Source: UNIVERSITY OF ARKANSAS submitted to NRP
ESTABLISHMENT OF IMPROVED METRICS USING GC-MS/MS AND NONVOLATILE TECHNIQUES FOR FUTURE DEVELOPMENT OF NOVEL AND SUPERIOR US AROMATIC RICE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
Reporting Frequency
Annual
Accession No.
1032439
Grant No.
2024-67014-42710
Cumulative Award Amt.
$300,000.00
Proposal No.
2023-08181
Multistate No.
(N/A)
Project Start Date
Jul 1, 2024
Project End Date
Jun 30, 2026
Grant Year
2024
Program Code
[A1103]- Foundational Knowledge of Plant Products
Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703
Performing Department
(N/A)
Non Technical Summary
Currently flavor forward crop varieties are having a lot of economic success in the U.S. For example, older conventional crop varieties such as Thompson Seedless Grapes and Cascade hops are often sold for far less (i.e. < $1 and < $3-11 a pound, respectively) than new flavor forward crop varieties such as Cotton Candy grapes and Citra® hops which command much higher prices (i.e. ~$3.33-4.99 and ~$15-34 per pound, respectively) (sources: https://tinyurl.com/CCGrapePrice and https://lupulinexchange.com/). Similarly, rice being imported from Thailand and India has a profound impact on the U.S. rice industry, and U.S. rice imports are projected to represent ~32% (or ~1.98 billion USD) of domestic rice use in 2022/23 which is an all-time high.Overall, this is because flavor plays a major role in consumer preference, and consumer demand has grown for Asian aromatic varieties such as Jasmine from Thailand and Basmati from India.To compete effectively with the imported Jasmine rice market, U.S. rice breeders need to develop cultivars with similar but superior and possibly novel flavors. GC-MS/MS along with some other techniques will be used to investigate the variation in the volatile/ nonvolatile profiles across a variety of different rice cultivars from many different U.S. breeding programs (University of Arkansas, Louisiana State University, and the California Cooperative Rice Experiment Station) as well as samples from the USDA-ARS World Rice Collection and subsets of genetically diverse genome-mapped accessions. After analyzing these different varieties multivariate statistics will be used to select a collection (~10-20) of the most diverse samples for sensory analysis. In this way, we will establish a tool so that breeders can better design flavor-forward aromatic rice varieties and set indicators for the key performance of U.S. aromatic rice performance. It is essential to establish infrastructure to facilitate the evaluation of the chemical characteristics in crops which are important for driving unique flavor characteristics in a cost-effective manner and the results of this seed project will help to develop new relationships which will lead to other future proposals which will be submitted to USDA AFRI programs such as A1141 Plant Breeding for Agricultural Production.
Animal Health Component
25%
Research Effort Categories
Basic
25%
Applied
25%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011530200025%
2061530200025%
2061530309025%
2061530108125%
Goals / Objectives
The main goal of this project is to generate a better and more comprehensive understanding of rice flavor so that domestic rice varieties remain competitive with imported aromatic varieties such as Thai Jasmine.To achieve this goal there are four main;Develop an understanding of the key volatiles important for different aromas in rice using GC-MS/MS and the nonvolatile starch characteristics using titration and differential scanning calorimetry impacting taste and mouthfeelCharacterize the different aromas/ flavors of rice using descriptive sensory analysisEstablish ranges for these key volatiles in important US varieties as well as across a wide diversity of rice samples using collections such as the USDA world populationGenerate preliminary data to identify the genes underlying the production of various flavor compounds by association mapping with chromosomal location markers
Project Methods
Volatile analysisVolatile detection will be performed on a 1.5 g of paddy/brown/milled rice in a 10 mL amber screw cap vial using a Shimadzu Nexis GC-2030 system equipped with a triple-quadruple mass selective detector. The volatiles in the headspace will be absorbed using an AOC-6000 Autosampler equipped with 1 cm long SPME fiber coated with Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS). A ZB-5MSplus (30 m x 0.25 mm x 0.25 μm) capillary column will be used and a splitless injection will be performed at an inlet temperature of 240 °C, at absorption and desorption times of 10 min and 3 min, respectively. Helium will be used as the carrier gas at a flow rate of 1 mL/min. The inlet pressure will be 47.6 kPa. The initial oven temperature will be set to 35 °C, held for 5 min, then raised to 150 °C at a rate of 5 °C/min, then raised to 280 °C at a rate of 8 °C/min and held for 5 min.11 The MS will be operated in full scan mode (41-400 m/z) and multiple reactions monitoring (MRM) for a total of 0.300 second with an interface and ion source temperatures of 290 and 240 °C, respectively. Unknown compounds will be identified on Shimadzu LabSolution software based on a library search. Linear retention indices will be created using an alkane standard mix solution (C7-C20) to confirm the molecule identifications. Deuterated standards (hexanal-d12, 2-acetyl-pyrroline-d5, β-myrcene-d6, ethyl hexanoate-d11, phenylacetaldehyde-d5, 1-octanol-d17, and linalool-d5) at 20 ng/μL will be added to each vial as internal standards at a fixed volume of 5 μL. For key compounds, MRM optimization will be executed by running product ion scan of each molecule of interest at collision energy increase steps of 3 kV. Then, Shimadzu MRM Optimization Tool will be used to choose the best combination of transitions and collision energies for each molecule for a total of three: one quantitative and two qualitative transitions. A calibration curve will be executed with all molecules of interest ranging from 0.5 ng/μL to 1000 ng/μL, increasing concentration at each point, for a total of 7 calibration points.Nonvolatile analysisFor apparent amylose analysis and gelatinization properties, paddy rice will be dehusked and ground. Apparent amylose will be determined by colorimetric assay as performed by Juliano (1971)12 and measured in a UV-Visible spectrophotometer (Shimadzu) at 620 nm. The gelatinization properties will be measured using a differential scanning calorimeter (DSC, model 4000, Perkin-Elmer, Norwalk, CT) based on Patindol, Jinn, Wang, Siebenmorgen 13 Eight mg of rice flour will be weighed into a stainless-steel pan and added with 16 μL of DI water. The hermetically sealed pan will be equilibrated for one hour at room temperature before scanning from 25 to 125°C at a rate of 5°C/min. The onset temperature (To), peak temperature (Tp), and end temperature (Te) will be obtained by Pyris data analysis software.Sensory analysisMultivariate statistics14 (as described below) will be used to select (10-20 rice varieties to perform sensory analysis on). For sensory analysis~1 kg of these rice varieties will be needed. Therefore, depending on the availability of the samples a seed increase request may be required. Sensory analysis will be performed in collaboration with the Sensory Science Center (Dr. Han-Seok Seo). In general, 11-13 sensory panelists will be recruited and trained (over 5-10 sessions) in advance of data collection on the key characteristics of the selected rice samples for sensory analysis. Over these sessions the panel will blindly and randomly evaluate all the samples to establish, by consensus, the sensory terms, and corresponding reference standards, which best describe the differences between the samples. The samples will then be evaluated over three replications in a randomized and balanced Latin Square order.Data analysisANOVA, three-way ANOVA (including the factors: judge, sample, and replication, as well as corresponding two-way interactions), multiple comparison analysis (Fisher's least significant difference, p < 0.05), Pearson correlation analysis, principal component analysis (PCA), multiple factor analysis (MFA), multiple linear regression, and graphical construction will be carried out using XLSTAT 2021.3.1 (Addinsoft, NY, USA).14 Initially multivariate statistics will be used to determine the most variable samples based on their volatile and nonvolatiele profiles (~10-20) for sensory analysis. These tests and graphs will then also be used to evaluate the significant differences in the aroma, taste, and mouthfeel profiles between the different rice varieties and to assess the associations between the chemical and sensory data. In addition, we will work with our collaborators to generate potential linkages to genomic data for key impact compounds, work to describe the general ranges of targets for key features in aromatic rice, and asses if GxE and/or year effects are having a major influence on the findings. These preliminary findings will then be used for future grant submissions.

Progress 07/01/24 to 06/30/25

Outputs
Target Audience:This project engages a diverse range of target audiences spanning research, education, policy, and agricultural stakeholders. While the central focus is on aromatic edible rice, the research also explores nonvolatile traits critical to rice's use in brewing and fermentation, creating new value-added opportunities for Arkansas-grown rice. 1. Academic and Government Research Communities: Our method development for rice aroma analysis--including volatile profiling using SPME-GC-MS and targeted MRM quantification has been fainlized and we will begin to share this with academic stakeholders. These stakeholders benefit from reproducible protocols for analyzing aroma-active volatiles in edible rice, helping to standardize approaches across institutions. The outcomes directly support scientific efforts to link biochemistry with consumer-relevant traits in breeding programs. 2. Breeding Programs and Rice Geneticists: Through chemical phenotyping and genome-wide association study (GWAS) preparation, this work offers new insights into how both volatile and nonvolatile traits vary across globally sourced rice cultivars. Breeders from USDA and commercial seed providers--who supplied germplasm--have been engaged throughout the project. Importantly, the inclusion of brewing-relevant traits such as gelatinization temperature, amylose content, and extract yield allows for the identification of dual-use cultivars that serve both traditional culinary markets and value-added applications like fermentation. 3. Brewing and Fermentation Industry Stakeholders: The project has reached a growing audience of brewers, maltsters, and beverage developers seeking domestic alternatives to barley and other starch sources. New protocols for rice malting, extract efficiency optimization, and enzyme characterization were presented at events including the Anheuser-Busch Discovery Day and the Arkansas Rice Industry Alliance. These events included technical brewers, rice growers, and processors interested in linking rice genetics to processing outcomes. Two peer-reviewed publications from this work have provided data-driven insights into rice's brewing potential: "Unveiling Cultivar and Agricultural Factors Influencing Extract Yield from Milled Rice" (Aitkens et al., 2025) "Investigating the Incorporation of Milled Rice in Brewing Non-Alcoholic Beer to Enhance Sensory Quality" (Manuscript DOI: 10.1080/10942912.2025.2520907 Journal: LJFP - International Journal of Food Properties 4. Undergraduate, Graduate, and Postdoctoral Trainees: A core component of the project has been hands-on training and mentorship of future food and agricultural scientists. Three undergraduate students were trained in rice handling, malting, moisture management, and extract yield testing. Two graduate students led analytical method optimization and published their findings in peer-reviewed journals. A postdoctoral fellow helped develop sensory descriptor mapping and supervised large-scale sample processing and data quality control. These trainees gained advanced skills in cereal chemistry, metabolomics, and fermentation science, aligning with NIFA workforce development goals. 5. Agricultural Economic Development Stakeholders: Arkansas agricultural and policy leaders represent a key audience for this work. The project supports ongoing efforts to diversify markets for Arkansas rice, especially through premium value chains like craft brewing and functional beverages. Findings were featured in legislative discussions and aligned with the passage of a new Arkansas House bill incentivizing brewers to use local rice. Support from the Arkansas Research Alliance and the Anheuser-Busch Foundation has enabled follow-up studies targeting rice extract efficiency and cultivar development, illustrating strong regional interest in the commercialization potential of rice-based brewing ingredients. 6. Future Genomics and Crop Improvement Researchers: The project is assembling a multi-omic dataset--including volatile compound profiles, physicochemical parameters (e.g., amylose content, gelatinization temperature), and enzyme activity--for over 350 rice cultivars. This dataset will support genome-wide association studies and future collaborations with plant geneticists and bioinformatics teams. It is expected to inform both basic genetic research and applied breeding strategies aimed at improving flavor, processing quality, and market resilience. In summary, this research has engaged a wide spectrum of audiences, from students and scientists to brewers and policymakers, with practical tools and findings that promote innovation in rice breeding, expand market applications, and support economic development in the Arkansas region. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided direct training for six early-career researchers: Three undergraduate students received hands-on experience in sample handling, moisture control, small-scale malting, extract testing, and data management. Two graduate students led major research components in volatile analysis and rice malting quality, resulting in co-authorship on peer-reviewed publications. One postdoctoral fellow developed and validated targeted analytical workflows (MRM), mentored students, and coordinated integration of chemical and sensory data. Trainees gained expertise in cereal chemistry, aroma metabolomics, fermentation science, and multivariate analysis. They also presented research findings at stakeholder events and internal seminars, developing both technical and communication skills. How have the results been disseminated to communities of interest?Results have been shared with both scientific and applied stakeholder communities: Peer-reviewed publications: "Unveiling Cultivar and Agricultural Factors Influencing Extract Yield from Milled Rice" (Aitkens et al., 2025) "Investigating the Incorporation of Milled Rice in Brewing Non-Alcoholic Beer to Enhance Sensory Quality" What do you plan to do during the next reporting period to accomplish the goals?Planned activities for the next reporting period include: Complete volatile compound quantification across the USDA panel using the expanded MRM method. Finish DNA extraction and submit all genotypes for sequencing, followed by GWAS analysis. Initiate trained descriptive sensory panels using the established aroma lexicon to evaluate a subset of high-priority cultivars. Complete physicochemical analysis of the remaining samples for amylose content, gelatinization temperature, and grain morphology. Integrate multivariate datasets (volatiles, nonvolatiles, sensory descriptors) to uncover correlations and candidate breeding targets. Engage stakeholders with targeted outreach, including reports for breeders, processors, and policymakers, highlighting dual-use rice opportunities (table + brewing). Continue manuscript development and presentations at national conferences, including AACC International and ACS, to enhance scientific dissemination and visibility. These efforts will bring the project closer to delivering predictive models and trait-based breeding strategies for premium-quality, flavor-forward U.S. rice cultivars.

Impacts
What was accomplished under these goals? Goal 1: Identify key volatile and nonvolatile compounds relevant to rice aroma and taste GC-MS-SPME protocols were optimized for volatile analysis, preserving aroma integrity while increasing throughput. A targeted MRM method is being expanded to quantify ~170 volatiles, including 30 newly selected compounds associated with key rice aroma traits. Amylose content and gelatinization temperature--two critical nonvolatile contributors to texture and mouthfeel--have been analyzed in 216 and 260 samples, respectively, using titration and differential scanning calorimetry (DSC). Goal 2: Characterize aroma and flavor using descriptive sensory analysis A curated database of aroma descriptors was assembled using Flavornet, Good Scents Company, ASBC, and published literature. This will guide sensory panels to evaluate aroma and flavor attributes across genetically distinct rice types. Goal 3: Establish ranges of these traits in U.S. and global varieties A genetically and geographically diverse panel of 358 rice genotypes was assembled through collaboration with the USDA. This collection includes: 126 genotypes received in 2024 232 genotypes to be grown and analyzed in 2025 The USDA collection focuses on cultivars from regions historically known for aromatic breeding (e.g., India, Pakistan, Thailand, Afghanistan, Japan) and those exhibiting extreme grain morphology (e.g., long, slender, or bold grains with color variation). Samples originate from 82 unique countries, including: 23 genotypes each from India and China 14 each from Japan and the United States 12 from Afghanistan Preliminary analysis of extract yield, grain size, and amylose levels has revealed high variability across this panel, informing potential breeding directions. Goal 4: Generate data for association mapping DNA extraction is in progress for all 358 genotypes, and sequencing will begin next period. Volatile and nonvolatile trait data are being prepared for genome-wide association study (GWAS) to identify markers linked to aroma, starch characteristics, and brewing-relevant traits. This project also supported two peer-reviewed publications linking rice composition to brewing applications, expanding the relevance of aromatic rice beyond table use into the value-added food and beverage sector.

Publications

  • Type: Peer Reviewed Journal Articles Status: Accepted Year Published: 2025 Citation: Schubert, C. et. al. Investigating the incorporation of milled rice in brewing non-alcoholic beer to enhance sensory quality. International Journal of Food Properties
  • Type: Peer Reviewed Journal Articles Status: Accepted Year Published: 2025 Citation: Aitkens, M. et. al. Unveiling Cultivar and Agricultural Factors Influencing Extract Yield from Milled Rice. JASBC, 2025.