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ARS Home Southeast Area New Orleans, Louisiana Southern Regional Research Center Commodity Utilization Research Research





- Shockey, Jay
- He, Zhongqi
- Klasson, K Thomas
- Cao, Heping
- 2023
- 2022
- 2021
- Publications
- Product Quality and New Uses (306)

Research Project: Development of Novel Cottonseed Products and Processes **Location:
                            Commodity Utilization Research** 2022 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**
                                

A primary goal of this project is to increase the commercial potential of cottonseed oil. One trait that would improve the health profile and frying oil characteristics of cottonseed oil is increased oleic acid content. High oleic acid traits from a non-commercial cotton variety are being bred into standard fiber production upland cotton (Sub-objective 1a). Initial breeding in previously released lines doubled the amount of oleic acid compared to standard cottonseed oil. Working with ARS researchers in Starkville, Mississippi, we created additional breeding populations and identified lines that contain triple the normal levels of oleic acid. These lines were planted for seed increase, then screened for high performance plants. The high oleate trait was maintained in several lines. These lines were used for self-pollination and crossed to upland cotton a second time, to advance the breeding process.

The increase in oleic acid is likely driven by 2-4 genes (Sub-objective 1b). To identify these genes, DNA from pools of high oleate or moderate oleate cotton plants was sequenced. Unique changes in the DNA sequence were detected in certain regions of the high oleate lines. Additional analysis and ranking of the likely importance of these sequence differences is ongoing.

Cottonseed oil also contains a rare fatty acid with three-membered carbon ring structures. Published dietary studies suggest that some types of ringed fatty acids improve liver and heart health, while other types of these ringed fatty acids have negative effects. How plants produce these unusual fatty acids is a mystery. ARS researchers at New Orleans, Louisiana, studied the production and functions of these lipids. Active plans include design of cotton plants engineered to produce either less (Sub-objective 1c) or more of the carbon ringed fatty acids (Sub-objective 1d). The subset of active genes that drive the first step in this pathway are now known. Two sites within these genes have been identified that will allow for gene editing to reduce their activity. Gene editing was tested in engineered cress plants, which act as a proxy for cotton plants. Working with collaborators at the University of North Texas, we are currently producing engineered cotton plants that express the gene editing constructs.

Successful enhancement of ringed fatty acids will require identification of all the additional components necessary for production. None of these are currently known. Cottonseed and closely related citrus may both contain helper proteins that assist in ringed fatty acid production. Libraries of potential helper proteins from both species have been tested. Candidate proteins have been identified, and are being re-tested for accuracy. Candidates that pass this test will be included in future cotton engineering experiments.

For sub-objective 2a, statistical analysis of the seed fracture resistance of four cotton species has been started. This analysis appears to indicate that upland cotton seed is weaker than pima seed. However, it is possible to identify some exceptions to this trend, as there is some overlap in the data distributions. Greenhouse space limitations suggested the multiple replications from single plants. However, this method was not sufficient to achieve the clearest comparisons. Additionally, G. arboreum and G. herbaceum seed appear to be considerably more rupture-resistance than either upland or pima cotton seeds, despite their smaller size.

Fracture resistance testing for the remaining sets of National Cotton Variety Tests (NCVT) seeds was completed (Sub-objective 2b). By including some seed sets from other studies a sufficient number of environments has likely been achieved; hence, no additional seed sets were collected. The data is now being evaluated and models that partition the variance are being compared.

ARS researchers at New Orleans, Louisiana, worked on a rapid method to determine seed integrity (Sub-objective 2c). Initially, a paint can shaker was proposed as a seed damage tool. Alternatively, seeds were passed through a Bauer mill with the grinding plates set at a wide opening. These seeds were then soaked in solvent to allow for seed oil extraction followed by UV-visible spectrum measurements. The results showed that seed samples had progressively higher absorbance spectra after one or two passes, compared to untreated seeds. Additional passes did not yield stronger spectral readings, suggesting that there is a limit to this approach for estimating seed damage. Another approach involves creating individual damaged seeds in different ways, such as creating holes in the seeds using straight pins. These samples are being used to determine if a clear relationship can be established between the level of some extracted kernel components and seed damage. These studies are continuing. Success in this approach will also require development of a reliable method to detect damage in small seed sample sizes, and in seed stored under different conditions (Sub-objective 2d). The most promising approach under development is wet milling of cottonseed kernels followed by production and quantification of fatty acids, which is an indication of oil degradation during storage.

We are also interested in developing cottonseed protein (CSP) as a protein drink supplement (Sub-objective 3a). CSP isolates were used to fortify apple, grape, and orange juices, and a commercial soda. Protein stability has been studied in the juices and drinks and in buffers under different temperature and pH conditions. Samples have been re-extracted from the drinks and used for assessment of protein quality and quantity. These results will assist in determination of ideal chemical conditions for CSP solubility in fruit juices.

ARS researchers in New Orleans, Louisiana, refined processes to use glandless cottonseed kernels to make food products similar to peanut butter (Sub-objective 3b). Roasting is a process used in production of many agricultural food products. Butter products produced with longer roasting times contained a smoother surface than the products generated using shorter roasting times. Long-roasted butter products had slightly higher oil separation rates. Oil separated less in stored cottonseed products compared to peanut and walnut butters. These results suggest higher stability of the cottonseed butter products. The evaluation of other butter properties is ongoing. This work also investigated the identities and abundance of extractable components in glandless cottonseed kernels during roasting. A large set of unique bioactive compounds were identified. This work will be helpful in developing different heat treatments for selective preservation or extraction of useful bioactive compounds during cottonseed food product preparation.

In collaboration with ARS scientists from Peoria, Illinois, we continued to develop cottonseed protein (CSP) as packaging film (Sub-objective 3c). As expected, CSP alone gave weak physical properties. Attempts were made to strengthen CSP-based films, using several approaches, including heat, pH adjustments, incorporation of plasticizers and additives, polymer blending, and the layering approach. The most promising results came from the layering approach. Elements of the other approaches are still being used, such as plasticizers, pH adjustments, and blending, for further work. Through polymer blending, a second polymer is added to CSP such that the final material can show improved end-use properties. This second polymer needs to be chosen with care. It should be biobased, biodegradable, and acceptable in food packaging applications. An attractive polymer is polylactic acid, which meets all of these criteria. However, it is difficult to cast films from mixed solutions of CSP and polylactic acid because these two materials do not dissolve in the same types of solvents. Instead, the layering approach has been used, with blended films consisting of two layers, one with polylactic acid, and one with CSP. Measurements of the mechanical, optical and barrier properties of these films is nearly complete.

Also in support of Sub-objective 3c, and together with the Rochester Institute of Technology, we are working on a promising alternative approach to packaging films. This approach involves combining the use of paper and cottonseed protein (CSP). Paper is biobased, inexpensive, commercially available, and compatible for food packaging. A mixture of CSP and cotton gin trash was used as a coating layer on paper. This type of coated paper provides heat seal capacity for food packages and reduces air and water vapor transmission. Study of the mechanical and barrier properties of this material indicate that it is a viable paper coating. These results show that CSP and gin trash provide moderate performance as a food packaging material. The results revealed possible future approaches that can further improve the performance of CSP-coated paper for food packaging.

In related work, we have tested cottonseed protein (CSP), alone and in combination with lignin-containing cellulose nanofibers (LCNFs), as additives to paper. The addition of CSP alone provided improved dry strength to paper, while the CSP/LCNF combination gave further improvements.

We made progress on agreement (No. 70641) with Cotton Incorporated and we analyzed samples from feeding studies, performed by a collaborator. Results from gossypol analysis were shared with the collaborator and updates were provided to Cotton Incorporated in quarterly reports. **Accomplishments**
                                

1. Identification of stable non-GMO high oleic acid trait in cottonseed oil. ARS researchers in New Orleans, Louisiana, seek to increase the commercial potential of cottonseed oil, which has lost some market share to other vegetable oils, especially in the frying oil market. The main cause of this decline is the relatively low stability of cottonseed oil at frying temperatures. To increase use of cottonseed oil for frying (sub-objective 1a), high oleic acid traits from a non-commercial pima cotton variety called GB713 are being bred into standard fiber-production upland cotton. Initial breeding led to increases from ~15-16% oleic acid in standard upland cotton seed oil to 32-35% in previously released germplasm lines HOa1 – HOa4. High oleic acid (52-56%) seed lines from the first backcross were planted for seed increase and analyzed on a plant-by-plant basis. The high oleate trait was maintained in several lines. These lines were used for both self-pollination and for a second backcrossing to advance the breeding process. 2. Improved use of cotton byproducts in food packaging. Cottonseed protein (CSP) and cotton gin trash are underutilized by-products generated from cotton production. ARS scientists at New Orleans, Louisiana, in collaboration with Rochester Institute of Technology, have investigated the possibility of using cottonseed protein and gin trash as paper coating for food packaging applications. Relevant data of the coating were obtained, including the density of the CSP and gin trash, adhesion to the paper, heat sealing strength, mechanical strength, and oxygen and water vapor barrier. The CSP-based coating exhibited noticeable adhesion to paper and proved viable as coating to paper. Gin trash is a valuable addition to CSP coating, especially in enhancing heat sealing strength and oxygen barrier. For a food packaging application that allows the food item in the package to “breathe”, CSP/gin trash coating on paper can be used with moderate performance as food packaging. This work has also pointed out possible future approaches that can further improve the performance of CSP-coated paper for food packaging. 3. Storage protein distribution in glanded and glandless cottonseeds was quantified. Glandless cottonseed was developed to remove or reduce the toxic compound gossypol present in cottonseed glands. ARS scientists at New Orleans, Louisiana, evaluated the level and composition of the protein fractions from glanded and glandless cottonseeds. The resulting information provides a description of the effects of gossypol gland depletion on major seed protein accumulation, quantity, and diversity in cottonseed. These results will help guide ARS researchers and food product manufacturers in future efforts to develop specifically tailored foods, such as spreadable butters, or other types of functional or bioactive cottonseed protein-derived products. **Review Publications**
                            *Cao, H., Sethumadhavan, K., Wu, X., Zeng, X., Zhang, L. 2022. Cottonseed extracts regulate gene expression in human colon cancer cells. Scientific Reports. 12:1039. https://doi.org/10.1038/s41598-022-05030-3.*
                                *He, Z., Liu, Y., Kim, H.J., Tewolde, H., Zhang, H. 2022. Fourier transform infrared spectral features of plant biomass components during cotton organ development and their biological implications. Journal of Cotton Research. 5:11. https://doi.org/10.1186/s42397-022-00117-8.*
                                *He, Z., Nam, S., Zhang, H., Olanya, O.M. 2022. Chemical composition and thermogravimetric behaviors of glanded and glandless cottonseed kernels. Molecules. 27(1). Article 316. https://doi.org/10.3390/molecules27010316.*
                                *Cao, H., Sethumadhavan, K., Wu, X., Zeng, X. 2021. Cottonseed-derived gossypol and ethanol extracts differentially regulate cell viability and VEGF gene expression in mouse macrophages. Scientific Reports. 11:15700. https://doi.org/10.1038/s41598-021-95248-4.*
                                *He, Z., Cheng, H.N., Nam, S. 2021. Comparison of the wood bonding performance of water- and alkali-soluble cottonseed protein fractions. Journal of Adhesion Science and Technology. 35(14):1500-1517. https://doi.org/10.1080/01694243.2020.1850612.*
                                *He, Z., Zhang, D., Cheng, H.N. 2021. Modeling and thermodynamic analysis of the water sorption isotherms of cottonseed products. Foundations. 1:32-44. https://doi.org/10.3390/foundations1010005.*
                                *Shmulsky, R., Dowd, M.K., Lopes, D.J.V., Miller Jr., G.D., Entsminger, E.D. 2021. Production of yellow poplar interior plywood with cottonseed-based protein adhesives. Wood and Fiber Science. 53(3):206-215. https://doi.org/10.22382/wfs-2021-20.*
                                *Thompson, C.M., Hendon, B.R., Mishra, D., Rieff, J.M., Lowery, C.C., Lambert, K.C., Witt, T.W., Oswalt, S.J., Bechere, E., Smith, W.C., Cantrell, R.G., Kelly, B.R., Imel-Vise, R.K., Chapman, K.D., Dowd, M.K., Auld, D. 2019. Cotton (Gossypium hirsutum L.) mutants with reduced levels of palmitic acid (C16:0) in seed lipids. Euphytica. 215(112). https://doi.org/10.1007/s10681-019-2423-4.*
                                *He, Z., Zhang, D., Mattison, C.P. 2022. Quantitative comparison of the storage protein distribution in glandless and glanded cottonseeds. Agricultural and Environmental Letters. 7(1). Article e20076. https://doi.org/10.1002/ael2.20076.*
                                *Regmi, A., Shockey, J., Kotapati, H.K., Bates, P.D. 2020. Oil-producing metabolons containing DGAT1 use separate substrate pools from those containing DGAT2 or PDAT. Plant Physiology. 184(2):720-737. https://doi.org/10.1104/pp.20.00461.*
                                *Shockey, J. 2020. Gene editing in plants: assessing the variables through a simplified case study. Plant Molecular Biology. 103:75-89. https://doi.org/10.1007/s11103-020-00976-2.*


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