Location: Commodity Utilization Research
2020 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. The progress described will first discuss efforts made during the final year of the project followed by a summary of the accomplishments achieved over the course of the five-year project cycle. To increase cottonseed oleic acid levels (Objective 1), a back-cross was made between a nematode-resistant plant variety that had been previously identified as having higher than normal levels of oleic acid and plants of an early generation (F2) of a cross made between GB713, a wild plant that contains high levels of oleic acid, and SG747, a publicly available cotton line. This was done to try to increase the seed oil oleic acid level beyond the levels in the recently released germplasm lines (named HOa1-HOa4), which were around 33-35% oleic acid. Prodigy seeds of these crosses were self-pollinated to yield seed from the first segregating generation. These seeds were then field planted in Starkville, Mississippi. One-hundred and fifty-one plants were obtained, and seeds of each plant produced oleate levels between 32 and 56%. This range indicates that significantly higher oleic acid levels are possible beyond that obtained from the nematode resistant-released lines, and justifies the continuation of this breeding strategy. In addition, DNA was extracted from the lowest (32-34%) and highest oleic acid plants (51-56%) and is being sequenced to look for the genetic differences that contribute to the elevated oleic acid trait. The highest oleic acid plants (53-56%) will undergo a second back-cross with the HOa1 line to further the breeding process.Cottonseed oil also contains small amounts of cyclopropane and cyclopropene fatty acids (a class of unsaturated cyclic fatty acids) that may have detrimental and beneficial physiological effects but their synthesis is poorly understood. ARS reseachers at New Orleans, Louisiana, have made DNA strands by gene editing and these will be used to support editing the cotton genome to answer questions about the synthesis of the acids and to determine how best to tailor the levels of these acids toward the beneficial forms. Collaborative projects with Texas A&M University and University of North Texas to further this work are being established. The transformation of cotton with the first constructs will begin soon. Experiments were conducted to look at the effects of hull material on cottonseed oil extraction and color. Gossypol has been blamed for causing crude cottonseed oils to be difficult to bleach to a low color. Extractions with seed of different gossypol levels has not shown the effect, leading to the thought that the variable presence of hulls during extraction might be the cause of the problem. Adding different levels of hulls to kernel tissue that was carefully cleaned of all hull and other debris did not exhibit increased oil color after oil extraction, or after refining and bleaching of the oil. However, extraction of glandless seeds and seeds of the recently developed ultra-low gossypol plant lines showed dramatic color differences despite having separated all of the hulls prior to solvent extraction and both samples having very low and similar gossypol levels (about 200 ppm). The difference indicates that it is the glands themselves, which are absent in the glandless seed but present in the ultra-low gossypol seed, that are responsible for the dark oil color and bleaching difficulties. Work is continuing to identify the components that contribute to the effect. Experiments on cottonseed proteins (Objective 3) focused on the adhesive performance of different protein fractions. Cottonseed protein was separated into water- and alkali-soluble fractions. Strength testing showed better bonding with alkali-soluble proteins than with the water-soluble proteins. Infrared spectroscopy of the glue line indicated better wettability for the alkali-protein formulations than for the water-protein formulations. In addition, adhesive experiments blending cottonseed protein and polyester plus different promoters are in progress. The polyester/cottonseed protein blends showed good results, with some formulations improving the dry adhesive strength by 70% and hot water adhesive strength by 50%. To improve water resistance, adding phosphoric acid and calcium salts to protein formulations was tested, and the results showed that combination enhanced the water resistance of the adhesive in comparison with other modifiers, due to calcium ion presenting a better synergy with the acid.
A modest amount of work was also conducted to develop uses for gin trash and cotton derived xylan (Objective 5). When 10 and 20% weights of modified gin trash were added to polypropylene, the composites gave the same or slightly higher tensile strength and higher modulus, but lower elongation than the pure polymer. Up to 60% weight of gin trash could be added to a slightly different polymer. These materials showed reduced tensile strength and elongation at break but enhanced stiffness compared with the polymer without the gin trash addition. No significant improvement in mechanical properties was observed when the gin trash was modified with either acetic or succinate acid to improve its compatibility with the polymer. Additionally, acetate and succinate derivatives of xylan were prepared. By changing the catalyst, dosage, and temperature, the derivatives could be prepared with different degree of substitution.
Progress was also made on a few satellite efforts. Under a reimbursable cooperative agreement (#58-6054-9-007) to identify genes that limit high level production of industrially useful fatty acids, six candidate genes have been identified. These will be over expressed in cotton to test the gene activity in modified plants. Work also continues on a reimbursable agreement (#58-6054-9-009) to evaluate cottonseed genotypes for differences in cyclopropyl fatty acid content and to evaluate the fatty acid profiles of commercial seed known to have germination difficulties.
Finally, cottonseed strength studies were started due to recent concerns that the ginning of weak seed is leading to greater levels of seed coat neps, i.e., fibers with attached hull fragments. A genetics-environment study is continuing, as well as a study comparing the seed strengths of Upland (Gossypium hirsutum L.) and Pima (Gosspyium barbadense L.) varieties. The Australian cotton industry has expressed some interest in this work, as they are also having regional ginning difficulties.
Summarizing the accomplishments over the five year project cycle, substantial progress was made on the development of cottonseed lines with higher levels of seed oil oleic acid (Objective 1). The proposed approach for this was initially delayed because of the identification of germplasm (bred to have nematode resistance) that also contained higher than normal levels of oleic acid. This material ultimately resulted in a germplasm release of plant lines (HOa1-HOa4) with double the normal levels of oleic acid. Additionally, the use of some of the nematode resistant parental material helped overcome the limited flowering of the wild high oleic cotton accessions, which helped re-establish the schedule for the originally planned breeding effort. Our first back-cross made from the planned breeding process resulted in plants with oleate levels considerably higher than expected (discussed above). Continuing with the breeding strategy should yield stable upland germplasm with 50% oleic acid or more, i.e., greater than our initial goal of 42%. Toward the second cottonseed oil effort, work to better understand the synthesis of the cyclopropyl fatty acids in cotton is progressing, although there is still much to learn. The molecular tools needed for these studies were developed, which was what was to be accomplished in this project cycle.
Regarding our cottonseed processing objective (Objective 2), it was learned that the high oleate lines have different physical properties that suggest some niche market uses. Also, it was found that the bleaching problems that occur with some crude cottonseed oils are due to components from the cottonseed pigment glands and not the hulls or gossypol itself. However, attempts to refine low-gossypol crude oils with physical methods were not successful due to the presence of an unknown phosphorus component that has proved difficult to remove.
The protein adhesive work of Objective 3 identified a number of formulation additives that improved cottonseed protein adhesive strength and water resistance. Two patent applications have been filed; to date, one patent has been issued and the second application is currently being evaluated.
Efforts (Objective 4) to identify additional bioactivity compounds has been less successful. While some crude ethanol extracts exhibited some bioactivity; no specific compounds were identified, and the degree of activity was insufficient to warrant additional consideration.
The final objective of the project (Objective 5) was to investigate the hemicellulose components from the seed. This material could only be isolated in small yield (6%) from cottonseed hulls and was more difficult to purify than are hemicelluloses from other woody materials. It would not be economic to recover this material at a practical scale.
Accomplishments
1. Improved cottonseed based adhesive formulations. The elimination of formaldehyde from the synthetic adhesives used in the wood product industry is highly desired for environment and worker safety. One approach for this is the use of bio-based protein adhesives, but these preparations have a number of limitations. ARS researchers at New Orleans, Louisiana, have found specific compounds containing catechol and carboxylic functionalities that enhance the adhesive performance of cottonseed protein. The use of these compounds improved the water resistance of the adhesive joints. A provisional patent application has been filed, and the work is of interest to the wood products industry as a method to help eliminate formaldehyde from their processes.
2. Development of higher oleic acid cottonseed oils. Vegetable oils with higher levels of oleic acid tend to be more oxidatively stable at elevated temperatures than oils with higher level linoleic acid. These oils are favored as frying oils, as the improved temperature stability allows the oils to be used for longer periods. ARS researchers at New Orleans, Louisiana, have conducted breeding experiments and have found that seed oil oleic acid levels can be increased to levels greater than what is currently available. This will be useful for developing cottonseed oils with higher levels of oleic acid than are currently available and will have improved stability when used for deep fat frying. These oils are important to the fats and oil communities.
3. Use of calcium salts and phosphates for improved cottonseed based adhesive formulations. To improve adhesive water resistance, ARS researchers at New Orleans, Louisiana, have been exploring the addition of additives to protein adhesive formulations to improve their water resistance. The addition of phosphoric acid and calcium salts showed that the combinations enhanced the water resistance of cottonseed protein based adhesives, due to calcium ion release from the chloride presenting a better synergy with the acid. The work is a step toward expanding the potential use of proteins as wood adhesives in place of current synthetic formulations.
Review Publications
Cao, H., Sethumadhavan, K. 2020. Regulation of cell viability and anti-inflammatory tristetraprolin family gene expression in mouse macrophages by cottonseed extracts. Scientific Reports. 10:775. https://doi.org/10.1038/s41598-020-57584-9.
He, Z., Cheng, H.N., Klasson, K.T., Ford, C., Barroso, V.A.B. 2019. Optimization and practical application of cottonseed meal-based wood adhesive formulations for small wood item bonding. International Journal of Adhesion and Adhesives. 95:102448. https://doi.org/10.1016/j.ijadhadh.2019.102448.
Dowd, M.K., McCarty Jr, J.C., Shockey, J., Jenkins, J.N. 2020. Registration of four upland cotton germplasm lines with elevated levels of seed oil oleic acid. Journal of Plant Registrations. 14(1):64-71. https://doi.org/10.1002/plr2.20017.
Cheng, H.N., Girolami, G.S. 2020. H.S. Gutowsky and the use of nuclear magnetic resonance in chemistry. In: Strom, E.T., Mainz, V.V., editors. Pioneers of Magnetic Resonance. American Chemical Society Symposium Series Volume 1349. Washington, D.C.: American Chemical Society. p. 21-31.
He, Z., Olk, D.C., Tewolde, H., Zhang, H., Shankle, M. 2019. Carbohydrate and amino acid profiles of cotton plant biomass products. Agriculture. 10(1):2. https://doi.org/10.3390/agriculture10010002.
Cao, H., Sethumadhavan, K. 2019. Gossypol but not cottonseed extracts or lipopolysaccharides stimulates HuR gene expression in mouse cells. Journal of Functional Foods. 59:25-29. https://doi.org/10.1016/j.jff.2019.05.022.
Ge, C., Cheng, H.N., Miri, M.J., Hailstone, R.K., Francis, J.B., Demyttenaere, S.M., Alharbi, N.A. 2020. Preparation and evaluation of composites containing polypropylene and cotton gin trash. Journal of Applied Polymer Science. 137(38):1-13. https://doi.org/10.1002/app.49151.
Li, J., Pardyawong, S., He, Z., Sun, X. S., Wang, D., Cheng, H. N., Zhong, J. 2019. Assessment and application of phosphorus/calcium-cottonseed protein adhesive for plywood production. Journal of Cleaner Production. 229:454-462. https://doi.org/10.1016/j.jclepro.2019.05.038.
Dowd, M.K., Manandhar, R., Delhom, C.D. 2019. Effect of seed orientation, acid delinting, moisture level, and sample type on cottonseed fracture resistance. Transactions of the ASABE. 62(4):1045-1053. https://doi.org/10.13031/trans.13109.
Dowd, M.K. 2020. Stability of the gossypol-amine adducts used for chromatographic measurement of total and isomeric gossypol. Journal of the American Oil Chemists' Society. 97(6):671-675. https://doi.org/10.1002/aocs.12355.
He, Z., Zhang, D., Olanya, O.M. 2020. Antioxidant activities of the water-soluble fractions of glandless and glanded cottonseed protein. Food Chemistry. 325:126907. https://doi.org/10.1016/j.foodchem.2020.126907.
