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ARS Home » Southeast Area » New Orleans, Louisiana » Southern Regional Research Center » Commodity Utilization Research » Research » Research Project #438713

Research Project: Development of Novel Cottonseed Products and Processes

Location: Commodity Utilization Research

2021 Annual Report

As directed by ARS research priorities, this proposal is focused on four broad objectives. Objective 1: Develop novel cottonseed oil products with traits to maintain and enhance market value. Sub-Objective 1a. Develop G. hirsutum cotton germplasm with 40% oleic acid in its seed oil. Sub-Objective 1b. Identify and characterize the additional genetic elements that contribute to the high oleic acid trait in GB713. Sub-Objective 1c. Modify cyclopropane synthase genes to reduce the levels of CPFAs in cotton tissues. Sub-Objective 1d. Modify and combine cotton and other plant genes to increase production of DHSA in lipids of roots, seeds, and other tissues. Objective 2: Explore reported seed quality concerns to improve seed quality. Objective 2a. Determine the magnitude and range of seed hull fracture resistance of the Gossypium species that produce usable cotton fiber. Sub-objective 2b. Determine the relative importance of genetics, environment, and their interaction on the fracture resistance of cottonseed. Sub-objective 2c. Develop a method to measure the propensity of cottonseed to be damaged during convening or ginning. Sub-objective 2d. Study the rate of deterioration in the quality of whole and damaged cottonseed under different storage conditions. Objective 3: Study the potential for using the whole seed and defatted protein meal of low-gossypol plant lines in food applications. Sub-objective 3a. Develop acidic juices and drinks fortified with cottonseed protein. Sub-objective 3b. Develop cottonseed-based butter and spread products. Sub-objective 3c. Develop cottonseed protein-based food-grade films to improve food shelf life. Objective 4: Develop new or modified processing methods to increase the value of processed products from cottonseed. Objective 4a. Recover the tocopherol and sterol components of deodorization distillate and add these back to deodorized cottonseed oil to improve its stability.

Several analytical, chemical, physical, microbiological, and genetic techniques will be employed to achieve the project goals. Genetic manipulation, molecular biology, and classical breeding methods will be used to study the synthesis of the cyclopropyl fatty acids and to increase seed oil oleic acid levels. Gas chromatography will be used to determine oil fatty acid profiles, which are needed in several objectives. Various physical and chemical techniques will be employed at the laboratory level to study seed durability and hardness. Some developmental work will be needed to develop a technique that can be used to test for seed durability. Chemical and physical techniques will be used to formulate ingredients and food products from seed kernels and to isolate high protein fractions to use to generate film products. Some of these potential products will also be evaluated by sensory panels. The processing objective will utilize a number of chemical fractionation methods to either eliminate unwanted components or to extract potentially useful components from deodorizer distillate.

Progress Report
This is the first annual report for the recently approved project. ARS researchers in New Orleans, Louisiana believe of the different fatty acids that make up the structure of vegetable oil triglycerides, oleic acid is a monounsaturated fatty acid that helps to increase the stability of oil at high temperature, e.g., during deep fat frying. To increase use of cottonseed oil for frying (Objective 1a), a breeding strategy is being used to transfer the high oleic acid mutant trait from a Gossypium barbadense cotton accession (GB713) into a G. hirsutum plant background. Plant-by-plant analysis of seeds from the prior generation of the two plants yielded eight plants with oleate levels above 50%, considerably greater than the 32-35% levels in the previously released HOa1-HOa4 cotton germplasm. Hence, the breeding strategy to increase cottonseed oil oleic acid levels is showing promise. During the year, the first backcross was made by ARS researchers in New Orleans, Louisiana with HOa1 (Milestone 1). Backcrossing is used to try to retain the genetic trait of interest but to reduce the presence of other unwanted traits from the G. barbadense parent. Seeds from the backcrossed plants with the highest levels of oleic acid (52-56%) were then grown and harvested by ARS researchers in New Orleans, Louisiana . These seed sets should exhibit the maximum amount of genetic segregation of the parental genes. With the help of ARS collaborators at Starkville, Mississippi, these prodigy seeds were field planted and will be harvested plant-by-plant and analyzed for oleic acid content. The plants will also be evaluated for elimination of other unwanted traits from the GB713 parent. To find the genes responsible for the elevated levels of oleic acid (Objective 1b), seeds of the six plants with the highest (52-56%) and lowest (30-32%) seed oleate levels were grown by ARS researchers in New Orleans, Louisiana. Their DNA was collected by ARS researchers in New Orleans, Louisiana to conduct a bulk segregate analysis (Milestone 2). Pooled DNA samples from the high and low oleate groups have been sequenced to look for differences that should be related to the different oleate levels. The evaluation of this data is on-going by ARS researchers in New Orleans, Louisiana. Cottonseed oil also contains small amounts of unusual fatty acids that contain either a three-carbon cyclopropane ring (CPFAs) or a three-carbon unsaturated cyclopropene ring (CPFEs) near the center of their fatty acid structures. Published animal studies suggest that the CPFAs are beneficial for liver and heart health, while the CPFEs may cause some detrimental physiological effects. To better understand the biosynthesis of these acids (Objective 1c), DNA constructs have been made by ARS researchers in New Orleans, Louisiana to try to alter cottonseed CPFA and CPFE content. One set of constructs has been designed to effect overproduction of CPFAs. A second set of constructs has been designed that will allow gene editing of the cotton genome, likely leading to lower levels of both CPFAs and CPFEs (Milestone 3). Collaborators at the University of North Texas have transformed the first constructs into cotton plants and have regenerated plants. These experiments should provide information about the biosynthesis of these unusual fatty acids and will assist in developing cotton plant with optimized levels of these acids. As a second approach to identify the genes and proteins involved in CPFA and CPFE biosynthesis, a two-hybrid bait plasmid experiment was developed by ARS researchers in New Orleans, Louisiana using the cotton cyclopropyl synthase gene as the ‘bait’ protein. This construct has been tested by ARS researchers in New Orleans, Louisiana in yeast and found to pass all requirements for functionality (Milestone 4). As the next step, a library of DNA constructs containing genes that interact closely with this gene will be recovered by ARS researchers in New Orleans, Louisiana to identify genes likely to be related to the bait gene. ARS researchers in New Orleans, Louisiana believe damage of cottonseed during ginning and transport is becoming an important issue. To better understand the factors that affect cottonseed damage (Objective 2), a materials tensile strength tester was used by to study seed hull fracture resistance. To determine hull strength of different cotton species (Objective 2a), seed of different varieties and genotypes of G. barbadense, G. hirsutum, G. arboreum, and G. herbacium were grown. Seeds were harvested plant by plant and tested to determine the maximum amount of force that the seed can withstand (Milestone 5). Results indicate that G. barbadense seed were generally, but not always, more rupture resistant than G. hirsutum seed. Also, the old-world seeds of G. arboretum and G. herbacium were considerably smaller and more fracture resistant than the seed of G. barbadense and G. hirsutum. Additionally, seed of several varieties were collected to determine the relative importance of genetics and environment on cottonseed hull strength (Objective 2b). For this, seeds were collected from plants grown and tested as part of the annual ARS National Cotton Variety Tests (Milestone 6). Approximately half of the genotypes included in the tests are commercial varieties, and the remainder are the best genotypes developed by private breeders. Seed from some locations for the 2016 to 2018 harvest years were available from other studies, and seeds from nine different locations were collected from the 2019 harvest year. Due to Covid restrictions, seeds from only three environments were obtained from 2020. Together with the 2016-2018 locations, seventeen environments are included in the study. This should be sufficient to gauge the relative influence of environment and genetics on hull strength. Hull strength testing is progressing, and the modelling needed for evaluation of the data is being developed. Objective 3 is to develop new food uses and products from the protein portion of the seed. This objective is dependent on the availability of low-gossypol cottonseed. Gossypol is a cotton secondary plant metabolite that can be toxic if fed to nonruminants or people. Its level in food products is controlled by the Food and Drug Administration (FDA). It was initially planned that this seed would come from plants genetically modified to inhibit gossypol biosynthesis. Because these seed are not yet available, glandless cottonseed (a natural mutant but also with low gossypol levels) will be used for this objective. Glandless cottonseed was obtained from a couple of sources. However, these samples were contaminated with varying levels of glanded seeds. As it was difficult to source better material during the pandemic, these samples were hand-picked to remove the glanded seed, tested for gossypol to ensure levels were below FDA standards, and then processed to prepared water-washed protein meals and protein isolates (Milestones 7 and 9). Sufficient material was processed to provide the material needed for Objective 3 of the project plan. Some characterization work has been completed on the glandless cotton samples. Mass spectrometric methods were used to characterize the peptides obtained by limited degradation of the proteins. Some of these peptides might show promising bioactivity. In addition, the moisture adsorption behavior of defatted cottonseed meal, water washed meal, and protein isolate (a protein fraction with >90% protein) was studied at different temperatures. This may contribute to the development of appropriate seed and meal storage and handling guidelines. For the protein drink fortification subobjective (Objective 3a), experiments are in progress to test the solubility of cotton protein in several types of acidic drinks. These experiments have been hampered by lack of laboratory availability but are now in progress. To produce protein-spread type products (Objective 3b), the low-gossypol seeds were roasted at temperatures to enhance flavor development (Milestone 8). Roasting was evaluated by different mass spectrometry and nuclear magnetic resonance spectroscopy techniques. Initial milling of roasted kernels with added sugar, salt, and oil added found that, unlike nuts or some other oil seeds, a single food processer could not produce a well-shaped butter-like spread product. A combination of a high-speed blender and a meat grinder was needed to produce smooth butter-like product. For Objective 3c, initial food-grade protein films were made from cottonseed protein isolate with several additives to help make the film more flexible (Milestone 9). Films containing modest levels of glycerol have shown the best elongation properties during tensile strength tests. Films prepared from water-washed cottonseed meal, which are lower in protein but are easier to prepare, are in progress. Objective 4 is to work on recovering valuable components from the deodorization distillate that is formed as a by-product of cottonseed oil refining. This objective requires a source of deodorization distillate, which was to be acquired from a collaborator. This was difficult to request during the pandemic, but some sample for preliminary characterization (Milestone 10) should be available by the end of summer. In addition to the project objectives, some subordinate efforts and collaborations have also been addressed. Under a reimbursable agreement (6054-41000-113-04R) with Cotton, Inc. (Cary, North Carolina) seeds of commercial cotton varieties were evaluated for their fatty acid profiles as part of an effort to better understand the causes of poor seed germination. Additionally, cottonseed oil CPFA levels were also determined for a selection of seed accessions. Cottonseed gossypol levels were determined for several seed and meals samples being used in animal feeding studies or being checked for maintenance of a low-gossypol trait.

1. Development of improved frying oils. ARS researchers in New Orleans, Louisiana, suggests cottonseed oil has been considered the ‘gold’ standard of frying oil. However, the oil has lost parts of this market to other oils that are more stable at the high temperatures of frying. ARS researchers at New Orleans, Louisiana, have identified cotton germplasm with seed oil composition that should perform better for frying. Together with ARS researchers at Starkville, Mississippi, these traits are being bred into cotton plants that also have good fiber properties. Upon completion of the breeding process, these cottonseed oils will be better for high temperature cooking applications and should regain some of this market share.

Review Publications
Cheng, H.N., Gross, R.A. 2020. Sustainability and green polymer chemistry – an overview. In: Cheng, H.N., Gross, R.A., editors. Sustainability & Green Polymer Chemistry Volume 1: Green Products and Processes. ACS Symposium Series, Vol. 1372. Washington, DC:American Chemical Society. 1372:1-11.
He, Z., Mattison, C.P., Zhang, D., Grimm, C.C. 2021. Vicilin and legumin storage proteins are abundant in water and alkali soluble protein fractions of glandless cottonseed. Scientific Reports. 11:9209.
He, Z., Nam, S., Fang, D.D., Cheng, H.N., He, J. 2021. Surface and thermal characterization of cotton fibers of phenotypes differing in fiber length. Polymers. 13:994.
Cheng, H.N., He, Z., Ford, C., Wyckoff, W., Wu, Q. 2020. A review of cottonseed protein chemistry and non-food applications. Sustainable Chemistry. 1:256-274.
He, Z., Zhang, H., Fang, D.D., Zeng, L., Jenkins, J.N., McCarty, J.C. 2020. Effects of inter-species chromosome substitution on cottonseed mineral and protein nutrition profiles. Agronomy Journal. 112:3963-3974.
Salimath, S.S., Romsdahl, T.B., Konda, A.R., Zhang, W., Cahoon, E.B., Dowd, M.K., Wedegaertner, T.C., Hake, K.D., Chapman, K.D. 2021. Production of tocotrienols in seeds of cotton (Gossypium hirsutum L.) enhances oxidative stability and offers nutraceutical potential. Plant Biotechnology Journal. 19:1268-1282.
Li, J., Pradyawong, S., Sun, X.S., Wang, D., He, Z., Zhong, J., Cheng, H.N. 2021. Improving adhesion performance of cottonseed protein by the synergy of phosphoric acid and water soluble calcium salts. International Journal of Adhesion and Adhesives. 108:102867.