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United States Department of Agriculture

Agricultural Research Service

Research Project: CHARACTERIZATION OF GRAIN BIOCHEMICAL COMPONENTS RESPONSIBLE FOR END-USE QUALITY

Location: Grain Quality and Structure Research Unit

2006 Annual Report


1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter?
The baking industry encounters tremendous loss in the areas of quality assurance to customers, consistency of product, processing parameters and product waste. It is estimated that as much as 10% of a company's product is relegated to waste due to lack of flour consistency. This variability in composition and quality of wheat flour is dependent upon genetic, environmental and supply chain factors. For example, not only will the protein content vary from lot to lot, but within a given protein content range (specified by end user requirements), the quality of the protein will vary considerably. Even when basic product specifications, such as ash, moisture, and protein content are met, the dough forming and baking characteristics of a given lot of flour are highly unpredictable.

Similarly, ingredient and processing variability translates into large economic losses in the baking industry. The U.S. baking industry annually uses about 42 billion pounds of flour, valued at about $4.2 billion, to produce an estimated $33 billion of baked goods. Variability in flour quality means that there are opportunities for large savings in the areas of raw material selection, processing, and distribution. Further, improved methodologies to better analyze the root causes of flour variability will open the door to the production of higher value products. The baking industry has limited tools at its disposal to deal effectively with the variability of wheat flour, its major raw material. All currently utilized industry tools for assaying and/or testing flour are highly empirical, offering little insight into the biochemical basis of (a) what constitutes a “quality” flour, (b) how a particular lot of flour can be characterized for its suitability for a given application, or (c) how a particular lot of flour should be processed to maximize product quality.

The objectives of this project are to: (1) Determine the roles and interactions of the major biochemical components of cereal grains (starch, storage proteins and enzymes) as they relate to food quality and functionality; (2) Define the role of the environment on functional properties of biochemical components that affect end-use properties; (3) Apply information generated in previous objectives towards development and refinement of methods to rapidly predict grain quality. Towards this end, investigation to determine role(s) of wheat starch granule size distributions on variations in functionality during bread-making will be performed. These approaches will utilize differential scanning calorimetry to provide thermal data on individual starch granule populations and starch fractions (pure A- and B- type granules) will be used for reconstitution experiments to determine how size classes affect bread-making quality. The mechanism(s) of action of oxidative enzyme addition to flour on storage proteins and non-storage proteins will be determined by addition of enzymes followed by functional analysis and examination of protein fractions and polymers using SDS-PAGE, size exclusion HPLC, and multi-angle laser light scattering. Environmental effects on starch and storage proteins will be determined using the above approaches with samples that have been exposed to controlled temperature and irrigation regimes during development. The potential of novel microfluidic devices will be thoroughly examined as a means to develop extremely rapid and highly reproducible separation of proteins for grain cultivar identification and quality prediction.

Advances in the knowledge of grain biochemical characteristics that determine the physical and functional properties critical to processing and end-product quality, and development of rapid, accurate methods to measure these quality determinants are essential to maintain a competitive position for U.S. grain in global markets. This project will provide the cereal food industry with the tools needed to define the end-use performance of cereal grains.

The research is under National Program 306 Quality and Utilization of Agricultural Products”, specifically on component 1 “Quality Characterization, Preservation, and Enhancement”. Multiple Problem Areas of this component are directly addressed in the objectives -- Problem Areas 1a (Definition and Basis for Quality); 1b (Methods to Evaluate and Predict Quality) and 1c (Factors and Processes that Affect Quality). The elucidation of fundamental biochemical processes and their role in determining product quality is paramount for the development of accurate methods for quality measurement.


2.List by year the currently approved milestones (indicators of research progress)
Year 1 (FY 2005):

Isolate large quantities of starch, separate into size fractions for baking and chemical testing. Continue testing the correction model for starch size distributions on the LDS. Identify wheat of different oxidation requirements and determine effect of oxidative enzymes and transglutaminase upon quality characteristics. Begin biochemical analysis on the effect of enzymes on protein interactions. Identify and begin to collect wheat samples to represent various growing environments. Begin isolating starch for analysis. Develop inexpensive lab-on-a-chip technology to extract and separate wheat gliadins.

Year 2 (FY 2006):

Isolation of large quantities of starch, separated into size fractions for baking and chemical testing. Continue testing the correction model for starch size distributions by laser diffraction sizing (LDS).

Determination of the effects of oxidative enzymes and transglutaminase upon quality characteristics. Biochemical analysis on the effect of enzymes on protein interactions.

Use LDS and our correction model to detect environmental differences in starch ratios. Begin isolating the starch fractions from different environments for chemical analysis.

Characterize by RP–HPLC and SEC-HPLC, the protein fractions of the various near- isogenic lines that are produced in year 1 study by our collaborators. Relate the amount of particular glutenin (polymeric) and gliadin (monomeric) proteins to the HMW-GS in the various near-isogenic lines.

Acquire representative samples for “lab-on-a-chip” analysis to develop a library of protein profiles of commonly grown U.S. wheat cultivars.

Year 3 (FY 2007):

Isolation of large quantities of starch, separated into size fractions for baking and chemical testing. Continue testing the correction model for starch size distributions by laser diffraction sizing (LDS).

Determine if particular starch granule ratios are markers for quality traits. Continue isolating the starch fractions from different environments for chemical analysis. Compare starch size distributions and chemical analysis to different environments. Use LDS and our correction model to detect environmental differences in starch ratios. Continue testing amylose/amylopectin ratios, pasting profiles, DSC temperatures and lipids of A-, B-, and C-type starch fractions.

Determination of the contribution of individual glutenin subunits on enzyme mediated crosslinking in gluten functionality. Characterization of enzyme effects on protein interactions. Correlate information obtained with data on bread or tortilla quality characteristics.

Continue characterization of enzyme effects on protein interactions between glutenin and albumins. Begin investigation of effect of HMW-GS contribution of wheat on enzyme mediated crosslinking.

Continue investigating lab-on-a-chip system to extract, separate and identify wheat varieties. Test unknown and difficult to classify samples against the library developed. Improve extraction methodologies and separation techniques incorporating novel methods available at that time.

Year 4 (FY 2008):

Isolation of large quantities of starch, separated into size fractions for baking and chemical testing. Continue testing the correction model for starch size distributions by laser diffraction sizing (LDS).

Bake studies using reconstituted gluten and starch fractions. Begin testing, amylose /amylopectin ratios, pasting profiles, differential scanning calorimetry (DSC) temperatures and lipids of A-, B-, and C-type starch fractions. Chemical analysis of the starch fractions. Correlation analysis comparing bake data, starch size distributions and chemical analysis.

Continue characterization of effect of HMW-GS contribution of wheat on enzyme mediated crosslinking. Determine the sizes of polymeric fractions and the MW distribution of polymeric proteins from various near-isogenic lines.

Compare starch size distributions and chemical analysis to different environments. Proceed with analysis of starch from wheat grown in different environmental conditions to determine correlations of environmental effects on starch size distribution.

Relate polymer sizes and molecular weight distributions to quality characteristics, provided by the HWWQL.

Lab-on-a-chip will be developed to identify quality-related proteins and unique proteins associated with specialty wheats. The effect of environment on proteins and quality will be analyzed.

Year 5 (FY 2009):

Complete bake studies and chemical analysis of the starch fractions. Determine correlations of starch size distribution to baking and chemical parameters.

Complete characterization of effect of HMW-GS contribution of wheat on enzyme mediated crosslinking.

Determine correlations of environmental effects on starch size distribution.

Determine if particular proteins are markers for quality traits.

Determine feasibility of lab-on-a-chip system for on the spot analysis.


4a.List the single most significant research accomplishment during FY 2006.
Isolation of large quantities of starch. We have developed a method to isolate large amounts of starch separated into size fractions for baking and chemical testing. The sonication procedure for starch isolation has been scaled up and tested allowing us to isolate starch from up to 40 grams of starch at a time. This procedure has decreased the time it takes to isolate starch from flour from ~1 hour per sample to ~10 minutes. The correction model is currently being applied to ~ 100 HRW and 98 HRS wheat obtained from FGIS. This data is being correlated to bake quality data to better understand the importance of starch size distribution. The research is under National Program 306 “Quality and Utilization of Agricultural Products”, specifically on component 1 “Quality Characterization, Preservation, and Enhancement”, problem areas 1a (Definition and Basis for Quality); and 1c (Factors and Processes that Affect Quality).


4b.List other significant research accomplishment(s), if any.
Microfluidics library development. Knowledge of cereal proteins is important both for predicting end-use performance and for identification. The development of new or improvement of existing methods for deriving models for predicting quality traits and/or quickly identifying grain cultivars will assist in determining their roles in relation to quality. Microfluidic systems (lab-on-a-chip) produce fast separations using very small liquid volumes. We have purchased a microfluidics instrument and begun to identify quality-related protein patterns and unique proteins associated with specialty wheats, particularly waxy wheats. The effect of environment on proteins and quality might be available through analysis of HMW-GS isoforms or through quantification of albumins and globulins. In addition, it is envisioned that the gliadin patterns, amounts of albumins and globulins, and HMW-GS isoforms of specific wheat varieties with known quality traits will be stored in a database and when one of those patterns (or combinations) is matched, it will tag the sample as a good quality sample. The research is under National Program 306 “Quality and Utilization of Agricultural Products”, specifically on component 1 “Quality Characterization, Preservation, and Enhancement”, problem area 1b (Methods to Evaluate and Predict Quality).

LDS correction model detects environmental differences in starch ratios. The environmental impact on the starch size distribution during grain filling of hard red winter wheat has been collected and collated. These samples were all grown at the Kansas State University Agronomy Study plots over a 6 year period of time. Statistical analysis of this data is nearing completion and correlations to some weather conditions have been noted. The larger starch size distributions (everything > 20 µm) are positively correlated to increased cumulative precipitation, while the 5-20 µm starch size distributions are negatively correlated to decreased cumulative precipitation and decreased cumulative and average evapo-transpiration rates. Decreasing soil and air temperatures also appear to have a negative correlation to the 5-20 µm starch populations, while increasing soil and air temperatures has a positive correlation to the 20-30 µm starch populations. The research is under National Program 306 “Quality and Utilization of Agricultural Products”, specifically on component 1 “Quality Characterization, Preservation, and Enhancement”. Problem areas 1a (Definition and Basis for Quality); and 1c (Factors and Processes that Affect Quality).


4c.List significant activities that support special target populations.
Collaboration with Kansas State University on research on Tef (Eragrostis tef) grain quality. The Tef project supports a USDA grant to the Kansas Black Farmers Association. Tef is native to Ethiopia where it is an important staple crop. It is drought tolerant and gluten-free thus has potential for patients with celiac disease.


5.Describe the major accomplishments to date and their predicted or actual impact.
Microfluidics system for grain analysis Knowledge of cereal proteins is important both for predicting end-use performance and for identification. The development of new or improvement of existing methods for deriving models for predicting quality traits and/or quickly identifying grain cultivars will assist in determining their roles in relation to quality. Microfluidic systems (lab-on-a-chip) produce fast separations using very small liquid volumes. We have purchased a microfluidics instrument and begun to identify quality-related protein patterns and unique proteins associated with specialty wheats, particularly waxy wheats. The effect of environment on proteins and quality might be available through analysis of HMW-GS isoforms or through quantification of albumins and globulins. In addition, it is envisioned that the gliadin patterns, amounts of albumins and globulins, and HMW-GS isoforms of specific wheat varieties with known quality traits will be stored in a database and when one of those patterns (or combinations) is matched, it will tag the sample as a good quality sample. Microfluidics system for cultivar identification is in early testing stages and we have held discussions with FGIS/GIPSA to obtain samples for library development and the resulting data will be shared with this agency.

Starch Analysis Starch constitutes the major weight portion of wheat endosperm (~ 75% v. ~15%) and contributes to food its own unique functional qualities such as volume, texture, appearance and staling rate. Varying ratios of large type-A and smaller type-B granules has been proposed to change the baking potential of bread. Bread made from reconstituted flour with 30% type-B to 70% type-A ratios gave optimum crumb grain scores and peak fineness values and second highest elongation ratios. As the proportion of type-B granules increased, it yielded bread with a softer texture and maintained that texture better during storage. Due to the unique properties of the different size starch granules in wheat, it is reasonable to consider starch size distribution as having a significant impact on baking performance.

Environmental changes not only affect protein quality but may affect starch size distribution and its chemical composition. Work has been initiated to contrast weather conditions (temperature and precipitation) to starch size distribution in developing wheat. With samples collected over multiple years it will be possible to trace the impact of changes in environmental growing conditions to critical growing phases in starch development. This may be useful in developing a data base incorporating environmental information during the growing season to ideal starch development for optimum end-use quality. A differential scanning calorimeter (DSC) has been purchased which will be used to study gelatinization profiles (melting) of isolated starches and differences within the various size populations. This information is important in detecting biochemical changes such as amylose/amylopectin ratios and lipid content in these isolated starches.

Major accomplishments of this project include optimization of starch granule sizing methods. This is a critical development in the study of wheat starch size distribution as it allows analysis with the speed of LDS but the accuracy of IA. This will provide determination of environmental effects on starch granule size distributions and end-use functionality.


6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
Lab-on-a-chip system for cultivar is being tested for wheat breeders who are supplying samples for development of a database of commonly grown wheat cultivars. The system has potential for rapid on-the-spot analysis of wheat cultivars.


7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
Chung, O.K., Dowell, F.E., Park, S.H., Lookhart, G.L., Tilley, M., Brabec, D.L., Ram, M.S., Seitz, L.M., Bean, S.R., Seabourn, Pearson, T.C., Bechtel, D.B., Casada, M.E., Hubbard, J.D., Downing, J.D., Maghirang, E.B., Wilson, J.D., Armstrong, P.R., Caley, M.S., Xie, F., Arthur, F.H., Lyne, R.K., and Xiao, S.Z. 2005. Wheat research in the U.S. Grain Marketing and Production Research Center. Annual Wheat Newsletter 51:193 - 210 (Review)

Park, S.O., S. Bean, J. D. Wilson and T. J. Schober. Investigation of conditions for rapid cereal starch isolation using sonication. AACC International meeting, Orlando, FL. Schober, T.J., and S.R. Bean Protein composition and rheological properties of spelt cultivars as a model of gluten quality. AACC International meeting, Orlando, FL.

Tilley, M. Glucose oxidase effects on wheat albumins and globulins. Presented at the 88th annual meeting of the American Association of Cereal Chemists, September 11- 14, 2005 Orlando FL.


Review Publications
Tilley, M., Bean, S.R., and Tilley, K.A. 2006. Capillary electrophoresis for monitoring dityrosine and 3-bromotyrosine synthesis. J. Chromatography A. 1103:368-371.

Tilley, M. 2005. Glucose oxidase effects on wheat flour albumins and gliadins. Abstract No. 238 in: 2005 AACC International Annual Meeting Program Book. p.144. Meeting Abstract.

Wilson, J.D., Bechtel, D.B., Todd, T., Seib, P.A. 2005. Measurement of wheat starch granule size distribution using image analysis and laser diffraction technology. Cereal Chem. 83:259-268.

Schober, T.J., Bean, S., Kuhn, M. 2006. Gluten proteins from spelt (Triticum aestivum ssp. spelta) cultivars: A rheological and size-exclusion high-performance liquid chromatography study. J. Cereal Sci. 44(2): 161-173.

Last Modified: 9/10/2014
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