Research Project: Development of Novel Cottonseed Products and Processes

Location: Commodity Utilization Research

2023 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 third annual report. The project focuses on issues related to the processing of cottonseed for oil, protein, and other components for food-based products, films, food packaging and other biobased products. Progress was made on all project objectives, which 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. Great potential exists in cottonseed oil for increasing the health profile and frying oil properties through increasing its oleic acid content. Non-commercial cotton varieties have been used for breeding with upland cotton used for fiber production (Sub-objective 1a). Recent breeding efforts generated lines that contain triple the normal levels of oleic acid. These lines still contained some tall stature and other wild characteristics associated with the original Pima cotton parent. To address this issue, the researchers crossed these lines to Upland cotton a second time. The high oleic acid trait is likely controlled by multiple genes, only one of which has been studied in detail, as described in a study published in the journal Plant Physiology and Biochemistry this year (Sub-objective 1b). ARS researchers in New Orleans, Louisiana, worked with scientists in the Cotton Fiber Bioscience Unit in New Orleans, Louisiana, to identify a second likely candidate gene variant (a gene very similar to the one we already studied). They are working on methods for using this variant as an identifier for screening plants with medium-oleate seed oil versus high-oleate seed oil. Genomic DNA sequence data will also be reevaluated for other possible gene variants that contribute to this valuable trait. Some parts of the cotton plant contain high amounts of chemicals with an unusual carbon ring containing fatty acids. Processed cottonseed oil retains a small amount of one of these fatty acids, and it has been linked to improved liver and heart health in humans and animal subjects. The other two types of ringed fatty acids have negative effects when consumed by most animals. How plants produce these unusual fatty acids and what they use them for is not known. ARS researchers in New Orleans, Louisiana, aim to understand both the production and functions of these lipids. For the first time, the researchers created cotton plants engineered to produce either less (Sub-objective 1c) or more of the carbon ringed fatty acids (Sub-objective 1d). They altered the DNA of these plants to allow for a process called gene editing. Collaborators at the University of North Texas recently supplied the first such engineered cotton plants; analysis of gene editing activity levels in cotton leaves and other tissues has been initiated. This work is partially supported by agreement 6054-41000-113-005I. Successful enhancement of ringed fatty acids will require identification of all the additional components necessary for production. None of these are currently known. Cottonseed should contain at least a few other enzymes or proteins that work together in ringed fatty acid production (Sub-objective 1d). Two rounds of library screening have been conducted by commercial companies, and candidate genes are being analyzed for authenticity. ARS researchers in New Orleans, Louisiana, conducted statistical modeling of seed strength data from five years of accumulated fracture resistance data (Sub-objective 2a). The researchers tested several statistical models to assess the possible sources of variability in the system. All the models showed that growth environment was an important factor in determining seed strength, more so than the seeds’ genetics. The results also showed that there was a small but significant difference in seed fracture resistance depending on if the samples were developed by a private or commercial breeder. The reduced seed strength found for the commercial seed types appears to have resulted from aggressive efforts of commercial breeders to breed for fiber traits such as fiber yield and gin turnout without considering seed quality traits. Seed hardness is an important trait in cottonseed that affects its overall value. ARS researchers in New Orleans, Louisiana, compared seed hardness between Pima cotton and Upland cotton varieties (Sub-objective 2b). A few Upland samples showed comparable or greater hardness, but most Pima cotton varieties are statistically stronger than seeds of Upland cotton varieties. A manuscript report on the comparison has been accepted by the Journal of Cotton Science. In addition to seed hardness problems, other types of seed damage can lead to poor rates of sprouting and reduce value. Methods for detecting seed damage prior to planting are not well-established. In support of Sub-objective 2c, ARS researchers in New Orleans, Louisiana, investigated several techniques to create their own damaged cotton seeds. The researchers developed a new analytical method to determine the extent of damage. The effectiveness of this method was tested by comparing intentionally damaged seeds to naturally damaged seeds, which they manually sorted into no damage, pinhole damage, low damage, and high damage seed categories. The new analytical method was also used to determine the minimum seed quantity needed to make damage assessments. The minimal sample size was determined as 50 seeds. In support of Sub-objective 2d, ARS researchers in New Orleans, Louisiana, generated damaged seeds based on the method developed in Sub-objective 2c. The storage of these seeds at three temperatures and four humidity levels were initiated. The researchers determined the initial concentration of free fatty acids (an indicator of seed degradation) for each condition, and samples will be taken over time to see the impact of the storage conditions. ARS researchers in New Orleans, Louisiana, are also interested in developing cottonseed protein (CSP) as a protein drink supplement (Sub-objective 3a). The researchers prepared protein isolates from glandless cottonseed and used them to fortify apple juice, grape juice, orange juice, and a carbonated soft drink. They studied the protein stability in the juices/drinks and buffers under different temperature and buffer pH conditions. Protein content measurements were used to identify optimized pH and temperature conditions for cottonseed protein solubility in fruit juices. They also evaluated the effects of the juices on cotton seed protein solubility and stability. To develop other new possible markets for cottonseed products, ARS researchers in New Orleans, Louisiana, continued our work to develop processes to use glandless cottonseed kernels to make food products similar to peanut butter (Sub-objective 3b). Oil content is a critical factor to create plant butter products with the proper consistency and stability. Raw cottonseed kernels are 35% oil by weight, but the researchers added additional cottonseed oil to create cottonseed butter products with six oil content levels (36, 43, 47, 50, 53 and 57%). The researchers found that butter firmness, spreadability, and adhesiveness decreased rapidly with increasing oil content. Higher oil content also decreased oxidative stability of butter products but did decrease the butter particle size slightly. In collaboration with ARS scientists from Peoria, Illinois, ARS researchers in New Orleans, Louisiana, continued to develop cottonseed protein (CSP) as packaging film (Sub-objective 3c). Such use of cotton byproducts could help to overcome environmental problems, such as those associated with plastic waste, and add value to the cotton industry. In this work, the researchers formulated biocomposites with polylactic acid, cottonseed oil, and three levels of washed cottonseed meal, oil, and plasticizing reagent glycerol. In some formulations, surfactant or carbohydrate polymer additives were included to evaluate their impacts on the formation and properties of cottonseed byproduct composites. Physical strength and measurement and microscopic analysis showed that cottonseed oil and washed cottonseed meal are compatible with polylactic acid for biocomposite production. The ideal sustainable biobased product should be both recyclable and biodegradable. Biocomposites are made up of multiple different natural materials, each of which adds to the properties of the novel finished product. ARS researchers in New Orleans, Louisiana, made biocomposites from the blends of polylactic acid, washed cottonseed meal, and cottonseed oil or glycerol used them in soil burial tests to evaluate their biodegradability. The weight decreased by 25% at the end of a 16-week soil burial. This work indicated that cottonseed byproduct biocomposites could be used for economical, biodegradable and high-quality food packaging materials, flower baskets, planting pots, and other applications. Raman spectroscopy (RS) is a specialized instrumental technique. Few other research groups have applied RS characterization to cotton fiber and cottonseed samples. ARS researchers in New Orleans, Louisiana, evaluated the potential of RS as a fast, reliable, and simple method for identification of fiber traits and other value-added traits in cotton crop. The experimental evidence confirmed the utility of combining RS with other types of analysis for rapid fiber and cottonseed nutrient determination. ARS researchers in New Orleans, Louisiana, made progress on agreement (No. 72545) with Cotton Incorporated. They analyzed samples from bovine feeding studies performed by collaborators. Results from gossypol analysis were shared with the collaborators.

Accomplishments
1. Identification of cotton genes that drive cyclopropyl fatty acid (CPFA) accumulation. The biochemical pathway that leads to ringed carbon fatty acid structures in cotton organs such as seeds and roots is not well understood. Better knowledge of the genes and enzymes that control this pathway would provide tools to manipulate the levels of these fatty acids in cotton and other crops. ARS researchers at New Orleans, Louisiana, tested two types of lipid synthesis enzymes in yeast cells and model plants and found that both help increase CPFA levels in plant seeds. Future creation of high CPFA cottonseed oil will economically benefit cotton growers and oil processors. Consumers will benefit from consumption of this oil, which will improve blood cholesterol levels.

2. Formulation of novel peanut butter-like products from glandless cottonseed. Plant-based (nut and seed) butters have steadily increased in consumer popularity. ARS scientists at New Orleans, Louisiana, made peanut butter-like food products from glandless cottonseed kernels. These cottonseed products were characterized for selected color, texture, and physical properties using both in-house instrumentation and outside collaboration. Observations from this work demonstrate the potential of glandless cottonseed in developing new food products. Cottonseed producers and food manufacturers will benefit from marketing and sales of spreadable cottonseed butters. These products will compete with peanut butter and offer lower risk of allergic reactions in some consumers.

3. Cottonseed protein-fortified fruit juice formations. Low gossypol protein isolates from glandless cottonseed were used to fortify apple juice, grape juice, orange juice, and a carbonated soft drink. Optimized pH and temperature of cottonseed protein solubility in fruit juices were identified. The effects of juices on protein stability under various juices were observed by gel electrophoresis. The information is useful for fortify cottonseed protein as a component in energy drinks. Formulation of highly soluble glandless cottonseed protein into fruit juices, sodas, and other drinks, will economically benefit cottonseed producers by providing a novel high-value use of the protein fraction, while benefiting consumers through commercial availability of palatable high-nutrition drink products.

Review Publications
Rahman, M., Klunga, J., Behera, J., Shockey, J.M., Kilaru, A. 2023. Biochemical properties of Acyl-CoA-dependent and Acyl-CoA-independent avocado acyltransferases positively influence oleic acid content in nonseed triacylglycerols. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2022.1056582.
Jiang, J., Zhu, Y., He, Z., Bing, X., Wang, K., Ma, H., Liu, F., Ding, J., Wei, J. 2023. Multiple spectral comparison of dissolved organic matter in the drainage basin of a reservoir in Northeast China: Implication for the interaction among organic matter, iron, and phosphorus. Heliyon. 9. Article e14797. https://doi.org/10.1016/j.heliyon.2023.e14797.
Shockey, J.M., Gilbert, M.K., Thyssen, G.N. 2023. A mutant cotton fatty acid desaturase 2-1d allele causes protein mistargeting and altered seed oil composition. BMC Plant Biology. 23. Article 147. https://doi.org/10.1186/s12870-023-04160-8.
Cao, H., Sethumadhavan, K. 2022. Identification of Bcl2 as a stably expressed qPCR reference gene for human colon cancer cells treated with cottonseed-derived gossypol and bioactive extracts and bacteria-derived lipopolysaccharides. Molecules. 27(7560):1-19. https://doi.org/10.3390/molecules27217560.
He, Z., Cheng, H.N., He, J. 2023. Initial formulation of novel peanut butter-like products from glandless cottonseed. Foods. 12(2). Article 378. https://doi.org/10.3390/foods12020378.
He, Z., Nam, S., Fang, D. 2023. Raman spectroscopic assessment of fibers and seeds of six cotton genotypes. Agricultural and Environmental Letters. 8. Article e20102. https://doi.org/10.1002/ael2.20102.
Son, Y., Shockey, J., Dowd, M.K., Shieh, J.G., Cooper, J.A., Paton, C.M. 2023. A cottonseed oil-enriched diet improves liver and plasma lipid levels in a male mouse model of fatty liver. American Journal of Physiology - Regulatory Integrative & Comparative Physiology. 324: R171–R182 https://doi.org/10.1152/ajpregu.00052.2022.
Jordan, J.H., Easson, M.W., Cheng, H.N., Condon, B.D. 2022. Application of lignin-containing cellulose nanofibers and cottonseed protein isolate for improved performance of paper. Polymers. 14(11), 2154. https://doi.org/10.3390/polym14112154.
He, Z., Liu, S., Nam, S., Klasson, K.T., Cheng, H.N. 2022. Molecular level characterization of the effect of roasting on the extractable components of glandless cottonseed by Fourier transform ion cyclotron resonance mass spectrometry. Journal of Food Chemistry. 403. Article 134404. https://doi.org/10.1016/j.foodchem.2022.134404.
Cheng, H.N., Kilgore, K., Ford, C., Smith, J., Dowd, M.K., He, Z. 2022. Adhesive performance of cottonseed protein modified by catechol-containing compounds. Journal of Adhesion Science and Technology. 36:1781-1793. https://doi.org/10.1080/01694243.2021.1984713.
Kaviani, S., Polley, K.R., Dowd, M.K., Cooper, J.A., Paton, C.M. 2021. Differential response of fasting and postprandial angiopoietin-like proteins 3, -4, and -8 to cottonseed oil versus olive oil. Journal of Functional Foods. 87:104802 https://doi.org/10.1016/j.jff.2021.104802.
He, Z., Nam, S., Liu, S., Zhao, Q. 2023. Characterization of the nonpolar and polar extractable components of glanded cottonseed for its valorization. Molecules. 28(10). Article 4181. https://doi.org/10.3390/molecules28104181
Behera, J.R., Rahman, M.M., Bhatia, S., Shockey, J., Kilaru, A. 2021. Functional and predictive structural characterization of WRINKLED2, a unique oil biosynthesis regulator in avocado. Frontiers in Plant Science. 12:648494. https://doi.org/10.3389/fpls.2021.648494.
Cao, H., Sethumadhavan, K. 2023. Plant polyphenol gossypol induced cell death and its association with gene expression in mouse macrophages. Biomolecules. 13(4). Article 624. https://doi.org/10.3390/biom13040624.
He, Z., Nam, S., Klasson, K.T. 2023. Oxidative stability of cottonseed butter products under accelerated storage conditions. Molecules. 28(4). Article 1599. https://doi.org/10.3390/molecules28041599.