KARL 92 flour was grown in a greenhouse at 20 ° C at 10 days post anthesis while another sample was grown in a greenhouse at 40° C at 10 days post anthesis.
• Fig. 27 verifies that temperature conditions present during kernel development have an effect on the ultimate bread-making quality of flours.
• the KARL 92 sample grown at 40° C had increased levels of dityrosine bonds formed during kernel development. As explained in Example 16, this increased level of dityrosine bonds formed during kernel development prevents or does not allow the proper formation of dityrosine bonds during the mixing and baking processes.
• the sample grown at 20 °C did not have significant differences when compared to NWS and KARL 92 control flours grown under normal environmental conditions. With respect to the sample grown at 40°C, the increase shown at the peak at approximately 21-22 minutes is not as significant as some of the other peak differences shown in the previous examples.
• KARL 92 is somewhat "heat-resistant” and can therefore be grown in a wider variety of environments and under a wider variety of environmental conditions without significantly compromising final wheat flour quality.
• wheat known to be “heat- resistant” can be selected for and perhaps genetically combined with less heat-resistant varieties in order to increase their heat-resistance.
• wheat varieties known to be heat resistant can be used in hotter climates.
• Example 18 This example compared dityrosine content of TAM 107 flour grown at various temperatures.
• KARL 92 wheat was grown at various temperatures and analyzed for dityrosine content.
• Fig. 28 illustrates that TAM 107 is less heat-resistant to tyrosine binding during kernel development than the KARL 92 flour shown in Fig. 27.
• the peak at 21-22 minutes demonstrates that the TAM 107 sample grown at 40° C at 10 days post anthesis was greatly effected by the heat as evidenced by the large increase in the amount of dityrosine bonds illustrated by that peak. Additionally, the sample grown at 20°C at 10 days post anthesis had a slight increase in its dityrosine peaks when compared with the two control samples.
• the peak at 25 minutes does not appear to change significantly with increased temperature.
• TAM 107 does not resist the influences of increased environmental temperatures as well as KARL 92.
• the TAM 107 samples decrease in bread-making quality due to the premature formation of dityrosine which occurs at a much greater rate than the KARL 92 samples grown under these various environments. This premature formation may inhibit or prevent the formation of dityrosine during dough processing and baking when it is needed to produce good quality products. Alternatively, the formation of dityrosine at this point may not leave enough available tyrosine residues for later binding during the dough processing and mixing stages. Thus, it appears that temperature-related stress and a subsequent decrease in bread making quality can be correlated with the wheat variety' s ability to resist over-formation of dityrosine bonds in the wheat kernels during development.
• Example 19 This example determines the effects of extrusion processing on dityrosine formation.
• the extruded product was very similar in structure and textural properties to a puffed "cheese curl" snack or to a piece of starch based packing material.
• Fifty (50) mg of flour or 50 mg of the extrudate that had been lyophilized and crushed in a mortar and pestle were hydrolyzed for amino acid analysis using the same procedure previously employed in this work.
• the HPLC method of amino acid analysis with fluorescence detection was also the same procedure used previously in this work.
• Fig.29 illustrates the HPLC chromatograms of this flour and the extruded product made from that flour.
• the flour sample exhibits a very small peak at 22.5 minutes, indicating very little dityrosine present in this control sample.
• Extruded samples processed with this flour contain an increased level of dityrosine.
• dityrosine must be formed during the extrusion process.
• the same conclusions that were drawn for the dough being mixed can be drawn from the flour being mixed and extruded using extrusion processes. It appears that dityrosine is being formed during this process at a rate higher than that detected in a standard dough that was mixed for ten minutes, however, the level of dityrosine in the finished product (extrudate) is only about one-third that of the dityrosine found in a full formula fully baked bread sample.
• dityrosine continues to be formed at all stages of the production process, regardless of whether this process is through traditional mixing and baking or through other methods such as extrusion.
• Example 20 This sample illustrates the detection of dityrosine in maturing wheat kernels. Kernels of wheat were harvested from wheat plants of the KSU experimental line KS97HW131, which is a quality noodle making variety of wheat but is not a good bread making variety of wheat. It is a cross between the cultivars Ike and KS91HW19.
• the above referenced wheat plants were grown in the green house and developing kernels extracted from the head of the plants at 5, 10, 15, and 25 days post anthesis (fertilization/flowering of the wheat plant) as well as after complete maturation of the grain.
• the kernels were kept on dry ice and then the bran layer and the germ were excised with a razor blade.
• the remaining endosperm chunks (where flour comes from) were hydrolyzed for dityrosine analysis as described previously. All dityrosine analyses were then performed as previously described. Results of this example are given in Fig. 30.
• Fig.30 demonstrates that there was very little dityrosine present at 5 days post anthesis. At 10 and 15 days post anthesis, an increased amount of dityrosine was present and the levels for 10 and 15 days were very similar. At 25 days post anthesis, a significant increase in dityrosine was detected. This is the point in the development of the kernel where the glutenin polymers have been shown to form. An increased level of dityrosine, which is responsible for crosslinking in these glutenin polymers is detected. At full maturity, the kernels have an even greater amount of dityrosine present. As noted above, this cultivar of wheat is not good for bread making.
• dityrosine present in the kernel has an increased level of dityrosine present in the kernel (as shown before with the KARL 92 and the TAM 107 grown under increased temperatures).
• the more dityrosine present initially in the flour the lower the mixing/bread making quality.
• dityrosine bond formation can be measured during wheat kernel development. This would allow a producer to manipulate the levels of dityrosine by any means and monitor the dityrosine formation and effects of manipulation in the wheat kernels as they mature. Analyses of these types will allow a grower of wheat to make the correct decisions about when to fertilize and how much to fertilize.
• these results, along with the results from Examples 16, 17 and 18 demonstrate that better bread making wheat varieties have less dityrosine in the developing kernels.
• this example also demonstrates that wheat varieties which are not good for bread making may be good varieties for other applications (i.e. noodle making).
• Example 21 This example compared tyrosine and phosphotyrosine levels from two different wheat varieties grown under various temperature conditions.
• the wheat varieties tested were KARL 92 and TAM 107.
• the hydrolysis tubes were evacuated and placed in a dry heating block for 3 hours. Hydrolysis longer than 3 hours resulted in a complete loss of phosphotyrosine. This loss was likely due to the heat labile phosphoester bond contained in phosphotyrosine.
• the hydrolysate was chilled at 4 ° C . 100 ⁇ l aliquots were taken from each hydrolysis tube and neutralized with an equal volume of 8N sodium hydroxide (NaOH). Each sample was then thoroughly mixed. After mixing, a 20 ⁇ l aliquot of the neutralized hydrolysate was added to 80 ⁇ l of borate buffer (Waters AccQ-FlourTM Reagent Kit). This mixture was completely mixed using the vortex genie. After mixing, the solution was centrifuged at 10 X 1,000 g for
• the supernatant (80 ⁇ L) was placed in a mini-HPLC vial and derivatized with 20 ⁇ l of 6-aminoquinolyl-N-hydrozysuccinimidyl carbamate (prepared according to directions provided in Waters AccQ-FlourTM Reagent Kit) and capped.
• the derivatized sample was allowed to stand for two minutes at room temperature. After the two minutes had passed, the samples were placed in a dry heating block (55 ° C) for 10 minutes to degrade byproducts of the derivatization reaction. Finally, the derivatized samples were ready to be analyzed by HPLC.
• the Hewlett-Packard model 1100 HPLC was used to perform all chromatographic analysis.
• the 1100 model contains a vacuum degasser, quaternary pump, auto-injector, thermostatted column, diode array detector, and a fluorescence detector.
• a linear gradient along with a Luna 5 ⁇ C(18)2 250 x 460 mm reversed phase column (Phenomenex, Torrance, CA, ) was used to separate the sample constituents. Injection volume was 20 ⁇ l.
• the solvents used in the linear gradient are denoted as A and B.
• Solvent A was a phosphate buffer prepared by dissolving 6 ml of 85% phosphoric acid in 1 liter of deionized water and titrated to a pH of
• Solvent B was a mixture of acetonitrile and deionized water (60%/40% respectively). The linear gradient begins with solvent B at 2% and increases from time zero to 100% over 45 minutes.
• the column temperature was set at 38°C.
• Fluorescence detection parameters included an excitation wavelength 250nm and an emission wavelength of 395 mn.
• Phosphotyrosine and tyrosine standards were used to determine elution times for each amino acid. Phosphotyrosine had an elution time of approximately 13.2 minutes, and tyrosine had an elution time of approximately 17.5 minutes. Results of this Example are given in Figs. 31 and 32.
• phosphotyrosine increases when the wheat plants are exposed to 40 °C temperatures at 10 dpa in both the KARL 92 and the TAM 107 samples.
• Fig. 31 shows the phosphotyrosine (13.2 min peak) and tyrosine (17.5 min peak) levels for the KARL 92 samples and
• Fig. 32 shows the phosphotyrosine and tyrosine levels for the TAM 107 samples.
• the first chromatogram is shifted slightly to the right.
• the PY and tyrosine (Y) peaks are marked on that chromatogram. This shift can occur during HPLC analyses between runs especially if it is the first sample analyzed in an experiment as this was.
• the level of PY in the TAM 107 control sample (field grown) is much greater than the level of PY in the KARL 92 control sample (field grown), while the levels of Y in both field grown samples are similar.
• the levels of PY and Y in the KARL 92 sample appeared to decrease when the sample was exposed to 20 °C at 10 dpa.
• the level of PY and Y present in the flour of KARL 92 increased significantly when the plants were exposed to 40 °C 10 dpa.
• the level of PY in the TAM 107 samples increased when the plants were exposed to
• Example 22 This example compares mixograph analyses taken from dough after 10 minutes of mixing and dough made with flour from each sample from Example 21. These analyses determined the mixing quality (and potential breadmaking quality) of each sample.
• the mixographs demonstrate that the KARL 92 (field harvest) has the best quality in terms of dough mixing parameters . There was a gradual increase in the curve until a high peak was reached and then there was a gradual decrease in the curve. The graph of the curve also stayed fairly wide as it decreased. This is a measurement of "tolerance" of a dough to overmixing.
• the KARL 92 (20 °C) sample showed a decrease in the peak height, making it a slightly lower quality mixograph.
• the mixing peak of the graph was not as high a the field harvest sample.
• the KARL 92 (40 ° C) had a fairly good front half of the curve and a nice peak, but the back half dropped off very quickly into a much thinner curve. This result means that the dough has less tolerance and is much poorer than the field grown sample or the 20 °C sample.
• the TAM 107 sample (field grown) started off with a poor mixograph. There was very little increase to the peak and the peak was low. Then it tapered off with a fairly thin tail.
• the TAM 107 (20 °C) sample hardly had a peak at all and was almost a flat graph. Because there is no distinct peak, these results showed very little elastic dough structure being built up and no strength.
• the TAM 107 (40 °C) sample was extremely poor. There was a very quick little peak that occurred, but it was low and it tapered off quickly to a very thin graph, exhibiting no tolerance.
• mixograph analyses confirm that mixing quality determined by these methods mirrors that determined by tyrosine and phosphotyrosine analyses.
• Example 23 This example compared dityrosine levels from doughs containing two different types of flour after the doughs were mixed for 10 minutes. The flour used to make the doughs came from the samples harvested in Example 21.
• TAM 107 and KARL 92 were grown under normal field conditions or in a greenhouse at 25 ° C during the day and 20 °C during the night until 10 dpa and thereafter each sample was grown at either 20 °C or 40 °C until harvest.
• Mixograph analyses (10 minutes of mixing) were performed for doughs made from each flour sample. The doughs were then placed in liquid nitrogen and lyophilized immediately after mixing. Dityrosine analyses were performed as described previously.
• doughs derived from TAM 107 grown under heat-stress contain much larger amounts of dityrosine as evidenced by the significantly larger peaks.
• flour derived from TAM 107 wheat samples contains more dityrosine than flour derived from higher quality wheat samples such as KARL 92. Additionally, these two wheat varieties react differently to heat stress in that TAM 107 samples exhibit increasing levels of dityrosine as temperature conditions present during wheat growth increase. Thus, these results verify that doughs made from these flours also differ in their dityrosine levels. Very little dityrosine forms during the mixing of the higher quality wheat variety (KARL 92) even when the plants from which the flour was obtained were exposed to increased temperatures during kernel development. However, increasing amounts of dityrosine form during the mixing of flour derived from TAM 107 wheat which is generally considered to be of lower bread-making quality.
• good quality flours should have little or no detectable dityrosine prior to mixing.
• there should not be too many dityrosine bonds already present in a good flour because a certain number of these bonds need to form during mixing and not before, to provide the right dough structure.
• too many dityrosine bonds should not form during the initial mixing stage for similar reasons pertaining to dough structure. This is because even more dityrosine bonds form during the rest of the bread-making process (i.e. kneading, fermentation or proofing, and baking). If too many dityrosine bonds are formed in the mixing process then fewer tyrosine residues are available for dityrosine bonding necessary in the other bread- making steps and a good-quality dough structure cannot be formed.
• Example 24 This example manipulated the phosphorous levels during plant development under normal conditions in order to effect dityrosine levels in glutenin proteins.
• Flours made with wheat grown in the presence of increased phosphorous levels have decreased levels of dityrosine in the kernel.
• Flours made with wheat grown without increased phosphorous levels have increased levels of dityrosine. Dityrosine formation for the groups receiving the highest phosphorous levels was substantially eliminated.
• Results showing the area under the dityrosine peak are given below in Table 1. These results show that the topical addition of fertilizer containing phosphorous at increasing levels to the ground adjacent growing plants decreases the levels of dityrosine in the developing kernels. As discussed previously, increased phosphorylation likely prevented overformation of dityrosine in the developing wheat kernels, therefore leading to a higher quality flour for bread- making.
• Example 25 This example compares several wheat varieties grown under heat stress conditions and the effect of phosphorylation upon dityrosine formation. These samples are analyzed for dityrosine, tyrosine, and phosphotyrosine levels.
• pots ofeachcultivar are moved to a greenhouse unit set at 40 °C at the following intervals: 5 dpa, 10 dpa, 12 dpa, 15 dpa, 18 dpa, 20 dpa, 22 dpa, 25 dpa, and 30 dpa.
• Each of these pots is fertilized with the same N-P-K fertilizer used previously every two weeks during the time they are exposed to the 40 °C temperature. All seed is then harvested at maturity and analyzed for the presence of dityrosine, tyrosine, and phosphotyrosine using methods previously described.
• results The increased phosphorous levels in fertilizer at both standard fertilization intervals during the initial growth of the plants (prior to anthesis), or during the kernel maturation process (after anthesis) has a protective effect upon storage protein (glutenin) and ultimate bread-making quality.
• the increased levels of phosphorous cause increased phosphorylation of the glutenins and prevent the over formation of dityrosine in kernels under heat stress.
• dityrosine levels of the growing wheat can be measured and subsequently controlled by the application of phosphorous. This allows the crop producer to take an active role in protecting wheat quality during environmental stress conditions and consistently produce high quality wheats.
• This example compares dityrosine formation in soft wheats which are generally regarded as being of low quality for breadmaking. Soft wheats do not form good quality doughs but rather form batters due to their inability to form gluten.
• Fig.35 shows the chromatograms from these analyses.
• the soft wheat flour has a very small amount of dityrosine and the dough mixed for 10 minutes made from the soft wheat flour has the same amount of dityrosine, indicating that there was no increase in the level of dityrosine during mixing as has been previously detected when mixing hard wheat (wheat suitable for bread-making) flour.
• the standard analysis from the NWS control dough is shown for comparison and exhibits a much higher peak. There is an increase in the second peak, which we have referred to as the "25" minute peak, during mixing of the soft wheat flour.
• dityrosine formation is integral to the formation of gluten.
• Hard wheat doughs form gluten and also show increased levels of dityrosine during all stages of the mixing and baking processes.
• Soft wheats do not form gluten and instead of forming a dough, they form a batter.
• dityrosine does not form appreciably during the mixing of soft wheat flour.
• this is entirely expected in the formation of batters since batters are very different from doughs which need to have certain visceolastic properties that are helpful in bread- making.
• Soft wheats such as these are typically used for making products such as cookies, crackers, sponge cakes and udon noodles.
• Example 27 This example compares dityrosine levels between Durum wheat flour and doughs made with Durum wheat flour.
• durum wheat flour and a 10 minute mix dough made from the durum wheat flour during standard mixograph analysis were examined for the presence of dityrosine using previously described methods.
• the results of this example are shown in Fig. 36.
• the durum wheat flour has a fairly high amount of dityrosine that has already formed in the kernel, however the dough made from the durum wheat flour and mixed for 10 minutes has approximately the same amount of dityrosine. This indicates that there was no appreciable increase in the level of dityrosine during mixing as has been previously detected when mixing bread wheat flour. This is in contrast to the normal increase in dityrosine levels seen during all stages of the mixing and baking processes of flours suited for bread-making. However, this is entirely expected because durum wheats are typically used for making products such as pasta, although there are some durum wheat varieties which have been bred for their bread-making potential.
• Durum wheats differ from bread wheat most significantly due to the fact that they contain only 2/3 of the genetic material that is contained in bread wheat. Wheat used for bread- making contains three genomes, denominated "A,” “B,” “D,” genomes, that were derived from three different wild grasses which were crossed resulting in the common bread wheat of today . Durum wheat is the result of only two of these grasses crossing and so it contains only genomes A and B rather than all three. Several of the proteins that form gluten are missing in durum wheat. The most notable of these are the high molecular weight glutenin subunits that are quoted for on the "D" genome, which is missing in these durum wheats.
• Example 28 This example measured the effects of adding a common reducing agent/free radical scavenger (cysteine) to doughs before mixograph analysis. These mixograph analyses were performed with a standard good bread-making quality control flour.
• a common reducing agent/free radical scavenger cyste
• Doughs incorporating five different levels of free cysteine were compared to a control dough and a cysteine standard after mixograph analyses.
• the cysteine levels included 40 ppm cysteine, 200 ppm cysteine, 0.5% cysteine, 2.5% cysteine, and 5.0% cysteine.
• doughs were placed in liquid nitrogen immediately after mixing and then lyophilized to dryness. Dityrosine analyses on the doughs were then performed as described previously.
• Fig. 37 shows the comparative dityrosine analyses from each dough incorporating cysteine, a control dough, and the cysteine standard.
• any incorporation of cysteine prevented the formation of dityrosine.
• the only dough exhibiting the presence of dityrosine was the control dough as shown at the peak at approximately 22 minutes.
• the peak which appears at approximately 16 minutes is likely a compound containing cysteine and tyrosine linked together.
• the presence of cysteine has a profound effect upon dityrosine formation in doughs. Therefore, the reducing agent/free radical scavenger cysteine, when incorporated into a dough system, prevents the ability of the dough to form dityrosine. Doughs containing cysteine do not form proper doughs and the mixograph analyses indicate very poor quality mixing properties.
• cysteine reducing agents/free radical scavenger
• cysteine are commonly used in the baking industry and are added to doughs when the doughs are too “bucky” or too elastic and do not relax properly. In cases such as these, the incorporation of cysteine prevents further dityrosine formation and helps to "relax" the dough.
• Example 29 This example compares the dityrosine analyses from dough samples which were mixed with varying levels of free glutathione. These samples were then compared to a dityrosine analysis from a control dough and the dityrosine analysis from a glutathione standard. Similar to Example 28, mixograph analyses were performed with a standard good bread-making quality control flour.
• the reducing agent/free radical scavenger (glutathione) was added in two different levels to the above referenced doughs and compared to the glutathione standard as well as the control dough. Levels of glutathione included lOOppm and 500ppm glutathione. After mixograph analyses, doughs were placed in liquid nitrogen and then lyophilized to dryness.
• the effect of this inhibition is that dityrosine formation is nearly entirely prevented.
• the reducing agent/free radical scavenger glutathione when incorporated into a dough system, prevents the ability of the dough to form dityrosine. Doughs containing glutathione do not form proper doughs and the mixograph analyses indicate very poor quality mixing properties.
• reducing agents/free radical scavengers such as glutathione are commonly used in the baking industry and are added to doughs when the doughs are too "bucky” or too elastic and do not relax properly. In cases such as these, the incorporation of glutathione prevents further dityrosine formation and helps to "relax" the dough.
• Example 30 This example sets forth methods for determining wheat protein (glutenin and gliadin) genes which are either up regulated or down regulated in response to heat exposure stress and/or differing phosphorous levels during wheat growth. As shown previously, genetically identical cultivars respond differently under these conditions, and this is correlated with the content of tyrosine, dityrosine and phosphotyrosine in the growing plants. Accordingly, knowledge of which genes are up regulated or down regulated permits control and manipulation of the genes through conventional methods so as to produce optimum wheat plants which can be consistently grown in given environmental conditions. The method makes use of differential displays of messenger RNAs (mRNAs) in order to demonstrate relative degrees of up or down regulation.
• mRNAs messenger RNAs
• Group A is grown under greenhouse conditions at 20 C C and the plants are fertilized at 2, 4, and 6 weeks after planting using either 10-60- 10, 10- 15- 10, or
• Group B is grown under greenhouse conditions at 40 °C and at the same three fertilizer concentrations.
• Group C is grown under normal environmental conditions and is fertilized using the same fertilizer procedure used for groups A and B.
• Wheat kernels are removed from the heads at 5, 10, 15, and 25 dpa and examined along with mature seed from the same batch of plants. Once the kernels are harvested, they are immediately immersed in liquid nitrogen and frozen at -80 °C. Once the wheat kernels are removed, differential mRNA displays are used to identify and isolate genes that are differentially expressed between the various groups of wheat samples. Additionally, kernels from each wheat sample are analyzed for the presence and level of tyrosine, phosphotyrosine, and dityrosine using previously described methods.
• poly A mRNA for each sample is first purified by treating it with DNase to remove contaminating DNA.
• the poly A mRNA is reverse transcribed to cDNA using reverse transcriptase and oligo dT12- 18 anchor primers which contain additional bases at the 3' end.
• the cDNA is then amplified using PCR with arbitrary primers and anchor primers used for reverse transcriptase.
• the resulting products are then subjected to electrophoresis side by side on polyacrymalamide gel. As the samples are run in parallel, the procedure allows for the simultaneous detection of differentially expressed genes. Additionally, gene expression over a time continuum can also be measured using this method.
• results from this electrophoresis are then compared and differentially expressed cDNAs are identified. Once these cDNAs of interest are identified, the bands of interest are excised. Next, the excised DNA is reamplified using primers from the primary reaction. Reamplified DNA is then reanalyzed on agarose gel and differential expression is confirmed using northern blot of RNA from the original sample populations using the labeled product DNA as a probe. Finally, the cDNAs of interest are cloned and sequenced using conventional techniques.
• transgenic wheats can be produced using known methods in order to optimize the response of the wheat to the known stress conditions.
• this same technique can be employed with other plants to achieve the same ends, response to exposure to heat stress and/or differing phosphorous levels.
• genes which are either up regulated or down regulated in response to these environmental stresses can be controlled and manipulated such that optimum wheat plants can be consistently grown despite environmental conditions.
• correlations between the levels of tyrosine, phosphotyrosine, dityrosine, and the environments and fertilizer regimes to which the plants were exposed demonstrates the protective effect that phosphotyrosine has on developing wheat kernels.
• Example 31 various peptides each containing a tyrosine pair (YY) were tested to determine their effectiveness in forming dityrosine linkages. Specifically, the different sequences were tested to determine their ability to form dityrosine linkages with high molecular weight glutenins.
• peptides of varying lengths were synthesized using conventional methods at the Kansas State University Core Biochemical Facility.
• the peptide sequences are as follows: QQGYYPTS, QGYYPTS, YYPTS, and YY (dipeptide, not dityrosine).
• Y free tyrosine
• Y free tyrosine
• tyrosine-containing peptides and derivatives were oxidized by horseradish peroxidase and dityrosine formation was investigated.
• the test peptides and derivatives used by Michon included the following: NAT ( N-acetyltyrosine), NATA (N-acetyltyrosine amide), PQQPY,
• Water soluble extract from flour (NWS control flour) (24 hr reaction).
• the water soluble extract is made by adding water to flour in a 5 : 1 ratio and mixing in a beaker on a stirring plate for 1 hr.
• the flour/water mix is then centrifuged and the supernatant is removed and used as the "water soluble extract.”
• KBrO 3 causes dityrosine formation even without any other factors which might be present in a dough or bread. If there were an agent in the flour that is required for dityrosine formation, dityrosine would not have formed in this experiment. Additionally, KBrO 3 was shown to significantly enhance dityrosine formation under these conditions because the control peptide formed no dityrosine when incubated under the same conditions but without the addition of
• This example describes site directed mutagenesis of DNA coding for QQGYYPTS repeats and variations thereof in expressed glutenin proteins in order to analyze the effect of the precise repeat amino acid sequence on dityrosine formation in doughs.
• dityrosine crosslinks form between and among glutenin proteins during dough mixing, and the ability to form dityrosine bonds effects product quality.
• High molecular weight glutenin subunit 1DX5 DNA This subunit is selected because it is known to be associated with good breadmaking quality wheat. It is to be understood that other glutenin subunits could also be isolated and treated as described below.
• Plasmids containing the coding region of Glu-1-Dx5 and other HMW-GS have been reported in the literature (Sugiyama et al (1985), Anderson et al. (1989), Galili (1989), and Halford et al (1992)). If such plasmids are not available, the gene encoding subunit Glu-1-Dx5 may be isolated from genomic/cDNA libraries.
• Genomic and cDNA libraries can be constructed or prepared by a suitable biotechnology company such as Stratagene. For commercial procedures, tissue or purified genomic DNA or mRNA can be obtained. The library(ies) are screened using a DIG labeled probe, e.g., a clone of PCR amplified repeat segment of the HMW-GS Dx gene. Following several rounds of screening/amplification of positive clones, in vivo excision of phage DNA to a phagemid (pBluescript SK+) is performed as described in the manual supplied with the library (Lambda Zap II library instruction manual, Rev. #127001c. The plasmids are prepared by the alkaline lysis method using a Qiagen miniprep kit (Valencia, CA), and the correct sequence is identified by restriction analysis and partial dideoxy DNA sequencing.
• a DIG labeled probe e.g., a clone of PCR amplified repeat segment of the HMW-GS Dx gene.
• Synthetic oligonucleotides incorporating the desired sites (5'-caacaaggttactacccaacttct- 3' codes for: QQGYYPTS) and also containing the appropriate flanking sequences are synthesized and phosphorylated at the 5' end.
• the flanking sequences can be 10-30 bp in size and can therefore increase the specificity of the insertion.
• an insertion of the (5'-caacaaggttactacccaacttct-3') sequence can be made if the primer used during PCR amplification also contains flanking regions that are specific for up to 22 bp on either side of the desired insertion location.
• Oligonucleotides with the following variations on the QQGYYPTS sequence are made: The actual mutated bases are underlined and shown in bold
• the protocols for site directed mutagenesis are based upon those published by Deng and Nickoloff (1992) and Lewis and Thompson (1990), and have been simplified by the commercialization of products such as Stratagene ChameleonTM Double-stranded site directed mutagenesis kit, and Promega Altered Sites® II in vitro mutagenesis system. These products allow double stranded plasmid DNA to be used for site-directed mutagenesis using selection by incorporation of a unique restriction site or antibiotic resistance in the altered strand of the plasmid. Generally, the mutagenesis protocol involves the following steps:
• oligonucleotide primer The 5 ' and 3 ' ends of the primer consist of the "clamp" sequences (homologous to the template DNA) and in between is the sequence to be inserted or mutated (5'-tactacccaacttct-3' or variations thereof). Other primers are synthesized for other regions of the sequence. This section describes only the synthesis of areas of the DNA containing the insertion sites. Primers for mutagenesis must be 5' phosphorylated. This may be chemically performed during synthesis or added to a primer using T4 ; polynucleotide kinase and ATP as phosphate donor.
• primers for selection will transform the Kpnl site in the plasmid polylinker to an Srfl site .
• Other plasmid vectors may involve change of antibiotic resistance.
• DNA polymerase and DNA ligase are added to synthesize mutant strand DNA and ligate the newly synthesized strands.
• dNTPs deoxy nucleotide triphosphates
• the mix is incubated at 37°C for 60-90 minutes.
• dNTPs deoxy nucleotide triphosphates
• Transform competent cells e.g. XL-1 Blue strain
• plasmids that have passed first selection criteria.
• the primer ends would be homologous to the regions of the protein just before and after the already existing 5'-caacaaggttactacccaacttct-3' sequence in the template. That portion of the template would not be amplified in that case. The resulting expressed protein would be missing that particular QQGYYPTS site.
• the column effluent is monitored by OD 280 and SDS-PAGE and fractions containing HMW subunit peptides dialyzed and lyophilized. Finally, fractions for N-terminal sequencing are separated by reversed phase (RP)-HPLC.
• RP reversed phase
• the freeze-dried extracts are dissolved at a concentration of 3 mg/mL applied to a Vydac C s semi-preparative column (10 mm X 25 cm) at 50°C using a System Gold (Beckman) HPLC with a model 126 solvent delivery system and a model 166 detector.
• Elution is with a linear gradient of water containing 0.07% (v/v) trifluoroacetic acid (TFA) (A) and acetonitrile containing 0.05% (v/v) TFA (B), from 18% B to 23% B over a 40 min period at a flow rate of 2.5 ml/min.
• TFA trifluoroacetic acid
• the column effluent is monitored at 210 nm and fractions containing the HMW subunit peptides are lyophilyzed.
• an excess of 4-vinylpyridine os added to the dissolving buffer and the reaction allowed to proceed for 10 min at 60°C prior to application to the column.
• N-terminal sequencing is performed on the peptides alkylated with the 4-vinylpyridine, using a pulsed-liquid amino acid sequencer (model 477A, Applied Biosystems) equipped with and online phenylthiohydantoin-amino acid analyzer (Model 120A).
• a pulsed-liquid amino acid sequencer model 477A, Applied Biosystems
• phenylthiohydantoin-amino acid analyzer Model 120A
• c. Purified peptides are re-oxidized in vitro to form disulfide crosslinks using potassium iodate. Re-oxidized samples are separated by SDS-PAGE using a multi- stacking system to check purity and identity.
• Mixing studies are conducted in triplicate with a 2-g Mixograph using a modification of the standard method for 35 g of flour scaled to the two gram size.
• Mixing parameters are determined using a modification of a previously reported MixSmart computer program. This modification automatically excises the portions of the recording during which mixing is halted. Multiple analysis of variance followed by the studentt-test is used to compare mixing parameters from different treatments using the MSUSTAT statistical program package. Least significant differences (L.S.D.) are determined at the 5% probability level. e. A reversible reduction/oxidation procedure for incorporating added peptides (5 mg) into glutenin is used. A special flour with 16.2% protein content milled from a Glu-Dl null line with a mixed Gabo/Olympic background, containing subunits lAxl, 1BX17 and lByl ⁇ , is used as a base flour.
• Flour is mixed with 1.0 mL water and 0.1 mL water containing 324 mM DTT for 30 s and allowed to react for 4 min.
• the reduced doughs are then treated with 0.1 mL oxidant solution containing 934 mM of potassium iodate. Mixing is continued for 30 s and the dough is then allowed to react for 5 min and then mixed for a further 10 min, as in a conventional Mixograph determination.
• Expressed protein 250 mg is also mixed into 100 gm of the base flour and baked into bread using the AACC 10-10B breadmaking procedure. Bread quality parameters are evaluated and compared in order to assess the effects of adding HMW-GS with the altered repeat sequences.
• Plots of wheat (Triticum aestivum L.) are grown at regular intervals, in 6 inch diameter plastic pots filled with Ready Earth (W.R. Grace & Co.) potting mixture, to maintain a steady supply of plant material. Plants are maintained in a growth chamber at 25 ⁇ 2°C and 20 ⁇ 2°C night temperatures under a 16 h photoperiod (150 ⁇ mol photon m "2 sec "1 ) provided by banks of fluorescent tubes and incandescent bulbs. All plants are watered eveiy second day and fertilized once a week with 0.4 g/1 of soluble greenhouse fertilizer (20:20:20).
• a. Preparation of scutella Wheat spikes are harvested 10-12 days post anthesis from greenhouse grown plants.
• Immature caryopses are removed from the spikelets and surface sterilized with 70% ethanol for 1 min and 20% (v/v) Javex bleach (1.2% sodium hypochlorite) for 20 minutes followed by five rinses with sterile double distilled water. Using a stereo dissecting microscope, the immature embryos are aseptically removed from the caryopses and the scutella isolated by carefully removing the embryo axis. The isolated scutella are then cultured with their abaxial (convex) surfaces in contact with the medium.
• the culture medium should consist of MS (Murashige and Skoog, 1962) medium supplemented with 2 mg l "1 2,4 dichlorophenoxyacetic acid (2,4-D) and 100 mg l "1 casamino acid, and herein after referred to as MS " " " " medium.
• the medium is solidified with agar (0.8%) and the pH adjusted to 5.8 prior to autoclaving.
• the cultures are incubated in the dark at 26 ⁇ 2°C for 1 week and then transferred to low light (10 ⁇ mol photons m "2 sec _1 ) under a 16 h photoperiod for another 2-3 weeks.
• DNA coating and bombardment Gold particles (1.0 ⁇ m) are coated with plasmid DNA using a procedure modified by Kleinet al.
• the isolated scutella is be precultured for 2 days on MS "1"1" medium in the dark prior to bombardment. Twenty precultured scutella are arranged in a circle of about 2 cm diameter in the center of a 60 x 15 mm petri plates containing 10 ml of agar solidified MS ++ medium. The petri plates are positioned at 115 mm target distance in such a way that the abaxial (concave) surface of each scutella was in the direct path of the micro projectiles. The microprojectiles are bombarded at a rupture pressure of HOOpsi using the DuPont helium driven Biolistics particle delivery system (PDS 1000). c.
• PDS 1000 DuPont helium driven Biolistics particle delivery system
• the scutella are cultured in the dark for 2 days on callus induction media containing 0.4 M mannitol for 4 h before and 20 h after bombardment.
• Bialaphos resistant embryos are selected on half MS media supplemented with 1 mg/L of bialaphos (Meiji SeikaKasha, Tokyo, Japan). The somatic embryos formed on this media are separated and to MS 4' media. The embryos capable of developing into green shoots within 3-4 weeks are characterized as putative transformants.
• the selected shoots are transferred to half MS for further growth and rooting.
• the plantlets developed on this media are transferred to environmentally controlled growth chambers and grown for further analyses. Protein is extracted from immature endosperm tissue and screening for the presence of the HMW-GS 1DX5 will be performed by SDS-PAGE analysis.
• Plants found to contain the modified HMW-GS are used to increase the amount of seed available by subsequent planting of seed from these plants. Once enough seed is available, all standard analytical tests as well as milling, mixograph and baking analyses are performed to assess the quality of the grain developed with the various modifications of the QQGYYPTS repeats. Dough and bread made from these kernels are also evaluated for the presence of dityrosine. A comparison of the levels of dityrosine present in the flour, mixed dough and baked bread, as well as the various quality parameters of these products, are used to assess the effects of modifying the YYPTS sequence on product processing quality. 5. Expression of the storage proteins in plants other than wheat.
• Any protein can be modified in transformed plants by the aforementioned techniques. Therefore, if modification of barley proteins, soybean proteins or any other protein is desirable, the above procedures can be performed using the sequence from the protein selected for modification as the template and simply inserting the 5'-caacaaggttactacccaactct-3' sequence or modifications thereof wherever the sequence is desired.
• Example 33 This example shows that common wheat substitutes, often used by people who cannot eat wheat, do not form significant levels of dityrosine during processing. This lack of dityrosine formation accounts for their production of inferior products because the substitute ingredients do not have the same properties as wheat flour and hence cannot form good quality doughs. Accordingly, dityrosine cross linking capabilities could be edited into these grains and their potential for wheat replacement in various products would be greatly enhanced.
• tyrosine bond formation may now be used to consistently produce optimum dough products.
• Standards for tyrosine bond content corresponding to different dough applications and different processing stages may now be found and used as a comparison or guide to direct dough processing.
• the starting tyrosine and dityrosine content in flour may also be found and used to both predict dough characteristics and precalibrate dough processing equipment such that waste is minimized and optimum products are consistently produced.
• Knowledge of this starting tyrosine and/or tyrosine bond content allows for prediction of the potential for tyrosine bond formation since the tyrosine present in the flour is the tyrosine used to form tyrosine bonds.
• a sample of flour exhibiting high levels of tyrosine and/or low levels of dityrosine should have the potential to form a higher number of tyrosine bonds. This in turn may contribute to a higher rate of tyrosine bond formation and therefore, shorter mixing times for dough formation.
• a high starting tyrosine content may also indicate the need for modification such as increasing the pH of the dough or the addition of metal chelating agents or free tyrosine to retard tyrosine bond formation.
• a flour exhibiting a lower level of tyrosine or higher level of dityrosine may need longer mixing times to form a high quality dough and/or some modification to promote tyrosine bond formation (i.e.
• the tyrosine and dityrosine content of flour may be measured through derivatized or underivatized amino acids, however, measuring the derivatized amino acids is a much more sensitive technique.
• An example of determining the tyrosine content of flour using HPLC of the underivatized amino acids is shown in Fig. 16.
• Tyrosine is represented by the peak which elutes at 12.380 minutes.
• environmental conditions were shown to have an effect on dityrosine content of flour and these conditions effected different wheat varieties to different extents. Thus, knowledge of environmental conditions will contribute to knowledge of what properties doughs made from such flours will exhibit.
• varieties which are better suited for particular environments may be selected for growth in these environments.
• amino acid profiles of lower quality varieties can be genetically manipulated by cross-breeding or DNA recombination/alteration in order to produce new higher quality varieties.
• Another alternative would be to alter the environmental conditions (such as adding phosphorous or phosphorous-containing fertilizers to the soil surrounding growing wheat) in order to inhibit premature dityrosine formation.
• dough production can be effected such that optimum dough products are consistently produced based on the knowledge of the tyrosine, dityrosine, phosphotyrosine and/or tyrosine bond content of the starting flour, assessment of tyrosine bonding during dough production and modification of tyrosine bond formation during dough production in response to the assessment in order to achieve an ideal tyrosine bond content based on the eventual end use of the dough produced. Additionally, levels of the same compounds can be monitored during growth of wheat plants and the resulting wheat kernels can be graded according to such levels.
• developing wheat plants can be analyzed to determine their levels of tyrosine, dityrosine and phosphotyrosine and knowledge of these levels in the developing plants contributes to methods of altering such levels in order to produce wheat plants having optimum characteristics.
• This alteration can be done in a variety of ways including applying phosphorous to the ground adjacent growing plants and altering the genome of the plant such that the number of base pair sequence subunits coding for preferred peptide sequences are either increased or decreased. Accordingly, similar alterations can also be performed in non- wheat plants in order to produce products having crosslinking characteristics similar to products produced by conventional wheat doughs.
• polymers and composite polymers can be modified such that the peptide sequences described above can be coupled with the polymers in order to effect crosslinking between and among polymer subunits.
• the peptides can be directly incorporated into the normal protein sequence or attached as a side chain or end cap to the protein.
• Crosslinking reaction conditions are dependent upon the specific polymers present, however, the rate of crosslinking can be affected by the addition of oxidizing agents, free radical scavengers, reducing agents, and free radical generators in a similar fashion to that found in dough processes.
• Galili G. 1989. Heterologous expression of a wheat high molecular weight glutenin gene in Escherichia coli. Proceedings of the National Academy of Sciences (USA) 86:7756-7760.

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