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ARS Home » Southeast Area » New Orleans, Louisiana » Southern Regional Research Center » Commodity Utilization Research » Research » Research Project #428791

Research Project: Increasing the Value of Cottonseed

Location: Commodity Utilization Research

2016 Annual Report

1a. Objectives (from AD-416):
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.

1b. Approach (from AD-416):
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.

3. Progress Report:
Work continued on the development of cotton varieties with different oil properties. Transferring a high oleic acid (a desirable monounsaturated fatty acid) trait from wild plants to cultivated varieties has not been successful. Alternative approaches will now be tried. These will involve either applying a virus known to induce flowering or growing the plants in a different environment. Costa Rica, which may be soon available as a replacement to the closed Cotton Winter Nursery, may be an option. Understanding the molecular basis for high oleic acid levels in cottonseed oil is progressing. In conjunction with ARS collaborators at the Genomics and Bioinformatics Research Unit (Stoneville, Mississippi) and the Food and Feed Safety Unit (New Orleans, Louisiana), the complete DNA (deoxynucleic acid) sequence was determined for a cotton plant that has unusually high levels of oleic acid in its oil. The entire gene family for an enzyme important in regulating the oleic acid level was identified. This gene family is being studied closely for changes in its DNA that could account for the high oleic acid character. This data set is also helping to identify genes that contribute to the unwanted production of cyclic fatty acids. The data is also helping identify regulatory genes that control oil synthesis in oilseed plants. The basic components of a new molecular technique (called cRISPER) for selectively targeting gene activities have been assembled. These components are undergoing functional testing in model plants and microbes. Successful preliminary findings with these tools have encouraged collaborators in the Cotton Fiber Bioscience Unit (New Orleans, Louisiana) to begin the process of creating cotton plants with a specific gene that helps to direct the location of gene changes. This will be useful for developing plants with specific genes blocked. Successful combinations of these modified plants, paired with the other gene components, will be tested in a hairy root tissue culture (which can be conveniently grown in the laboratory) for their suppressive effects on cyclic fatty acid production. Several small phosphate compounds were added to cottonseed proteins adhesive formulations and were tested for their adhesive properties. Several of these compounds increased adhesive strength and water resistance. The results appear to be specific to formulations composed of cottonseed proteins, as similar improvements were not found with soybean proteins. This difference may be related to the different levels of arginine (one of several amino acids found in proteins) in the two protein types. In addition, a re-examination of the use of protein denaturants (chemicals that cause proteins to unfold) has also found adhesive improvement at conditions not previously studied. Cottonseed meal was also studied as an adhesive. The effects of pH and storage time were considered. Under a completed Reimbursable Agreement with Kansas State University (Manhattan, Kansas), the production of water-washed cottonseed meal was scaled up to a 20 lb. pilot-plant procedure. This work provided a quantity of the washed meal for industrial adhesive applications and research efforts. Chemical analysis of the products provided the fundamental data for quality control of future mill scale production of these products. A CRADA (Cooperative Research and Development Agreement) has been established with the Department of Sustainable Products of Mississippi State University (Starkville, Mississippi) to produce particle boards with cottonseed protein-based adhesives. An initial sample of cottonseed protein isolate was prepared for this work. This material will be used to explore the conditions to make suitable boards. After reasonable pressing conditions are determined, guayule resin (a plant-based insect deterrent) will be added, and the boards will be tested for termite and fungal resistance. In addition, two invention disclosures have been written on the cottonseed adhesives work. The patent review committee has approved the filing of one patent application, which will include claims related to blended protein preparations, and the addition of carboxylic acids and phosphorus-containing chemicals to protein-based adhesives. To determine if cottonseed contained minor compounds that are active in biological systems, defatted glandless cottonseeds were extracted different solutions containing alcohols. Different concentrations of ethanol extracts from the seed coat and kernels were used to treat biological cell cultures. Cell viability assays showed that extracts from cottonseed retained cell survival while the control tests lost significant mitochondrial (energy production) activity over a 24 h period. High concentrations of purified gossypol (a well-known bioactive compound in cottonseed) resulted in significant cell death. RNA (ribonucleic acid) was extracted from the cells to analyze if gene expression was affected by the extracts, with an emphasis on genes related to immune system response. Peptides and proteins were also extracted from glanded and glandless seeds. The fractions were separated into individual components. These protein components are being further characterized by mass spectrometry. The function and bioactivity of these novel proteins are being tested in biological assays. Xylan (a carbohydrate plant polymer) has been isolated from cottonseed hulls (outer part of the seed) and cotton burrs (outer covering of the cotton bowl), and a number of treatments were used to improve the purity and properties of these preparations. The preparations were derivatized with different classes of compounds to alter their properties. Preliminary studies with the modified products have shown that when combined with cellulosic materials useful film-like materials can be prepared. The xylan extracted from the hull was also treated to form a more bio-based polyurethane polymer. The viscosity of these preparations, helpful for some product applications, was also determined. To better understand and control seed damage that occurs during ginning (the process used to separate fiber and seed), seeds have been studied for their mechanical strength. Several seed samples, including different commercial varieties and varieties grown in different conditions, were tested for tensile strength, maximum elongation before breakage, and breakage energy. Results show that significant differences exist among the samples, and both genetic and growing conditions appear to affect this property. This may provide a convenient method to test for the tendency of different seed types to be damaged. Because of the many decades of successful breading cotton varieties for increased fiber yield, it has generally been believed that cottonseed has become smaller. To understand these changes, a series of seed samples were characterized for their fiber-to-seed ratio, seed size, linter (short fibers that remain with the seed after ginning), hulls, and kernel tissue percentages, and kernel seed, protein, and gossypol composition. Results indicate that seed-to-fiber ratio has decreased from around 1.7 in the 1940s to an average of 1.45 today. Similarly, seed size has on average decreased around 20% over a similar time period. Smaller differences were found regarding the percentage of linters, but the amounts of hull and kernel tissue and the amounts of kernel protein and oil do not have appear to have been significantly changed by breeding for fiber yield.

4. Accomplishments
1. Improved protein adhesive formulations. ARS scientists at New Orleans, Louisiana, have found that by including additives in cottonseed based protein adhesives, improved adhesive performance can be obtained. Both adhesive strength and water resistance have been improved by the addition of small amounts of organic acids or phosphorous containing compounds. Improved water resistance is of particular interest, as most protein based adhesives exhibit poor water resistance.

2. Less expensive protein adhesives. ARS scientists at New Orleans, Louisiana, have found that by blending cottonseed proteins with other components in adhesive formulations, improved adhesive formulations can be obtained. Blending some cottonseed protein with soybean protein allowed for improved performance over soybean protein alone. Additionally, cottonseed-based protein adhesives retain much of their properties when blended with relatively cheap fibrous fillers. These blends may provide an opportunity to decrease the amount of protein used in adhesive formulations, thereby reducing cost.

3. Preparation of carbohydrate polymer products from cotton byproducts. ARS scientists at New Orleans, Louisiana, have extracted carbohydrate-based polymer fractions (i.e., hemicelluloses) from cotton and cottonseed processing waste materials, and improved the purity and properties of the resulting material. Biodegradable film materials have been made from these materials. These materials may also be useful as thickeners in a number of water-based product formulations.

4. Mutant cotton genotype sequenced. ARS scientists at New Orleans, Louisiana, and at Stoneville, Mississippi, have completed the genome sequence of a mutant Gossypium barbadense accession with high levels of oleic acid in its seed oil. This will allow the rapid assessment of genetic differences that result in this desirable trait, which may occur because of modification of one or several enzymes plus various regulatory elements. The work will be of particular interest to those working to expand the value and uses for cottonseed oil.

5. Genes identified for desaturating monounsaturated fatty acids. The full complement of cotton fatty acid desaturase 2 genes (also known as FAD2 genes) have been identified by ARS researchers at New Orleans, Louisiana. These proteins are critical during production of seed oils and other lipid components, and they control the balance between monounsaturates (which typically contain the best properties for human and animal health as well as frying stability) and diunsaturates (which are less desirable). Eight separate genes have been identified, and the set represents that largest known FAD2 gene family currently reported in the plant science literature. The work will help guide efforts to improve the properties and utilization of cottonseed oil.

5. Significant Activities that Support Special Target Populations:

Review Publications
Liu, Y., He, Z., Shankle, M., Tewolde, H. 2015. Compositional features of cotton plant biomass fractions characterized by attenuated total reflection Fourier transform infrared spectroscopy. Industrial Crops and Products. 79:283-286.
Cheng, H.N., Ford, C., Dowd, M.K., He, Z. 2016. Soy and cottonseed protein blends as wood adhesives. Industrial Crops and Products. 85:324-330.
He, Z., Chapital, D.C., Cheng, H.N. 2016. Comparison of the adhesive performances of soy meal, water washed meal fractions, and protein isolates. Modern Applied Science. 10(5):112-120.
Cheng, H.N., Ford, C., Dowd, M.K., He, Z. 2016. Use of additives to enhance the properties of cottonseed protein as wood adhesives. International Journal of Adhesion and Adhesives. 68:156-160.
He, Z., Klasson, K.T., Wang, D., Li, N., Zhang, H., Zhang, D., Wedegaertner, T.C. 2016. Pilot-scale production of washed cottonseed meal and co-products. Modern Applied Science. 10(2):25-33.
Zhu, L.-H., Krens, F., Smith, M.A., Li, X., Qi, W., Van Loo, E.N., Iven, T., Feussner, I., Nazarenus, T.J., Huai, D., Taylor, D.C., Zhou, X.-R., Green, A.G., Shockey, J., Klasson, K.T., Mullen, R.T., Huang, B., Dyer, J.M., Cahoon, E.B. 2016. Dedicated industrial oilseed crops as metabolic engineering platforms for sustainable industrial feedstock production. Scientific Reports. 6:22181.
Shockey, J., Regmi, A., Cotton, K., Adhikari, N., Browse, J., Bates, P.D. 2015. Identification of Arabidopsis GPAT9 (At5g60620) as an essential gene involved in Triacylglycerol Biosynthesis. Plant Physiology. 170:163-179.
Liu, S., Zhu, Y., Meng, W., He, Z., Feng, W., Zhang, C., Giesy, J.P. 2015. Release and transformation of carbon and phosphorus from aquatic macrophytes of lakes: Insight from solid-state 13C NMR and solution 31P NMR spectroscopy. Science of the Total Environment. 543:746-756.