Location: Commodity Utilization Research
2019 Annual Report
Objectives
The overall goal of the project is to improve the postharvest utilization of cottonseed and thereby increase the value of the U.S. cotton crop through improved understanding of cottonseed composition, properties and processing of the seed’s components. There are five total objectives in the project. Three objectives focus on studying and modifying the oil, protein, and hull components of the seed. One objective is directed toward the study of processing operations to improve the separation of these components and the last objective is directed toward isolation of minor components that may exhibit beneficial bioactivity.
Objective 1) Enable the development of new, commercial cotton varieties which express high levels of oleic acid in the seed.
Sub-objective 1a) Study FAD2 structure in naturally high oleic acid cotton accessions.
Sub-objective 1b) Use genes and other DNA regulatory elements associated with cyclopropyl fatty acid synthesis to silence production of these fatty acids in developing cottonseed.
Sub-objective 1c) Determine the compositional and functional property differences between naturally high oleic acid and normal cottonseed oils.
Objective 2) Enable new commercial process technologies that maximize the profitability of converting low-gossypol cotton seed into oil and meal products.
Sub-objective 2a) Determine conditions that result in low-color oils from the processing of glandless cottonseed.
Sub-objective 2b) Physically refine crude cottonseed oil from glandless cottonseed to produce commercial grade oil.
Objective 3) Enable the commercial production of new products from the protein fraction of cottonseed meal.
Sub-objective 3a) Improve water resistance of cottonseed protein meals, concentrates and isolates used as wood adhesives.
Sub-objective 3b) Explore the use of cottonseed proteins as functional additives in non-food commercial products.
Sub-objective 3c) Explore the use of cottonseed protein fractions to improve non-food product properties.
Objective 4) Enable the commercial production of new bioactive food ingredients from glandless (no gossypol) cottonseed.
Sub-objective 4a) Identify minor bioactive phenolic components from glandless cottonseed.
Sub-objective 4b) Identify bioactive peptides and proteins from glandless cottonseed.
Objective 5) Enable the commercial production of new products from the carbohydrate components in cottonseed burrs, hulls and kernels.
Sub-objective 5a) Isolate, characterize, and study the functionality of hemicellulosic components from seed processing byproducts.
Sub-objective 5b) Exploit the potential use of hull and other seed byproducts as fillers in composite materials.
Approach
Several analytical, chemical, physical, microbiological, and genetic techniques will be employed to achieve the project goals. To alter cottonseed oil composition, a combination of genetic manipulation and classical breeding will be used. Various physical and chemical techniques will be employed at the laboratory level to mimic processing steps and to fractionate meal (i.e., protein) and hull components. Chemical, enzymatic, and physical techniques will be used to modify these isolated components and to characterize the resulting products. Performance of these fractions for different potential applications will be achieved through a series of physical testing methods. Isolation of seed minor components will be achieved for bioactivity studies through chemical fractionation and chromatographic methods and several cell-based assays will be used to test for activity.
Progress Report
The objectives of the cottonseed project fall under National Program 306 and contain elements of both the Food and Non-food components. Under the Food component, the work addresses Problems 1A, to define, measure, and preserve/enhance/reduce attributes that impact quality and marketability; Problem 1B, to develop new bioactive ingredients and functional foods; and Problem 1C, to develop new and improved food processing technologies. Under the Non-food component, the work relates to Problem 2B to enable technologies for producing new marketable non-food biobased products derived from agricultural products and byproducts and estimate the potential economic value of the new products.
Vegetable oils with high levels of oleic acid are desirable because they are considered heart healthy and because these oils tend to be more stable to oxidation and useful for applications like deep fat frying. As part of Objective 1a, work continued to develop cotton germplasm with elevated levels of oleic acid in the seed oil. Continuing last year’s progress, thirteen high oleic acid germplasm lines, initially selected from nematode resistant plants found to contain the trait, were grown in duplicate field plots. Testing of the seeds from these plants indicated that the high oleate trait was genetically stable. Levels in these plants were between 30 and 35%, essentially double the level found in normal cottonseed oil. The four lines with the highest levels have been approved as a germplasm release. While these levels are lower than found in some modified oilseeds (sunflower, soybean), these plants represent a step in the right direction for cottonseed.
Continuing work started in 2018, an improved DNA marker that was developed to detect a mutated gene (called fad2-1d) was tested on the plant lines with different levels of oleic acid in their seed oils. All lines with over 30% oleic acid tested positive for the marker, confirming the involvement of this mutated gene in the trait. Additional testing conducted on the population of 2017 field-grown nematode-resistant plants revealed oleic acid levels between 13 to 38%. Plants with greater than 27% oleic acid tested positive only for the mutated gene and plants with less than 20% oleic acid appeared not to contain the mutated gene. Plants with intermediate levels of oleic acid appear to have a copy of both the mutated and non-mutated forms.
Because the upland lines do not have the levels of oleic acid observed in the wild plants, other genetic differences are likely contributing to the oleate level in the wild germplasm. Markers representing several other candidate mutant genes are being developed and tested. Larger scale genome sequencing and comparisons between high- and low-oleic acid individual plants within the breeding populations generated during the development of the high oleic acid lines (referred to as HOa lines) is also planned to search for the contributing genes.
Cottonseed oil also contains small amounts of cyclopropylene fatty acids, which is a class of cyclic unsaturated fatty acids that may be associated with detrimental effects when fed or consumed at large levels. Cottonseed lacking these fatty acids could be more valuable. Two members of a family of genes responsible for producing these fatty acids (called cyclopropane synthases, or CPS) are currently being targeted for inactivation using genome editing technologies. As part of Objective 1b, the necessary DNA constructs are being prepared to test the effectiveness of CPS elimination on cyclopropyl fatty acid synthesis.
Work continues under a reimbursable cooperative agreement to identify genes in host crops that limit engineering plants to produce high levels of industrially useful fatty acids. Plant germplasm with targeted mutations in four different genes have been completed, and these lines are being analyzed for changes in oil content and composition. One line, containing a combination of two mutated genes, is nearly complete. Four other lines with different gene targets are in development. This work is being conducted in Arabidopsis but the results will be applicable to cotton and other oil seeds.
Work also continued on understanding differences in the processing of low-gossypol seed (Objective 2). Extraction of oil from these seeds was not problematic and can be achieved by either pressing or solvent extraction. The color of the oil is lighter, which may help solve color development issues that can occur with cottonseed oil. Physical refining of the oil, however, has proven difficult as the phosphorus in the crude oil has been difficult to remove. Degumming reduced the phosphorus level from about 300 ppm to 50 ppm, but the level needs to be 5 ppm in order to continue with this refining strategy. The results suggest that there is a non-phospholipid phosphorus component in the crude oil. To try to determine the nature of this compound, phosphorus nuclear magnetic resonance (NMR) testing is being conducted on the degummed oil samples and the degummed residual. Additionally, a number of physical adsorbents are being tested to see if the phosphorus can be removed by an absorbent.
As a component of Objective 2a, properties of the low-gossypol meals were investigated to understand how cooking protocols will need to be altered to maximize the value of the protein for different purposes (e.g., aquaculture feeds, or food uses). Meals were prepared from low-gossypol seed by dehulling and milling to yield particles that would cook more evenly. Cooking protocols were applied using steam and dry heat at varying times and temperatures followed by hexane extraction under a standard protocol. The meals are being tested this summer for protein, residual oil, color, and protein digestibility.
A number of compounds were tested to improve cottonseed protein adhesive formulations (Objective 3). Catechin, gallic acid, and caffeic acid (phenolic compounds) were evaluated with cottonseed protein isolate (CPI). The addition of gallic or caffeic acid improved dry adhesive performance, hot water resistance, and the soak test, while catechin only showed marginal improvement. Thus, the carboxylic acid components of these compounds seem to be a factor for improving adhesive strength. Additionally, phosphoric acid, potassium phosphate, calcium oxide, and calcium hypophosphate were also added to CPI formulations and tested. These compounds also improved the dry, wet, and soak strengths of protein adhesive formulations. The best performance was obtained with phosphoric acid, which improved water resistance by 88%. Also, an optimized cottonseed protein isolate formulation was applied to pine plywood, and the water resistance of these samples passed industrial soak tests as Type II (interior) plywood.
Water-washed cottonseed meal, cottonseed oil and polycaprolactone (a type of biodegradable polymer) were used to formulate a bio-based plastic (Objective 3b). These blended materials showed satisfactory mechanical and adhesive properties. The findings suggest that cottonseed protein can be a viable raw material for the formulation of bioplastics, and possibly can be used as a hot melt adhesive.
As part of Objective 4a, extracts of cottonseed were prepared and tested for their activity on cell growth and gene expression with different cancer cells, mouse macrophages (white blood cells), and adipocytes (fat cells). The extracts decreased mitochondrial activity of some cancer cells but no activity was observed in macrophages or adipocytes. The extracts also increased gene expression in macrophages. For Objective 4b, cottonseed proteins were treated with trypsin, an enzyme that degrades protein to form peptides, but these peptides did not exhibit biological activity. Other proteases are being explored to produce different peptides for further testing.
In collaboration with scientists from the Rochester Institute of Technology (Rochester, New York), composites made from polypropylene and gin trash have been prepared and tested. Together with a coupling agent, (i.e., maleic anhydride-modified polypropylene [MAPP]), and ethylene-vinyl acetate copolymer, the mixtures were extruded into films. In general, the addition of gin trash reduced tensile strength and elongation but enhanced Young’s modulus (stiffness). Films were also made from gin trash that had been washed with toluene solvent and derivatized with MAPP, which improved mechanical properties (Objective 5). The advantages of gin trash as a filler include low cost, ease in absorbing dyes and pigments, greater gas permeability, and greater affinity for water. Thus, the composites may be useful where reduced cost or additional material stiffness are desirable.
Finally, a number of stakeholder initiatives have been either started or completed. Cottonseed oil high in tocotrienols has been extracted for further testing. These genetically-modified oils will likely be more stable to oxidation. Work was also started to produce an oil with an elevated level of dihydrosterculic acid (one of the cyclopropyl fatty acids in cottonseed oil). The oil will be used at the University of Georgia, Athens, Georgia, to confirm earlier tests of the benefits of this fatty acid in slowing the onset of fatty liver disease. Cottonseed oil naturally has small levels of this acid (0.2-0.3%) and this work may suggest a new direction for cottonseed oil research. Finally, an initial study was conducted on cottonseed hull strength. Ginners have been reporting problems with small and weak seed, which appears to have occurred because of decades of breeding cotton for maximal fiber yield. Damaged seed is detrimental to oil and fiber processors. The initial study found that a wide range in hull strengths exist within current commercial lines.
Accomplishments
1. Release of cotton germplasm with elevated seed oil oleate levels. Vegetable oils with elevated levels of oleic acid are desirable as these oils tend to last longer in deep fat fryers. ARS researchers from New Orleans, Louisiana, working with ARS researchers from Starkville, Mississippi, have identified cotton plants with levels of oleic acid in the seed oil approximately double the level of this fatty acid in normal commercial cottonseed oil. The trait has been found to be genetically stable. These plant lines will be released to allow cotton breeders to use the germplasm. Based on prior usage of cottonseed oil for frying, regaining this market would represent a substantial gain for the industry. Assuming a 25% higher premium price and a 25% market penetration, these elevated oleic acid oils would be worth $110 million more than standard oil.
2. Domestic production of industrial oils. Because of severe allergic reactions to the proteins of the caster plant, castor oil production is prohibited in the United States. Because the oil contains unusual fatty acids that make it an important industrial lubricant, large amounts of castor oil are imported into the United States. Working with University collaborators, ARS researchers in New Orleans, Louisiana, produced oilseed lines containing some of the highest reported seed levels of these fatty acids outside the caster seeds. These lines were engineered to express a combination of two genes from the oil synthesis pathway of the castor bean plant. The work is a step in the direction of being able to produce a castor-like lubricating oil within the United States.
3. Biodegradable plastics from cottonseed proteins. Biodegradable polymers are of current interest because of the need for sustainability, the environmental concerns of plastic contamination and disposal, and toxic hazards involved in the production and degradation of synthetic polymers. ARS researchers at New Orleans, Louisiana, have taken water-washed cottonseed meal and oil and polycaprolactone (a biodegradable polyester) to form a bioplastic. These blends showed mechanical and adhesive properties that will be useful for some applications. The findings suggest that cottonseed protein can be a viable raw material for the formulation of bioplastics, and a possible application as a hot melt adhesive. The market for global biodegradable plastics is growing and expected to reach $6 billion by 2025.
4. Additives improve adhesive performance of cottonseed proteins. Bio-based protein adhesives suffer from being less water resistance than petroleum-based adhesives. To improve the water resistance of protein based adhesive formulations, ARS researchers in New Orleans, Louisiana, studied the adhesive properties of cottonseed proteins mixed with different additives. Additives with a phosphate or carboxylic component improved the water resistance of the formulations. The work is a step toward the development of bio-based adhesives that are as water resistant as petroleum-based adhesives.
Review Publications
Cheng, H.N., Wyckoff, W., Dowd, M.K., He, Z. 2019. Evaluation of adhesion properties of blends of cottonseed protein and anionic water-soluble polymers. Journal of Adhesion Science and Technology. 33(1):66-78. https://doi.org/10.1080/01694243.2018.1495404.
He, Z., Guo, M., Sleighter, R.L., Zhang, H., Chanel, F., Hatcher, P.G. 2018. Characterization of defatted cottonseed meal-derived pyrolysis bio-oil by ultrahigh resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Journal of Analytical & Applied Pyrolysis. 136:96-106. https://doi.org/10.1016/j.jaap.2018.10.018.
Pradyawong, S., Li, J., He, Z., Sun, X.S., Wang, D., Cheng, H.N., Klasson, K.T. 2018. Blending cottonseed meal products with different protein contents for cost-effective wood adhesive performances. Industrial Crops and Products. 126:31-37. https://doi.org/10.1016/j.indcrop.2018.09.052.
Cheng, H.N., Gross, R.A., Smith, P.B. 2018. Green polymer chemistry: pipelines toward new products and processes. In: Cheng, H.N., Gross, R.A., Smith, P.B., Editors. Green Polymer Chemistry: New Products, Processes, and Applications. ACS Symposium Series. Washington, DC: American Chemical Society. p. 1-11. https://doi.org/10.1021/bk-2018-1310.ch001
Cao, H., Sethumadhavan, K., Bland, J.M. 2018. Isolation of cottonseed extracts that affect human cancer cell growth. Scientific Reports. 8:10458. https://doi.org/10.1038/s41598-018-28773-4.
Cao, H., Sethumadhavan, K. Cottonseed extracts and gossypol regulate diacylglycerol acyltransferase gene expression in mouse macrophages. Journal of Agricultural and Food Chemistry. 66(24):6022-6030. https://doi.org/10.1021/acs.jafc.8b01240
Cheng, H.N. 2019. Enzymatic modification of polymers. In: Kobayashi S., Uyama H., Kadokawa J. (eds). Enzymatic Polymerization towards Green Polymer Chemistry. Green Chemistry and Sustainable Technology. Singapore: Springer. p. 357-385. https://doi.org/10.1007/978-981-13-3813-7_12.
Cheng, H.N., Kilgore, K., Ford, C., Fortier, C., Dowd, M.K., He, Z. 2019. Cottonseed protein-based wood adhesive reinforced with nanocellulose. Journal of Adhesion Science and Technology. 33(12):1357-1368. https://doi.org/10.1080/01694243.2019.1596650.
Cheng, H.N., Ford, C.V., He, Z. 2019. Evaluation of polyblends of cottonseed protein and polycaprolactone plasticized by cottonseed oil. International Journal of Polymer Analysis and Characterization. 24(5):389-398. https://doi.org/10.1080/1023666X.2019.1598641.
Ling, Z., Wang, T., Makerem, M., Santiago Cintron, M., Cheng, H.N., Kang, X., Bacher, M., Porthast, A., Rosenau, T., King, H.A., Delhom, C.D., Nam, S., Edwards, J.V., Kim, S., Xu, F., French, A.D. 2019. Effects of ball milling on the structure of cotton cellulose. Cellulose. 26(1):305-328. https://doi.org/10.1007/s10570-018-02230-x.
