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Research Project: Development of Novel Cottonseed Products and Processes

Location: Commodity Utilization Research

2024 Annual Report

Objectives
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.

Approach
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 fourth annual report. The project focuses on issues related to the processing of cottonseed components for food-based products, films, food packaging and other biobased products. The project objectives fall under National Program 306, Component 1 – Foods, Problem Statement 1A: Define, Measure, and Preserve/Enhance/Reduce Factors that Impact Quality and Marketability; Problem Statement 1.B: New bioactive ingredients and health-promoting foods; and Problem Statement 1.C: New and improved food processing and packaging technologies. ARS researchers in New Orleans, Louisiana, seek to increase the commercial potential of cottonseed oil, protein, and other components of cottonseed processing. The genetic diversity of upland and pima cotton provides promising gene alleles that can be used to create vegetable oils with enhanced health profiles and improved frying oil properties. This goal requires increasing the seed oil content of monounsaturated oleic acid, which is the focus of Objective 1. In collaboration with ARS researchers in Starkville, Mississippi, the diverse genetics present in a noncommercial variety of pima cotton was exploited, resulting in multiple generations of breeding for cotton varieties that contain ~300% more seed oil oleic acid than standard upland cottonseed oil. The final stages of the breeding process are underway. Previous studies suggest that the high oleic acid trait is likely generated by interaction between 2-3 genes, only one of which has been identified. Additional work by ARS researchers in New Orleans, Louisiana, is focused on identifying these genes, a major goal in Objective 1. Two new candidate genes were identified, and the correlations between these genes and the high oleic acid trait are currently being studied in a new medium-to-high oleic acid breeding population. Cotton is one of a small group of plants that produces unusual fatty acids that contain cyclic carbon rings. Processed cottonseed oil retains a small amount of these fatty acids. Published studies conducted by researchers at the University of Georgia and ARS researchers in New Orleans, Louisiana, showed that these acids may suggest cottonseed oil as a possible treatment for fatty liver disease. Cotton plants tailored to produce specific profiles of these fatty acids would provide extra value to cotton crops. However, little is known regarding the synthesis of these unusual fatty acids. In support of Objective 1, ARS researchers in New Orleans, Louisiana, with collaborators at the University of North Texas, are working to produce engineered cotton plants that contain tailored profiles of the carbon-ringed fatty acids by either suppressing or overproducing the key enzyme that produces them. First generation plants of this type did not show significant changes, but second-generation plants are under development and will be available for analysis next year. Objective 2 is focused on other traits that contribute to seed quality. In addition to fiber yield and quality, and seed oil content, traits such as seed hardness also affect the overall value of the crop. There is growing concern in the cotton industry that prolonged efforts to breed for fiber traits without considering seed quality traits may be leading to smaller, weaker seeds. Harder, stronger seeds are better able to avoid damage during ginning and handling that increases costs for growers and other processors. ARS scientists in New Orleans, Louisiana, seek to determine the range of fracture force that different fiber-producing cotton seeds can withstand, and the relative effects genetics and growth environment have on seed hardness. Of the four cotton species that produce fiber, studies showed that upland cotton seed is generally weaker than pima seed, and seeds from other cotton species were significantly more fracture resistant than upland and pima seeds, despite their smaller size. Thorough testing comparing the effects of genetics and environment on fracture resistance has been completed. Both factors seem to have significant impacts on this trait; statistical analysis of the variances between these complex datasets is underway. In support of Objective 2, a suitable technique was developed to damage cotton seeds based on partially compressing and cracking seeds between two rollers. The damage was confirmed by the analytical procedure developed last year. This work was extended to include additional seeds from non-ARS sources. In collaboration with researchers at North Carolina State University, the procedure was verified with planting seeds that had been visually sorted based on extent of damage (undamaged, damaged, highly damaged). In addition, damaged seeds were generated based on a newly developed method. The storage of these seeds at three temperatures and four humidity levels was initiated. The initial concentration of free fatty acids (an indicator of seed degradation) was determined for each condition, and samples will be taken with time to see the impact of the storage conditions. To develop other new possible markets for cottonseed products, to support Objective 3, ARS researchers in New Orleans, Louisiana, continue to develop processes to use glandless cottonseed kernels to make cottonseed butter/spread. Roasting temperature is a critical factor that affects the oxidative stability of cottonseed food products. Batches of glandless cottonseed kernels were roasted in a convection oven for 15 min over a range of temperatures and the oxidation stability was tested with a rapid oxygen measurement technique. From these tests, the shelf life of cottonseed butter made from kernels roasted at moderately high temperatures (140 degrees) was estimated to be approximately 1 to 1.5 years depending on storage temperature. Addition of raw cottonseed oil further increased the oxidative stability due to other antioxidants in the raw oil. ARS scientists at New Orleans, Louisiana, also continued to develop cottonseed protein-based biocomposites for food packaging and agricultural applications to support the goals of Objective 3. Four-component biocomposites were formulated with polylactic acid, washed cottonseed meal, glycerol, and either cottonseed oil or polyethylene glycol. Instrumental analysis was used to test the degree of blending and chemical interaction for each of the different blends. Cottonseed oil effectively blended into the biocomposites. In another trial, biofilm was made from polylactic acid and cottonseed protein extracted from the washed cottonseed meal. The bilayer films were prepared by a “solvent casting” method. A cottonseed protein film was first made by mixing dissolved washed cottonseed meal with glycerol at basic pH, then pouring the mixture into a dish for drying at room temperature to form a film, showing the feasibility of making cottonseed biomass-based bioplastic products. In support of project objectives, under an agreement (Log 72545) with Cotton Incorporated, work was completed and analytical results of approximately 250 samples for animal blood plasma were provided to collaborators. The final report was submitted. In support of Objective 2, under an agreement (Log 74834) with Cotton Incorporated, discussions were held with collaborators about future work and information on the latest research by our research scientists were distributed at a scientific meeting. Objective 1 was also supported by Outgoing Agreement 6054-41000-113-009A and Incoming Agreement 6054-41000-113-005I. A postdoctoral researcher was hired and has begun work on the soft funds project entitled “Creation of Enhanced-Value Cotton Germplasm via Modulation of Cyclopropyl Fatty Acid Accumulation”. Methods for high throughput testing of gene editing processes in cotton leaves are being optimized; these preliminary tests will help to identify which plasmid DNA designs work more effectively. Only those that pass this test will be worthy of the extensive time commitment necessary to generate transgenic cotton plants. For the outgoing agreement, the first annual allotment of funds was sent to a collaborator for use in formulating experimental mouse meals containing cottonseed oil. Fundamental understanding of cottonseed oil production was also supported by rapid progress on the incoming agreement 6054-41000-113-008R, which relates to a soft funds project titled “Beyond Static Metabolic Maps - Understanding the Cellular Organization and Dynamics of Lipid Flux for Enhanced Seed Oil Production”. Two technicians have assisted in extensive studies targeting multiple lipid metabolic genes for gene editing in model oilseeds. Edited lines with progressively complex genetics are undergoing analysis for changes in seed oil composition. Project objectives were supported by an agreement (Log 74381) with Cotton Incorporated, titled “Formation and Characterization of Cotton Gin Waste/Cottonseed Meal (Protein) Biocomposite Particleboards”. This project is in its very early stages. ARS scientists in New Orleans, Louisiana, focused on planning and preparatory tasks to ensure laboratory readiness for this project. Three 25 lb bags of cotton gin waste samples with different particle sizes and a 25 lb bag of defatted cottonseed meal have been obtained. Another agreement (Log 74382) with Cotton Incorporated titled “Preparation of Polylactic Acid-Cottonseed Oil Composites” also supports the goals of Objective 3. ARS scientists in New Orleans, Louisiana, seek to study the properties of biocomposites containing cottonseed components. Preliminary experiments were conducted to optimize the compound molding conditions. The effects of melting/blending on the strength of the polylactic acid-cottonseed oil biocomposites were evaluated, and the molding protocol was optimized by re-setting the press pressure and temperature parameters.

Accomplishments
1. Determination of oil content impacts on the textural properties of cottonseed butter/spread products. As an essential ingredient in cottonseed butter products, the oil component not only provides nutrients and metabolic energy, but also plays important roles in the textural properties and butter stability. ARS researchers in New Orleans, Louisiana, observed that the butter products with 47-50% of total oil content showed favorable characteristics of smooth but firm butter products with reasonable storage stability. Such products with 53-57% oil content might serve as creamy spread products with shorter shelf-life times. The results of this work provided information for further optimization of formulating parameters of cottonseed butter products.

2. Valorization of cottonseed byproducts for bioplastic applications. With increasing awareness of plastic pollution in the environment and accumulation of micro-plastics in water, urgent research and development is ongoing to replace synthetic plastics in packaging and coatings. ARS researchers in New Orleans, Louisiana, explored the properties of blends of a biodegradable polymer (carboxymethyl cellulose) and washed cottonseed meal as possible agricultural-based, biodegradable, sustainable alternatives to plastics. Blends of these two components with glycerol as a plasticizer produced single-layer films from 50 to 90 micrometers in thickness. Such films may be useful as water-soluble food packaging and coatings and as dissolvable bags and pouches for detergents and agrochemicals.

3. Development of an efficient high-throughput transient cotton genome editing system. The powerful technology known as genome editing (‘CRISPR’) offers great possibilities for development of value-added non-GMO cotton breeding. However, this technology has not received much attention in cotton, mostly due to the long processing time needed for engineering of cotton and the risk of lost time due to use of untested genome editing experimental designs. ARS researchers in New Orleans, Louisiana, have developed a system for use of cotton leaves in a fast, high-throughput genome editing screening protocol. A one-week experiment can now better identify which editing strategies will be worthy of the 18-month commitment required for creation of intact engineered cotton plants.

4. Identification of new high oleic acid cottonseed oil genes. High oleic acid cotton varieties have been produced in the past, but until now, this was only achieved by use of transgenic modifications that complicate the regulatory approval process and do not fully explain the biochemical pathways that control seed oil composition. ARS researchers in New Orleans, Louisiana, have identified two novel gene versions, representing the two terminal steps of the fatty acid biosynthesis pathway that appear to be linked to the high oleate trait. These could be developed as useful markers for future marker-assisted breeding strategies aimed at combining the high oleate trait into commercial fiber producing varieties.

U.S. DEPARTMENT OF AGRICULTURE

Commodity Utilization Research: New Orleans, LA