Progress 06/01/24 to 05/31/25

Outputs
Target Audience:Major stakeholders are growers, food processors, and customers. If there were a larger market for pulse consumption in the USA, growers would be more likely to grow these crops for improved soil and potential financial health. For consumers and food processors, reducing off-flavors is critical for increasing consumer acceptance of pulses. Most American consumers do not know the nutritional value, cooking strategies, and benefits of including pulse crops in their diet. Pulses are unfamiliar to Americans because pulse crops are not traditionally included in American cuisine (except cowpeas and peanuts in the southern USA). Stakeholders are involved in three stages: (1) proposal development, (2) project implementation and data communications, and (3) recommendations for project sustainability. Two major listening sessions were conducted at Clemson University. The workshops involved pre-and post-tests after lectures, round table discussions, and cooking experiences in the research kitchen. The community responded 90% positively to growing pulses in winter and adding them to their diets. The PD has regularly met with regional growers and the protein food industry, identifying problems mainly related to industrial production and marketing. Changes/Problems:No changes are expected. What opportunities for training and professional development has the project provided?This project has allowed three doctoral students and one undergraduate student to work on several aspects of the research. Two doctoral students (Amod Maduraperumege and Nathan Windsor) won the Wade Stackhouse graduate fellowship awarded by Clemson University for their outstanding research contributions. These doctor students completed their flying licenses at the US Department of Transportation -Federal Aviation Administration and obtained their flying licenses. These students attended three professional conferences and presented their research. Another doctoral student (Chamodi Senarathne) who worked on this project completed the Certificate of Human Subjects Protections Course - Group 1 Investigators Conducting Social and Behavioral Science Research (SBR) at Clemson University (Record ID 63848996) and Certificate of Human Subjects Protections Course - Group 2 Investigators Conducting Biomedical Research at Clemson University (Record ID 6384997). Undergraduate research intern (Charles Kresser) completed the dry pea canning study and got an opportunity to present at three professional workshops and will be completing the manuscript in the summer of 2025. How have the results been disseminated to communities of interest?The results have been presented at professional conferences, and one peer-reviewed manuscript has been submitted for open access. Two peer-reviewed publications are being prepared for submission. First-year data will also be presented at the North American Pulse Improvement Association in November 2025. Further, data has been shared with the pulse community, including growers, food processors, researchers and breeders. What do you plan to do during the next reporting period to accomplish the goals?We will characterize the type and concentration of flavor compounds in the dry pea association mapping population for the Year-2 field season and select the most favorable accessions as parents for future breeding. We are also developing a standard research grant in 2026 to understand flavor chemistry towards pulse breeding to pulse-inclusive diets using trained sensory panels.

Impacts
What was accomplished under these goals? Flavor compounds characterization: Our primary goal is to identify the type and concentration of flavor compounds in dry pea seeds from cultivars and advanced breeding lines grown under organic production in South Carolina (SC) for two years and locations. Major off-flavors are generated from volatile and non-volatile compounds (i.e., aldehydes, ketones, alcohols, and saponins) that originate in the seeds due to genetic and environmental factors. This study aimed to measure flavor compounds in 25 dry pea cultivars and 19 advanced breeding lines grown in SC for two years and locations for advanced testing trails for organic production. The experimental design was a randomized controlled block design with two replicates, two locations, and two years. 100 g of dry seeds were collected from the Clemson University Pulse Breeding Program (these trials were done in 2019 and 2020, and seeds were collected from cold storage). A combination of chromatography techniques, including gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC), was used to identify and quantify key flavor-related compounds, including fatty acid esters, organic acids, terpenoids, and phytosterols. The results showed that linoleic acid esters (1518.52 ± 59.4 mg/100 g) had the highest concentration among unsaturated fatty acids, followed by oleic (1137.62 ± 39.31 mg/100 g), linolenic (295.58 ± 9.20 mg/100 g), palmitic (585.03 ± 16.76 mg/100 g) and stearic acid (259.63 ± 89.18 mg/100 g) esters. Among the organic acids, citric acid concentration was the highest for all cultivars (27.58 ± 2.60 mg/100 g) followed by malic + succinic acids (15.98 ± 1.05 mg/100 g) lactic + quinic + shikimic acids (1.59 ± 0.94 mg/100 g), gallic acid (9.01 ± 0.9 mg/100g), and tartaric acid (5.47 ± 0.81 mg/100 g). Terpenoids, including β-amyrin (0.65 ± 0.011 mg/100 g) and γ-tocopherol (0.56 ± 0.01 mg/100 g), and phytosterols like stigmasterol (1.07 ± 0.004 mg/100 g) and γ-sitosterol (4.25 ± 0.06 mg/100 g) were also identified, with some compounds playing a role in modifying texture and mechanical properties in food applications. Statistical analysis, including ANOVA and Pearson's correlation, revealed significant genotype and environmental effects on the composition of these flavor-associated metabolites. Some cultivars exhibited higher concentrations of unsaturated fatty acids, which correlated positively with phenolic compounds like gallic acid and γ-tocopherol. In contrast, high concentrations of saturated fatty acids, such as stearic and palmitic acids, negatively correlated with desirable flavor traits. The findings suggest that selective breeding strategies can be implemented to suppress off-flavors while enhancing sweet and palatable notes, thereby improving consumer acceptance. This manuscript has been written and will be submitted in May 2025. Field Study: A diversity panel of 363 unique pea accessions was grown under field conditions on an organic farm in Pelion, SC. The field trial consisted of two replications with checks blocked for maturity for 755 plots. This panel will be grown in 2024 and 2025 to identify the flavor compounds in the pea diversity panel. This diversity panel represents 11 species and sub-species of peas from 16 countries. The accessions in this panel are seeds from the USDA's Pea Single Plant (PSP) collection and Spain's Institute for Sustainable Agriculture (IAS). 2025 field trials are underway, and data will be collected on the flavor traits of this diversity panel. The experiment followed an alpha lattice design at a single location in 2024. Chemical analyses were performed using advanced analytical instruments to quantify secondary metabolites associated with flavors. Gas Chromatography-Mass Spectrometry (GC-MS) was used to quantify fatty acid esters (oleic, linoleic, and linolenic acid esters) responsible for beany and grassy notes, along with terpenoids and phytosterols (β-amyrin, γ-tocopherol, stigmasterol, and sitosterol), which contribute to bitterness and texture. Ion Exchange Chromatography (IEC) also quantified organic acid (lactic, shikimic, quinic, malic, succinic, tartaric, and citric acids) concentrations influencing astringency. The results indicated total fatty acid ester concentrations of 2237.00 ± 16.76 mg/100 g, total organic acid concentrations of 67.13 ± 3.64 mg/100 g, total terpenoid concentrations of 1.48 ± 0.01 mg/100 g, and total phytosterol concentrations of 4.09 ± 0.07 mg/100 g. We explored the potential of Fourier Transform Mid-Infrared (FT-MIR) spectroscopy as a high-throughput phenotyping tool for quantifying flavor-related metabolites in the pea diversity panel. Flavor extracts from dry pea samples were analyzed, and chemometric models were developed using Partial Least Squares Regression (PLSR). These models, utilizing 70% of samples for calibration and 30% for validation, successfully predicted total phytosterol, terpenoid, and fatty acid concentrations contributing to off flavors. This approach presents a promising, cost-effective alternative for breeders with limited access to large sample sizes and sophisticated analytical instruments, allowing for a more efficient selection of dry pea varieties with improved flavor profiles. Further analysis has been in progress for two years to quantify all the flavor compounds in the pea diversity panel and identify the candidate genes responsible for different flavor profiles. Two manuscripts are being prepared for the field study. Canning dry peas: We tested 12 advanced organic dry pea breeding lines and 7 cultivars from our organic pulse breeding program for canning quality. The dry pea seeds were prepared using a 1:1 water ratio and processed using a PRX-900 Pilot Sterilizer at 118 ºC and 1.97 atm for 9 minutes and 37 seconds, maintaining an F-value of 6.0. The packaged material was polypropylene (PP) and polystyrene (PS) to ensure optimal sealing and preservation. Nutritional analysis was conducted to assess protein, starch, dietary fiber, and protein digestibility using Fourier Transform Mid Infrared Spectroscopy (FT-MIR). Visual parameters were evaluated through color testing and texture analysis to measure the canned pea's hardness, chewiness, and overall texture. The results indicated protein varied from 20-25 g/100g. Starch concentration increased from 47 to 52.92 g/100g, which was attributed to gelatinization and resistant starch formation during the canning process. Dietary fiber was not changed (22 g/100g). Protein digestibility showed no significant change, indicating that the canning process maintained the bioavailability of protein. Surface morphological analysis was conducted using Hitachi 3400 Scanning Electron Microscopy (SEM) to compare raw seeds, slow-cooked, pressure-cooked, and canned peas. The SEM analysis revealed distinct morphological differences between cooking methods. The pressure-cooked samples exhibited ruptured cell structures and leached starch granules with varying morphologies, highlighting the impact of intense heat and pressure on cell integrity. Overall, these canned organic dry peas samples retained desirable nutritional traits and contributed to improved texture and visual quality.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Nathan Windsor, Lucas Boatwright, Richard Boyles, William Bridges, Diego Rubiales, Diego, Dil, Thavarajah, 2024. Biofortifying Dry Pea (Pisum sativum L.) for Improved Performance and Nutrition. Legume Science, 6(3), e250. https://doi.org/10.1002/leg3.250.
• Type: Other Status: Under Review Year Published: 2025 Citation: Gamlath Senarathne, Dil, Thavarajah, 2025. Interdisciplinary approaches to enhance sensory properties and consumer acceptance in pulse crops. Plants, People, Planet-PPP-R-2025-01258.
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Charles Kresser, Chamodi Senarathne, Amod Madurapperumage, Scott Whiteside, Pushparajah Thavarajah, Dil Thavarajah, 2025. Canning quality of Organic Dry pea (Pisum sativum L.) advanced breeding lines adapted to South Carolina. Clemson Fresh, Food, Packaging and Sustainability Summit, March 3-5, 2025.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Chamodi Senarathne, Pushparajah Thavarajah, Dil Thavarajah, 2024. Flavor chemistry towards biofortification and palatability of pulse-based food. CAFLS Research Symposium, Garrison Expo Center, August 19-20, Clemson, SC.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Thavarajah Dil, 2024. Invited Webinar, Pulse crops toward Middle Eastern food security, USAID, MERC program webinar, Oct 1, 2024.
• Type: Other Status: Other Year Published: 2024 Citation: Thavarajah Dil, 2024. Organic pulse breeding for human health. Invited lecture Stone Barn Center, Blue Hill Farms, NY, May 23, 2024.
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Thavarajah Dil, Leong Tang, Pushparajah Thavarajah, 2025. Unlocking Agricultural Potential: Fast, Cost-Effective and Non-destructive Analysis with FT-MIR Spectroscopy, Aglilent and Separation Science Global Webinar, March 25 and 26, 2025.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Thavarajah Dil, 2024. Organic Pulse Breeding and Nutrition, Institute of Chemistry, Sri Lanka. Nov 30, 2024.