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 National Program this project is aligned to is NP 306, Quality and Utilization of Agricultural Products. Wheat is a vital food component for humans worldwide. In 2005 (most recent year with available data), 2.1 billion bushels of wheat were harvested in the United States (2 percent decrease from 2004), representing a total farm-level value of $7.1 billion (USDA Agricultural Statistics: 2006). With slightly 48 percent of its wheat going to export, the United States leads the world in international wheat sales (25% of the World's traded wheat), followed by Australia (14%), Canada (13%), the European Union (13%), and Argentina (12%). For soybeans, these numbers are similarly impressive ($16.9 billion value, 43% of the export market share, followed by Brazil and Argentina at 35% and 14%, respectively). Thus, the United States is a key player in the production and sale of these commodities. Research within USDA-ARS, including that performed under this project, is designed to maintain the country's preeminence in the capability of delivering quality wheat and soybeans for domestic and international consumption. This project is divided into three components, or "objectives," each of which is addressed in the following paragraphs. First, to maintain a supply of high quality grain for domestic consumption, as well as for overseas export, the United States must develop new, rapid, and objective procedures and instruments that will assess product quality. Inspection is also performed for the purpose of food safety in that inspectors are required to examine the grain for mold, filth, and insect damage. In spite of the increased need for quality and safety assessment of grain, inspection today is much the same as it was 50 years ago. It is overly reliant on human visual analysis, which is slow (several minutes per sample) and subjective. The planned research is focused on the development of an automated system that can be used in official grain inspection procedures for detection of moldy and damaged wheat kernels, and ideally, in commercial operations. The system will be based on visible and/or near-infrared (NIR) spectroscopy and image analysis. By law, all grain that is destined for export must undergo official inspection. Inspection is also performed on domestically traded grain upon request. Grain inspection traditionally is reliant on human analysis, usually by appearance and odor. As such, the procedure is time consuming. The Federal Grain Inspection Service (FGIS) of the USDA Grain Inspection, Packers and Stockyards Administration (GIPSA), in its Fiscal Year 2007 prioritized list of research needs for ARS, has reiterated the desire for the development of wheat functional assessment tests and methodologies that are accurate, consistent, rapid, easy to use, inexpensive, and rugged. Among other areas, GIPSA has asked ARS to focus on the use of NIR or Mid-IR spectroscopy for resolving issues of gluten functionality, water absorption, and starch quality. As a second component of this project, a rapid and objective NIR method for classifying or measuring the degree of waxiness in wheat is being developed. Plant breeders are currently developing waxy wheat varieties in the United States for the purpose of filling a new market niche. Because of the hexaploid nature of bread wheats (Triticum aestivum L.), the waxy condition, characterized by a very low or non-detectable level of starch amylose (with elevated levels of amylopectin, the complementary component), is regulated by the genes from all three (A, B, and D) genomes. For tetraploid species such as durum wheat, amylose production arises from single genes from the A and B genomes. The traditional wet chemical method for characterizing waxiness of starch is too slow and expensive for breeders to use in all but the most advanced generations. Lack of reasonable early generation screening procedures for waxiness hampers the efficiency of new variety release for bread wheats and semolina wheats. A rapid assessment procedure is needed to permit selection for waxiness at the early stages of new variety development. Differences in bond strengths of C-H and O-H groups of amylose and amylopectin (arising from differences in molecular structure and hydrogen bonding) may be sufficiently large for these macromolecules to be distinguishable in the NIR region. The development of a rapid, objective procedure to identify and quantify the waxy condition in wheat lines is directly relevant to the plant breeder. Conventional methods for identifying waxy and partial waxy genotypes, such as SDS-PAGE, which separates the isoforms of the enzyme that regulate starch amylose synthesis, are expensive, complicated, and time consuming. To a lesser extent, the same is true for enzyme linked immunosorbent (ELISA) tests; however, this procedure requires a preliminary starch extraction procedure. With this new tool, the breeder will be able to screen crosses with greater efficiency, thus leading to the speedier release of high quality low- or no-amylose wheat varieties whose potential uses extend to the food and non-food (papermaking, adhesives) industries. A third component of the project is to examine the use of NIR spectroscopy for rapid assessment of soybeans in soybean breeding programs. This objective arises from the expressed assertion of the United Soybean Board (USB), whose check-off program called the "Better Bean Initiative" aims to improve the compositional makeup and quality of new soybean releases in the United States. In addition to the traditional goals of augmentation of protein and oil contents, the Initiative, and hence this objective, seeks to develop rapid NIR-based procedures for measurement of profiles of fatty acids and amino acids, and phytate. These specific targets are also mentioned in USDA-GIPSA's 2007 prioritized research needs for ARS (see above). 2.List by year the currently approved milestones (indicators of research progress) Milestones – The milestones listed in this section pertain to a research project that began in fall 2004. Year 1: Objective 1 - Improve integration of diode array spectrometer with computer controlled positioning system. Refine hyperspectral imaging system for small grain analysis. Collect scab damaged wheat samples and verify damage with USDA GIPSA, Federal Grain Inspection Service, Board of Appeals and Review. Objective 2 - Develop parametric and nonparametric classification models for granule bound starch synthase gene classification, using existing data. Obtain and scan breeders’ protein quality samples. Objective 3 - Collect and spectrally screen Better Bean Initiative (BBI) breeders' soybean samples. Conduct phytate analysis of soybean meal samples. Augment BBI samples with new crop year. Year 2: Objective 1 - Optimize diode array system design. Develop 2-4 wavelength NIR image model for scab detection. Collect NIR, FT-IR, and Raman spectra on ground wheat and develop DON calibrations. Objective 2 - Compare field application study classification results with actual proportions of progeny. Develop amylose content models. Collect spectral data on Nebraska-grown glutenin/gliadin samples and California samples. Objective 3 - Obtain reference values for fatty acids and develop calibrations. Collect FT-NIR and FT-mid-IR spectra on BBI samples and perform 2-D correlation analysis. Develop NIR phytate classification models. Year 3: Objective 1 - Test imaging and diode array systems for scab on FGIS market samples. From diode array tests, identify one to four wavelengths useful for inexpensive scab detection instrument. Refine DON calibrations. Objective 2 - Perform 2-D correlation spectroscopy (NIR vs. mid-IR) on protein quality data to better understand spectral sensitivity. Develop classification models using all GBSS genotypes. Objective 3 - Transfer BBI technology to United Soybean Board. Refine NIR phytate classification models. Year 4: Objective 1 - Using hyperspectral machine vision imaging system, develop classifications for other molds and kernel damage categories. Develop multispectral equivalent to the above system. Transfer technology to official inspectors and Industry. Objective 2 - Finalize development of GBSS gene classification and amylose content models for distribution to plant breeders. Objective 3 - Transfer technology of NIR phytate to breeders. Year 5: Objective 1 - Refine multispectral system for other damaged categories. Objective 2 - Transfer technology to breeders and the FGIS. Objective 3 - Completed. 4a.List the single most significant research accomplishment during FY 2006. Near-infrared Analysis of Phosphorus in Soybeans - Because of potential nutritional deficiencies associated with food and feed with high phytic acid that is caused by a decreased absorption of essential minerals, soybean breeding programs aimed at targeting low phytate releases are currently underway. As near-infrared (NIR) spectroscopy is a common tool for macronutrient analysis in breeding programs, its application to low phytate line development is foreseen as readily accepted, provided such modeling is possible. A study was completed that utilized 191 recombinant inbred soybean lines. Spectral data collection consisted of diffuse reflectance (1100-2500 nm) of ground meal and single-bean transmittance (600-1900 nm) of whole seed. Partial least squares regression models indicated that best performance was obtained with diffuse reflectance data. Performance declined when averages of single-bean spectra were used to represent a sample, and further declined when single-bean spectra alone were used. While NIR phosphorus modeling errors are comparatively larger than those for common macronutrient regressions such as those for protein and oil, this technique will be useful in the screening of soybean lines for low phytic acid. This accomplishment is aligned with Problem Area 1b (Methods to Evaluate and Predict Quality) of National Program 306. 4b.List other significant research accomplishment(s), if any. Classification of the State of Waxiness in Tetraploid wheat - Currently, wheat programs in North America and elsewhere are exploring the development of these types of waxy durum wheats for possible new and unique uses. Plant breeders must rely on slow chemical techniques to identify waxy seed during development of new lines. A study was completed that examined the potential of an spectroscopy-based method for determination of the waxiness state (wild type, waxy, and two states of partial waxy). The results indicated that identification of waxy seed could occur at greater than 95% accuracy; however, discrimination among the three other classes was not possible. Fortunately, in waxy breeding programs, the identification of the waxy seed is the most critical. Therefore, this technique stands as a demonstrated tool for use in breeding programs and eventually in identity preservation. This research is also aligned with Problem Area 1b (Methods to Evaluate and Predict Quality) of National Program 306. 4c.List significant activities that support special target populations. none. 4d.Progress report. Evaluation of Spectral Pretreatment Procedures in Near-Infrared Calibrations – In our work of previous years, a systematic procedure was developed for the evaluation and optimization of near-infrared (NIR) calibration equations used in the prediction of biochemical components in wheat and forages, but which could be generalized to any calibration. This addressed the needs of the NIR analyst who, during the development of calibrations for constituent concentration, is constantly faced with the challenge of determining the best preprocessing to apply to the spectral data before actual multivariate regression analysis. In cooperation with another USDA Beltsville scientist, begun in the previous year, statistical tests were finished on identifying those pretreatments that are significantly superior to other pretreatments. Patterned after a comparison of means procedure in an analysis of variance, this procedure has the ability to comb through hundreds of pretreatment combinations, inclusive of scatter correction, smoothes, derivatization, and variance scaling. This development serves to improve the accuracy of NIR calibrations and removes much of the subjectivity of pretreatment selection during calibration development. The next four paragraphs serve to document research conducted under a Specific Cooperative Agreement (SCA) between Cornell University and USDA-ARS. Work in the past year has resulted in experimental methods that allow for unambiguous separation of delayed elastic effects and plastic flow in gluten. The first method is a "rapid" shear creep-recovery test that allows for determination of the equilibrium delayed recoverable elasticity of gluten. This is a fundamental measure of the reversible extensibility of gluten. The second method is a large deformation tensile test that results in roughly 50% plastic flow and 50% recoverable extensibility. Gluten in dough is subject to a wide range of shear and extensional stresses and strains in practical uses such as mixing, sheeting, oven rise, and hand-pulling of noodles. These two tests are designed to provide an objective measure of the viscoelasticity of gluten defined as simultaneous reversible extensibility and plastic flow. Preliminary experiments have shown that glutens obtained via the Glutomatic standard washing procedure have similar wet gluten contents and gluten index, but have substantially different rheological properties as determined by the above two methods. This suggests that elastic structures responsible for the recoverable extensibility of gluten forms during the deformation itself, and thus meaningful rheological tests must have a recovery mode after prior deformation. It was also noted in preliminary experiments that the patterns of creep-recovery obtained for Glutomatic glutens using a fundamental controlled stress rheometer were qualitatively similar to those obtained for gluten using a Brabender Glutograph-E. In both cases, glutens with similar Glutomatic properties were distinguished in creep-recovery. Thus, it appears that the combination of the Glutomatic and Glutograph-E could form the basis of a practical rapid method of objective characterization of the intrinsic, native viscoelasticity gluten in a way not currently utilized. Future work will involve characterization of the creep-recovery and tensile properties of a series of well-characterized glutens, with the ultimate objective being adoption of standard methods for the characterization of the viscoelasticity of gluten. This paragraph describes the research conducted through a reimbursable agreement between USDA-GIPSA and USDA-ARS. This agreement dovetails with the SCA between ARS and Cornell and also involves the cooperation of Oklahoma State University. The development of a rapid assessment method for gluten functionality that can be used in international commerce is the overall goal of this research. During this first year of the four-party study, ten pure variety wheat samples (approximately 150 lb per sample) were procured by USDA-GIPSA. Each sample was milled, mixed, and stored for upcoming tests. In a preliminary study to test the suitability of commercially available gluten dough assessment equipment, three separate samples were tested. All three of them gave varied gluten deformation patterns. While wet gluten quantity or its dry gluten or gluten protein contents of the samples remained roughly constant, the gluten strength varied. These findings point out the shortcoming of much of the existing gluten assessment instruments, which is further justification for the development of a consolidated gluten strength testing method. 5.Describe the major accomplishments to date and their predicted or actual impact. This research project began in fall 2004. Therefore, the accomplishments described herein relate to the first two years of the five-year project. They address the program components "Quality Characterization, Preservation and Enhancement" of ARS National Program 306 - Quality and Utilization of Agricultural Products. Likewise, they address the following Performance Measures in the ARS Strategic Plan:. 1)1.2.1 - "Provide producers with scientific information and technology that increase production efficiency, develop improved germplasm, safeguard the environment, improve animal well-being, and reduce production risks and product losses;" and. 2)3.1.2 - "Develop and transfer to Federal agencies and the private sector systems that rapidly and accurately detect, identify, and differentiate the most critical and economically important foodborne microbial pathogens." A wheat kernel damage detection system has gone through as series of developmental stages, starting back in the previous five-year period. Early work was based on a static system in which kernels were precisely aligned and at rest during scanning. Several wavelengths, mathematically combined in different trials, demonstrated the ability to identify Fusarium-damaged wheat at 95 percent or better accuracy. From there, the restriction on kernel alignment was partially removed (keeping the concept of axial alignment intact), with accuracies being on par with the previous work. This led to testing under conditions approaching that of commercial high-speed sorters. Unfortunately, accuracy suffered as a consequence of the thousand-fold increase in inspection speed. For example, 50 percent of visibly damaged kernels would typically be recognized and removed during sorting, as compared to a better than 90 percent operation involving an analytical spectrometer tuned to an equal number of wavelengths (typically two). The most recent research has attempted to probe this discrepancy. Single kernel inspection systems, involving the in-flight analysis of single kernels in freefall, have been developed. With these systems, we intend to better understand the sources of variability in high-speed detection systems. Breeders, inspectors, and mills are the intended beneficiaries of this work. Research at Beltsville several years ago demonstrated the success of using near-IR spectroscopy to identify the fully waxy condition in hexaploid (bread) wheat, but also the difficulty in identifying the intermediate genetic stages of the waxy state, known as partial waxy. With the advent of the current project, this research approach was adapted to tetraploid (durum) wheat, which, because of its relative genetically simpler background, should make it easier to identify the limits of accuracy of a near-IR based classification system. We have recently finished the analysis using conventional linear quantitative and qualitative analyses, with results again pointing to the difficulty in distinguishing between partial waxy genotypes and between the partial waxy and wild type conditions. Current research is demonstrating some additional success when non-linear classification algorithms are utilized. 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? In the course of the last two years, information on the detection and sorting of mold-damaged wheat has been shared directly with manufacturers of high speed commercial sorters and inspection-level instruments. The greatest challenge lies with ramping up throughput speeds to that required by milling operations. Our work continues to address throughput speed. As the result of our research on exhaustive analysis of spectral pretreatments on partial least squares (PLS) calibrations, a statistical method for selecting the best pretreatments is now available and directly ties into PLS software that we have previously made available by website download. Finally, during the past year, the techniques developed for analysis of the waxy condition were applied to a food ingredient adulteration problem of a large domestic manufacturer. 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). Reeves, J.B., III, Delwiche, S.R., Daughtry, C.S., Luthria, D.L. Near-infrared (NIR) research at the Beltsville Agricultural Research Center. NIR News. Part I in 1616:9-12; part II in 1618:12-13. (2005). Review Publications Delwiche, S.R., Gaines, C.S. 2005. Wavelength selection for monochromatic and bichromatic sorting of fusarium-damaged wheat. Applied Engineering in Agriculture. 21(4):681-688. Delwiche, S.R., Pearson, T.C., Brabec,, D.L. 2005. High-speed optical sorting of soft wheat for reduction of deoxynivalenol. Plant Disease Journal. 89(11):1214-1219. Himmelsbach, D.S., De Haseth, J.A., Delwiche, S.R. 2005. Prediction of SDS sedimentation volumes for wheat via FT-NIR and FT-Raman spectroscopy. Proceedings of the United States-Japan Cooperative Program in Natural Resources Food and Agriculture Panel. 34th Annual Meeting, Susono, Shizzoka, Japan. pp. 74-77. Delwiche, S.R., Pearson, T.C., Brabec, D.L. 2005. High-speed bichromatic sorting of fusarium-damaged wheat kernels. AACC Pacific Rim Conference Abstracts. Available: http://www.aaccnet.org/meetings/2005/abstracts/o-64.htm. Delwiche, S.R., Pearson, T.C., Brabec, D.L. 2005. Toward bichromatic optical sorting of scab-damaged wheat. In: Proceedings of the 2005 National Fusarium Had Blight Forum, December 11-13, 2005, Milwaukee, WI. East Lansing, Michigan State University. p. 176. Reeves III, J.B., Delwiche, S.R., Daughtry, C.S., Luthria, D.L. 2005. Near-infrared (nir) research at the beltsville agricultural research center. NIR News. 1616:9-12, part 1 split into two issues. 1618, part 2. p. 12-13. Delwiche, S.R., Graybosch, R.A., Hansen, L.E., Souza, E., Dowell, F. 2006. Single kernel near-infrared analysis of tetraploid (durum) wheat for classification of the waxy condition. Cereal Chemistry. 83(3):287-292.