Location: Commodity Utilization Research
2018 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
Progress was made on all five of the project objectives, all of which fall under National Program 306 and contains 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.
To promote the use of cottonseed oil as a frying oil, higher levels of oleic acid than are normally present in the oil (~17%) are desirable. As part of Objective 1, the wild GB-713 cotton plant that we had previously identified as having a high oleic acid trait was also found to exhibit nematode resistance. Because of this other trait, the GB-713 genotype has been used to transfer nematode resistance into an agronomic short-staple cotton variety (SG-747). Seeds from different steps in this breeding program were tested for oil oleic acid level, and elevated levels (30-40%) were found throughout the breeding sequence. Four series of plants from this program were planted in the summer of 2017 and harvested in the fall. Two of the plant lines were finished lines (i.e., the plants are considered genetically stable), and two lines were intermediate in the breeding process. Testing these plants indicated elevated levels of oleic acid in some plants from all four plant lines. Seeds from plants of the two finished lines exhibiting the highest oleic acid levels were planted in the greenhouse and harvested to increase the available seed. These seeds were then used to plant field rows this summer. If these plants are found to be uniformly high in oleic acid (around 30% is expected for the one finished plant line and ~35% is expected for the second plant line), then the genetic components that contribute to these increased levels will be considered stable, and these plant lines will be made available in germplasm releases for commercialization. These will represent the first stable agronomic Upland cotton cultivars with elevated seed oil oleic acid levels.
Also in support of achieving Objective 1, an improved DNA marker was developed for a mutant desaturase gene (called fad2-1d) present in the wild GB-713 plants, because our first marker exhibited false positives. This gene is responsible for at least part of the high oleic acid trait and is useful for helping to select individual plants for the high oleic acid trait. The improved marker will be used to select field plants for the mutant desaturase. As the marker was found to be present in all of the plants from one of the finished lines, which all had about 30% seed oil oleic acid, the gene appears to account for about half of the elevated oleate trait present in GB-713 plants. This suggests that additional mutations exist in the wild plant. A list of candidate mutant genes has been determined. Marker testing protocols for the top candidates in this list are being developed, and testing will begin soon.
In order to study the properties of cottonseed oils with high levels of oleic acid, bulk seeds from GB-713 plants (which were produced at the ARS Winter Nursery) were extracted to recover a high-oleate cottonseed oil. The oil was then refined and bleached. This oil will be used in the upcoming year for solvent fractionation experiments to compare the fractionation properties of the two oil types.
Reducing the level of cyclopropyl fatty acids in cottonseed is also desirable as these acids contribute unwanted biological activity to the oil. Using the genome sequence of the cotton wildtype plant (GB-713), the family of cyclopropyl genes (called cps) has been identified, and the DNA elements that control the expression of these genes have been cloned. These elements have been attached to a reporter gene that produces a brightly colored chemical in the plant tissues where it is active. The DNA fusion molecules are being prepared for expression in cotton hairy root cultures to study which of the cps genes likely contributes the most to cyclopropyl fatty acid production.
During oil refining, cottonseed oil can sometimes develop high color, a process referred to as color set, which can affect its value. Glandless cottonseed genotypes have reduced levels of phenolic compounds. Because phenolic compounds can oxidize to form dark oxidation products, oil from glandless plants may exhibit reduced color formation during refining. To better understand these effects, crude cottonseed oils from glandless plants were refined by different techniques (Objective 2). Several batches of crude oil were prepared by dehulling, flaking, cooking and extruding the kernel tissue followed by extraction with hexane, a commercial solvent. These crude oil samples were used to study different refining strategies. The normal chemical refining with sodium hydroxide (a base that reacts with some oil impurities) produced oils with very low Lovibond red color (0.7 to 0.9 red values). Although not as low as the color of cottonseed oils prepared by pressing in earlier experiments, these red color levels are still well below the standard for prime cottonseed oil (2.5 red value). Hence, crude oils from glandless plants when processed by chemical refining should yield refined oils of much lower color, and the color set problems that affected cottonseed oil in the past would be eliminated. The physical refining of vegetable oils is an alternative refining process that purifies the oil without sodium hydroxide. This refining process, however, does not appear to work with cottonseed oil. Repeated attempts have been made, but the initial degumming of the oil did not reduce phosphorus levels sufficiently to allow for continuation of the refining process. The physical oil refining process of cottonseed oil might be easier with pressed oils, which have less impurities than solvent extracted oils. At present, obtaining pressed oil from glandless seed has been difficult due to the unavailability of the processing equipment, which is owned by an outside group. It is unclear when we will obtain access to this processing equipment.
Under Objective 3, new uses for cottonseed protein, which at present is used solely as low-value feed ingredient, are being evaluated. With the help of researchers at the ARS National Center of Agricultural Utilization Research in Peoria, Illinois, large-scale (10 Kg) production of cottonseed protein isolate (>90% protein) was achieved. This material was needed to support collaborative work with the Department of Biorenewable Resources, Mississippi State University at Starkville, Mississippi, to produce bio-based termite-resistant plywood boards. The first boards were prepared late in 2017, and an optimization process is underway to determine the best conditions for adhering the veneer layers. The first stage of the optimization process analyzed boards made with different amounts of protein and water. This experiment indicated that a range of both protein and water levels can be used to produce boards. Experiments to determine the best pressing temperature and time are in progress.
Several additional protein adhesive experiments have been conducted utilizing either cottonseed protein isolate or cottonseed meal. Experiments on the preparation of cottonseed protein meals for use as wood adhesives showed that the drying method employed during meal preparation had an impact on adhesive performance. High-temperature oven drying in adhesive formulations requires the use of higher temperatures during pressing to form adhesive joints with adhesive strength equal to that developed from protein meals dried at lower temperature. The effect of smaller particle sizes on the adhesive properties of the protein meal was found to be minimal but some decrease in water resistance was noted with the smaller particles. Replacing up to 40% of the urea-formaldehyde adhesive with cottonseed meal showed doubled water soaked bonding strength, compared to either pure synthetic resin or cottonseed meal adhesives alone. Additionally, cottonseed protein isolate, when added to nonwoven cotton products, increased the dry strength of the products. Eleven percent protein tripled the tear strength and burst strength compared to untreated nonwoven fabric.
As part of Objective 4, protocols were developed to isolate bioactive extracts from cottonseed, and these extracts were tested for their activity on different human cancer cells. Gossypol (a polyphenolic metabolite present in cottonseed) and ethanol extracts of glanded and glandless cottonseed kernels significantly decreased the mitochondrial activity (energy production) of breast cancer cells and pancreas cancer cells.
In addition, cottonseed extracts and gossypol affected the expression of DGAT enzymes (an enzyme associated with lipid biosynthesis). Results suggest that this is an inducible gene that responds to stimulators, such as polyphenols, whose enzyme product plays an important role in fat biosynthesis. The results indicate that gossypol and ethanol extracts from glanded cottonseed kernels are stimulators of DGAT2 gene expression and that they may be novel agents for intervention of lipid-related diseases.
Different xylan (a carbohydrate polymer) preparations were isolated from cotton plant byproducts, including cottonseed hulls and cotton burrs (Objective 5). This complex carbohydrate was treated to make positively and negatively charged polymers, and these novel products were characterized by a number of analytical techniques. Mixed solutions of the differently charged polymers were found to increase the dry strength of paper by 75%.
Accomplishments
1. Cottonseed oil with desirable elevated levels of oleic acid. 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 that combine two beneficial traits: elevated (30%) oleic acid in seed oil and broad spectrum root resistance to soil worm pests. Additional agronomic and chemical tests are ongoing but so far the traits appear to be genetically stable, and this plant line will be submitted for germplasm release in a year. These will be the first agronomic cotton plants to exhibit elevated levels of oleic acid in the seed oil, and the oil from these plants should be better able to compete with other vegetable oils used for frying. Based on prior usage of cottonseed oil for frying, regaining this market would represent a substantial market 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. Use of cottonseed protein as a strength additive for nonwoven cotton. Nonwoven fabrics are being used in a wide range of consumer products. Cotton-based nonwovens are of interest because of their ability to be recycled, resulting in more environmentally friendly products compared with petroleum-based counterparts. ARS researchers in New Orleans, Louisiana, have used cottonseed protein as an additive to increase the dry strength of cotton-based nonwovens. If these strengthened products increase the use of cotton in nonwoven materials by 10%, the value of the additional cotton fabric used in would be around $30 million dollars. These results will be helpful to enhance the usage of cotton nonwovens and cottonseed protein in several applications.
3. Improved protein-based wood adhesive formulations. Researchers in New Orleans, Louisiana, have developed improved wood adhesive formulations by including different chemical additives. The inclusion of small amounts of polymer additives, for example, improves both the dry adhesive strength and the hot water resistance of the protein-based formulations. The results will be of interest to the wood products industry that is seeking to eliminate formaldehyde from current wood adhesive formulations.
4. Low color cottonseed oils. Dark oil color is a sporadic problem associated with cottonseed oil refining that reduces the value of the oil. For some seed, oils have to be handled that bleach to less than ideal color levels and these oils often can only be managed by blending with light color oils. ARS researchers in New Orleans, Louisiana, have been studying the refining and bleaching of cottonseed oils prepared from new seed varieties that do not have the pigment glands of regular cottonseed. The bleachable color of the oil extracted from these seed is much lower than the color of oil from regular glanded seed. The result should eliminate this occasional processing complication and will be of interest to cottonseed oil processors.
Review Publications
Zheng, L., Shockey, J., Bian, F., Chen, G., Shan, L., Li, X., Wan, S., Peng, Z. 2017. Variant amino acid residues alter the enzyme activity of peanut type 2 diacylglycerol acyltransferases. Frontiers in Plant Science. 8:1751.
Zheng, L., Shockey, J., Guo, F., Shi, L., Li, X., Shan, L., Wan, S., Peng, Z. 2017. Discovery of a new mechanism for regulation of plant triacylglycerol metabolism: The peanut diacylglycerol acyltransferase-1 gene family transcriptome is highly enriched in alternative splicing variants. Journal of Plant Physiology. 219:62-70.
He, Z., Cheng, H.N., Olanya, O.M., Uknalis, J., Zhang, X., Koplitz, B.D., He, J. 2018. Surface characterization of cottonseed meal products by SEM, SEM-EDS, XRD and XPS analysis. Journal of Materials Science Research. 7(1):28-40.
Cheng, H.N., Villalpando, A., Easson, M.W., Dowd, M.K. 2017. Characterization of cottonseed protein isolate as a paper additive. International Journal of Polymer Analysis and Characterization. 22(8):699-708.
Alam, M.S., Watanabe, W.O., Carroll, P.M., Gabel, J.E., Corum, M.A., Seaton, P., Wedegaertner, T.C., Rathore, K.S., Dowd, M.K. 2018. Evaluation of genetically-improved (glandless) and genetically-modified low-gossypol cottonseed meal as alternative protein sources in the diet of juvenile southern flounder Paralichthys lethostigma reared in a recirculating aquaculture system. Aquaculture. 489:36-45.
He, Z., Cheng, H.N., Klasson, K.T., Olanya, O.M., Uknalis, J. 2017. Effects of particle size on the morphology and water- and thermo-resistance of washed cottonseed meal-based wood adhesives. Polymers. 9(12):675. https://doi.org/10.3390/polym9120675.
Liu, M., Wang, Y., Wu, Y., He, Z., Wan, H. 2018. "Greener" adhesives composed of urea-formaldehyde resin and cottonseed meal for wood-based composites. Journal of Cleaner Production. 187:361-371.
Cheng, H.N., Doemeny, L.J., Geraci, C.L., Schmidt, D.G. 2016. Nanotechnology overview: Opportunities and challenges. In: Cheng, H.N., Doemeny, L.J., Geraci, C.L., Schmidt, D.G., editors. Nanotechnology: Delivering the Promise Volume 2. ACS Symposium Series, Washington, DC: American Chemical Society. p. 1-12.
Klasson, K.T. 2018. QXLA: Adding upper quantiles for the studentized range to Excel for multiple comparison procedures. Journal of Statistical Software. Journal of Statistical Software, Code Snippets. Vol(85):1-9.
Cheng, H.N., Ford, C., Dowd, M.K., He, Z. 2017. Effects of phosphorus-containing additives on soy and cottonseed protein as wood adhesives. International Journal of Adhesion and Adhesives. 77:51-57.
Paton, C.M., Vaughan, R.A., Alpergin, E.S.S., Assadi-Porter, F., Dowd, M.K. 2017. Dihydrosterculic acid from cottonseed oil suppresses desaturase activity and improves liver metabolomic profiles of high-fat-fed mice. Nutrition Research. 45:52-62.
He, Z., Cheng, H.N. 2017. Evaluation of wood bonding performance of water-washed cottonseed meal-based adhesives with high solid contents and low press temperatures. Journal of Adhesion Science and Technology. 31(23):2620-2629
Cheng, H.N., Ford, C., Dowd, M.K., He, Z. 2017. Wood adhesive properties of cottonseed protein with denaturant additives. Journal of Adhesion Science and Technology. 31(24):2657–2666.
Li, N., Prodyawong, S., He, Z., Sun, X.S., Wang, D. 2017. Effect of drying methods on the physicochemical properties and adhesion performance of water-washed cottonseed meal. Industrial Crops and Products. 109:281-287.
He, Z., Chiozza, F. 2017. Adhesive strength of pilot-scale-produced water-washed cottonseed meal in comparison with a synthetic glue for non-structural interior application. Journal of Materials Science Research. 6(3):20-26.
Dowd, M.K., Pelitire, S.M., Delhom, C.D. 2018. Seed-fiber ratio, seed index, and seed tissue and compositional properties of current cotton cultivars. Journal of Cotton Science. 22:60-74.
