Research Project: Grain Composition Traits Related to End-Use Quality and Value of Sorghum

Location: Grain Quality and Structure Research

2025 Annual Report

Objectives
OBJECTIVE 1: Determine and quantify grain components linked to ‘health-promoting’ benefits and commercial quality of sorghum foods and feed. • Subobjective 1.A. Determine the mechanism related to the reduced protein quality of cooked sorghum flour. • Subobjective 1.B. Characterize protease inhibitors in sorghum and their role in modulating digestibility in sorghum flour. • Subobjective 1.C. Evaluate and identify bioactive compounds in sorghum linked to anti-cancer and other health promoting properties. OBJECTIVE 2: Develop and improve methodologies for rapid prediction and measurement of sorghum grain attributes linked to valuable end-use quality traits. • Subobjective 2.A. Utilize UHPLC (ultra-high-performance liquid chromatography) size exclusion for characterizing sorghum polymeric protein complexes related to end-use quality of sorghum. • Subobjective 2.B. Develop near infrared spectroscopic methods to predict grain composition and quality traits of sorghum.

Approach
Sorghum is an important drought tolerant crop in the central U.S. where water is limited and rainfall unpredictable. Sorghum has been primarily used for animal feed in the U.S. and is consistently used by the biofuel industry and increasingly used in human foods. As for any cereal, grain composition plays an important role in its utilization. To support utilization of sorghum grain, research is needed that identifies grain components linked to functional and nutritional quality of sorghum products. One issue for sorghum utilization is how processing, especially cooking, impacts sorghum flour nutritional and functional properties. It is known that heating increases sorghum protein cross-linking, which in turn affects both protein and starch functionality and digestibility. The exact mechanism of how this occurs is not known; nor is it known how protein and starch changes influence the role of digestive inhibitory compounds in sorghum. Identifying the mechanism behind these changes will provide avenues to improve sorghum flour quality as well as provide new targets to improve sorghum grain composition at the genetic level. Likewise, sorghum is known to have high levels of bioactive compounds that have potential human-health promoting benefits. However, much of the past research on bioactive compounds in sorghum has been based on chemical assays. To further define and identify the health-promoting benefits of sorghum, research using additional methods such as cellular based assays are needed. Such research will help define the value of sorghum in human foods and provide targets for the genetic improvement of sorghum.

Progress Report
This project has completed its 5-year plan, and this is the final summary report for this project cycle. Related to Objective 1.A “determine the mechanism related to the reduced protein quality of cooked sorghum flour” research was conducted to determine changes to specific fractions of sorghum proteins when sorghum flour was cooked. This research identified changes to sorghum protein fractions that were related to the decrease in protein digestibility of sorghum when cooked. Sorghum varieties containing lower levels of these protein fractions could be useful in breeding sorghum for improved cooked protein digestibility. Related to this objective, research was conducted in collaboration with Texas Tech University to investigate changes to sorghum protein content, composition, and digestibility in a chemically induced mutant population of sorghum. Sorghum lines with improved protein digestibility were identified in this research. Related to factors that can influence protein quality in sorghum, analysis of protein content, composition, and protein digestibility was completed on samples treated with different levels of nitrogen fertilization. Samples from sorghum lines that varied in planting date were also analyzed for protein content, composition, and digestibility to determine how planting time impacts grain composition and protein quality. These studies showed how agronomic practices could influence sorghum protein quality and provide additional tools for improving end-use value of sorghum grain. Also related to protein digestibility in sorghum, research investigated hydrolyzing sorghum flour with granular starch hydrolyzing enzyme to prepare a high digestible protein for food applications. Results showed protein digestibility increased in the residual material left after fermentation. Decortication of sorghum grain effectively increased protein content and a combination of decortication and starch hydrolysis via granular enzymes could be used to produce sorghum protein isolates with improved digestibility. In similar research, protein digestibility was investigated in the production of a fermented sorghum beverage. Fermentation resulted in improvements in overall protein digestibility Related to Objective 1.C. “Evaluate and identify bioactive compounds in sorghum linked to anti-cancer and other health promoting properties” research was conducted to encapsulate sorghum phenolic extracts in vesicles for greater bioavailability/delivery, potentially enabling new high value uses for sorghum. Research also evaluated the effect of sorghum phenolic extracts on reducing inflammation. A project was also completed improving to develop a new high throughput polyphenol extraction method to maximize the yield of 3-deoxyanthocyanidins and other flavonoids. Historically, acidified solvents have been used to extract 3-deoxyanthocyanidins, but certain high throughput systems do not allow the use of acids. Collaborative research with researchers at Kansas State University found that using small amounts of acid post high-throughput extraction maximized the identification and quantification of 3-deoxyanthocyanidins. This will lead to environmentally friendly extraction methods because significantly smaller amounts of corrosive acids need to be used to extract target molecules from large samples. Progress was also made on the development of a novel method to separate polyphenolic compounds based on degree of polymerization. This methodology is an important step in characterizing the bioactivity of sorghum tannins and oligomeric polyphenols, which will lead to better breeding of sorghum lines as a functional food. Additionally, the effects of varying pH on sorghum polyphenols were evaluated during wet cooking. It was demonstrated that shorter cooking times and lower pH were associated with higher extractable polyphenols and higher bioactivity in the extracts. This finding will form the basis for more robust research looking into sorghum processing methods with a focus on polyphenols and bioactivity. Research was also conducted to make in-depth comparison of polyphenols in three popular gluten free grains, sorghum, corn and rice, using commercially available high phenolic genotypes. In addition to research on grain, a study evaluating expression of polyphenols in sorghum plant tissue in five sorghum genotypes throughout the growing season was conducted. Sorghum leaf tissue can have high levels of phenolic compounds and can serve as another source for bioactive compounds with potential human health benefits. Related to Objective 2 “Develop and improve methodologies for rapid prediction and measurement of sorghum grain attributes linked to valuable end-use quality traits”, ~ 24,000 grain samples were analyzed by near-infrared spectroscopy (NIR) for high-throughput grain phenotyping to support development of sorghum varieties with improved grain composition and end-use value over the course of this project. Samples were analyzed for collaborative projects with Kansas State University, Texas A&M, Texas Tech University, University of Nevada, ARS research groups in Texas, Nebraska, and Puerto Rico as well as stakeholder groups and seed companies. NIR calibrations for grain traits including moisture, protein, lysine, starch, amylose, crude fat, and total phenolics were developed and improved over the last 5 years. Discriminate calibrations to classify sorghum lines as having waxy starch or tannins were also developed and preliminary research was carried out to develop calibrations for predicting protein digestibility and levels of mold that can occur on the grain during wet conditions. Calibration curves were also developed for predicting protein content in sorghum grain using a handheld battery powered NIR instrument that could be used in the field or as an inexpensive instrument for sorghum breeding programs. Collaborative research with other ARS scientists at Manhattan, Kansas, resulted in development of a single kernel near-infrared sorting instrument capable of analyzing sorghum grain. Related to the development of single kernel sorting instruments, methods were modified to analyze protein content in single sorghum grains. Additionally, a collaborative project with ARS scientists in Lubbock, Texas, was initiated to develop near-infrared calibrations for analyzing dhurrin levels in sorghum plant tissues using a handheld near-infrared spectrometer. Additional collaborative research with other ARS at Manhattan, Kansas, developed automated methods for analyzing images of sorghum grain to measure grain structure and a high throughput image analysis instrument has been used to generate preliminary profiles for analyzing sorghum grain.

Accomplishments
1. Phenolic extracts of sorghum reduce inflammation in mice. Recently, sorghum has received attention as an excellent source of bioactive components such as polyphenols that exhibited protective effects against multiple chronic disease models. Inflammatory bowel disease has increased globally and is linked to the incidence of colon cancer and other intestinal chronic diseases. ARS scientists in Manhattan, Kansas, and collaborators at the University of Tennessee, and Kansas State University determined the ability of sorghum ethanolic phenolic extracts to mitigate inflammation induced by dextran sulfate sodium in mice. Since sorghum phenolic compounds were found to reduce inflammation in mice, sorghum phenolics represent a potential alternative to mitigate colonic inflammation and colitis. This research benefits both the sorghum industry by demonstrating the value of sorghum phenolic compounds to human health and to the general population by providing additional foods for improving health.

2. Analysis of total starch and amylose in single sorghum kernels. Starch and amylose content greatly affect the value of sorghum as a trading commodity and quality in industrial applications, which makes their measurement important in the whole supply chain from seed breeding to end-use applications. To facilitate development and utilization of waxy sorghum, ARS scientists in Manhattan, Kansas, and colleagues at Kansas State University established near-infrared (NIR) spectroscopy models to quickly estimate amylose and starch content in single sorghum kernels. Reliable procedures for measuring starch and amylose in single kernels were established that had low errors. Next, NIR models were developed that accurately predicted starch and amylose content in single sorghum grains. Overall, this study successfully established methods to accurately measure amylose and starch contents in single sorghum kernels and NIR models to predict starch and amylose contents in single sorghum kernels, which will be a useful tool to check starch/amylose profiles of sorghum kernels for sorghum breeding programs and sort kernels by their composition for sorghum application research. Therefore, this research benefits U.S. sorghum producers and industry by increasing capacity to develop new, more valuable sorghum varieties.

3. Deep learning for sorghum yield forecasting using unmanned aerial systems and lab-derived imagery. Sorghum is an important grain crop in the central plains of the United States and is used for feed, fuel, and food. Sorghum has a wide degree of genetic and phenotypic variability which can be exploited to improve the agronomic performance and end-use quality and value of the crop. Grain yield is a primary trait that the sorghum breeding industry is working on improving as increased yields directly relate to the value of the crop. Therefore, to take advantage of the genetic diversity of sorghum and develop new lines with improved yield, methods for rapidly determining and predicting yield are necessary. ARS scientists in Manhattan, Kansas, collaborated with researchers at Kansas State University and Texas Tech University by providing laboratory images and measurements of sorghum grain used in broader research to evaluate the use of deep learning algorithms to predict yield from images of sorghum and found that yield could be forecast using deep learning processing of images. This research will benefit sorghum breeders and seed industry by facilitating higher throughput measurements of grain yield in breeding programs leading to the development of sorghum germplasm with improved agronomic performance and value.

4. Effect of molecular weight on the bioactivity of sorghum polyphenols. Sorghum contains a diverse array of phenolic compounds which have important human health benefits including anti-cancer properties. However, the differences in bioactivity among the different phenolic compounds in sorghum is not well known. To address this, ARS scientists in Manhattan, Kansas, and scientists at Kansas State University developed a method to effectively separate phenolic compounds in sorghum by molecular weight. Polyphenolic compounds with higher molecular weight were found to have higher bioactivity than smaller compounds. This information can be used to help screen sorghum lines for improved bioactivity and develop products from sorghum polyphenols with improved bioactivity and human health properties. This would benefit the U.S. sorghum industry by increasing use of sorghum in high-value markets and development of novel products for U.S. consumers.

5. Comparison of methods for isolating waxy sorghum starch. Waxy sorghum starch has special functional properties that could be used as value-added food ingredients. ARS scientists in Manhattan, Kansas, and collaborators at Kansas State University compared methods for starch isolation to develop an efficient process to isolate starch from waxy sorghum. Research was also done to compare the properties of waxy sorghum starch isolated using different methods. Using an enzyme-based method combined with flour made from decorticated sorghum was found to be an effective method for producing quality waxy sorghum starch. This research provides a new market opportunity for waxy sorghum.

6. High-throughput phenotyping of sorghum grain composition and physical kernel properties. Understandably, yield is the primary trait targeted for improvement in the development of sorghum varieties and directly benefits sorghum farmers. However, incorporating grain composition traits that increase the demand and/or value of sorghum grain can also benefit sorghum farmers and industries that rely on sorghum. To help address this, ARS scientists in Manhattan, Kansas, utilized near-infrared spectroscopy (NIRS) to analyze grain composition in over 10,000 samples to support collaborative research with various research programs including ARS scientists in Lubbock, Texas; Texas A&M; Kansas State University; Texas Tech University, and the University of Reno, Nevada. This research provides the necessary information to identify sorghum varieties with improved end-use quality as well as determining the genetic mechanisms that control sorghum grain composition. This effort directly supports the development of new sorghum lines with enhanced value for the U.S. sorghum industry.

7. Use of ultrasound tempering of sorghum grain for improved sorghum flour. Sorghum is an important grain crop for the Central U.S. and is primarily used as feed and biofuel production. However, sorghum is increasingly used in human food products, especially for the gluten-free industry. Development of sorghum foods represents new market opportunities for sorghum and thus increased value for the U.S. sorghum crop. However, sorghum flour lacks the functionality found in wheat flour with regards to production of baked goods such as bread. To investigate methods for improving the quality of sorghum flour, ARS scientists in Manhattan, Kansas, and collaborators at Kansas State University investigated how ultrasound tempering of sorghum grain prior to milling impacted the properties of the resulting sorghum flour. Ultrasound tempering of sorghum grain was found to alter the properties of flour made from the treated grain which led to some improvements in texture of sorghum breads made from the flour. Ultrasound tempering could be used as an effective tool for enhancing the properties of sorghum flour and its desirability in the gluten-free food market, thereby improving its value for the sorghum industry and providing a better product for U.S. consumers.

8. Effects of new sorghum waxy genes on sorghum grain quality. In sorghum grain, starch is an important source of carbohydrates and plays a key role in human food, livestock feed, and ethanol production for the brewing and biofuel industries. Starch has two main components, amylose and amylopectin. Sorghum grain with low levels of amylose is called waxy sorghum and has unique functional properties in foods, ferments faster than non-waxy starch, and has higher digestibility compared to non-waxy starch. While the genetic changes resulting in waxy sorghum have been identified, their broader effects on seed development and grain quality remain poorly understood. To address this gap in knowledge, ARS scientists in Manhattan, Kansas, and collaborators at Texas Tech University studied novel waxy sorghum lines during seed development. The waxy sorghum lines had increased kernel hardness, larger starch granules, and higher protein levels. This study provides additional information on waxy sorghum grain quality overall and provides a comprehensive molecular framework for developing improved waxy sorghum varieties, providing sorghum producers and the sorghum industry with new tools for developing value-added sorghum lines.

9. Starch development in sorghum grain. Starch is the major component in sorghum grain and starch content and structure impacts the end-use quality and value of sorghum. Because of this, understanding the process of starch formation and all factors that influence starch in the final grain can ultimately help improve the end-use quality of sorghum. ARS scientists in Manhattan, Kansas, and partners at Kansas State University and Texas Tech University investigated the formation of starch in sorghum kernels in growing sorghum kernels. Starch formation was noted as early as 5 days into kernel development and changes to starch structure were seen throughout kernel growth. This research benefits the scientific community working on improving sorghum grain quality, and the sorghum industry, and industries that utilize sorghum for food, feed, and fuel.

Review Publications
Belaineh, Y.G., Bean, S.R., Aramouni, F.M., Wu, X., Liu, S., Tesso, T.T. 2025. Genotype and grain pretreatment effects on digestibility of sorghum proteins in the Ethiopian fermented bread. Cereal Chemistry. 102(1):102-116. https://doi.org/10.1002/cche.10821.
Norton, A., Kim, M., Peiris, K.H., Cox, S., Tilley, M., Smolensky, D., Bean, S.R. 2024. Synthesis and characterization of hybrid gold-coated cereal particles from sorghum bran flour and wheatbran flour. Cereal Chemistry. 102(1):23-33. https://doi.org/10.1002/cche.10840.
Pulivarthi, M.K., Bean, S.R., Pordesimo, L.O., Siliveru, K. 2024. Influence of ultrasound tempering on flour quality of white and sumac sorghum milled on a roller mill. Journal of the ASABE. 67(5):1337-1351. https://doi.org/10.13031/ja.15965.
Edache, D.O., Beyene, T.J., Baruch, J., Shi, X., Sanderson, M.W., Nagaraja, T., Smolensky, D., Cernicchiaro, N. 2024. Sample type and processing plant differences in the proportion of enterohemorrhagic Escherichia coli O157 and non-O157 serogroups in feces and on hides of cull dairy cattle at slaughter. Foodborne Pathogens and Disease. 21(11):698-707. https://doi.org/10.1089/fpd.2024.0017.
Li, D., Jiao, Y., Wu, X., Bean, S.R., Shi, Y. 2025. Morphology, composition and structure of starches during sorghum seed development. Cereal Chemistry. 102:256-265. https://doi.org/10.1002/cche.10875.
Weiss, T., Hong, S., Xiao, R., Wu, X., Li, Y., Tilley, M., Wang, D. 2025. Assessment of granular starch hydrolysis enzyme on ethanol yield from partially swollen sorghum starch and analysis of extracted protein properties. Journal of Agriculture and Food Research. https://doi.org/10.1016/j.jafr.2024.101621.
Sleem, I., Rodriguez, A., Chen, B., Perumal, R., Peterson, J.M., Smolensky, D., Dia, V. 2025. Phenolic ethanolic extracts of specialty sorghum ameliorate intestinal colitis and inflammation induced by dextran sulfate sodium in mice. Biofactors. 51. Article 70028. https://doi.org/10.1002/biof.70028.
Santana, A.L., Peterson, J.M., Perumal, R., Hoon, Y., Lee, S., Siliveru, K., Smolensky, D. 2025. High molecular weight polyphenols from Sorghum bicolor retain higher glycation inhibition and cancer cell inhibition in vitro, compared to small molecular weight polyphenols after separation using Sephadex LH-20. ACS Food Science and Technology. 5:1010-1023. https://doi.org/10.1021/acsfoodscitech.4c00851.
Peterson, J.M., Santana, A., Cox, S., Perez-Fajardo, M.A., Covarrubias, J., Perumal, R., Bean, S.R., Wu, X., Wang, W., Smolensky, D. 2024. Impact of heat and high-moisture pH treatments on starch digestibility, phenolic composition, and cell bioactivity in sorghum (Sorghum bicolor L. Moench) flour. Frontiers in Nutrition. 11. Article 1428542. https://doi.org/10.3389/fnut.2024.1428542.
Khan, A., Bean, S.R., Yerka, M.K., Yinping, J. 2024. Transcriptome and metabolome analysis reveals regulatory networks associated with nutrition synthesis in sorghum seeds. Communications Biology. 7. Article 841. https://doi.org/10.1038/s42003-024-06525-7.
Pontieri, P., Troisi, J., Calcagnile, M., Aramouni, F.M., Tilley, M., Smolensky, D., Guida, M., Del Giudice, F., Merciai, A., Samoylenko, I., Chessa, A.L., Aletta, M., Alifano, P., Del Giudice, L. 2024. Nutritional composition, fatty acid content, and mineral content of nine sorghum (Sorghum bicolor) inbred varieties. Foods. 13. Article 3634. https://doi.org/10.3390/foods13223634.
Edache, D.O., Baruch, J., Kreikemeier, W., Nagaraja, T.G., Renter, D.R., Smolensky, D., Cernicchiaro, N. 2024. Investigation of feedlot-level use of a direct-fed microbial on fecal shedding of E. coli O157:H7. Journal of Food Protection. 87(11). Article 100370. https://doi.org/10.1016/j.jfp.2024.100370.
Mcdowell, R., Banda, L., Bean, S.R., Morris, G.P., Rhodes, D.H. 2024. Grain yellowness is an effective predictor of carotenoid content in global sorphum populations. Scientific Reports. 14. Article 25132. https://doi.org/10.1038/s41598-024-75451-9.