Project : USDA ARS

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Research Project: Increasing the Value of Cottonseed

Location: Commodity Utilization Research

2017 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 genome of a wild Gossypium barbadense cotton accession (GB-713) was sequenced as part of Objective 1a. This accession has a higher than normal level high-oleic acid and a lower than normal level of linoleic acid in its seed oil. From the genomic data, a gene was identified (referred to as FAD-2d1) that appears to be abnormal. The abnormality appears to be adjacent to the gene, which we believe prevents correct localization of the enzyme within the cell leading to reduced activity. A DNA (deoxyribonucleic acid) construct was generated of this region to help identify this sequence in breeding experiments. Additionally, although GB-713 cotton is photoperiodic (i.e., it does not readily flower outside its native environment), plants of this accession did flower for a short period in our greenhouse, and the pollen was used to make crosses to two G. barbadense varieties (long-stable cotton) and two G. hirsutum varieties (short-staple cotton). The seed from these crosses can be used to start a breeding effort to move the high oleic acid trait into agronomic cotton. However, not enough seed was generated to be sure that we will get the desired combination of traits from both parents. Fortunately, another avenue now appears more promising. During the preparation of these crosses, it was learned that GB-713 had previously been used in another ARS breeding effort in the Genetics and Sustainable Agriculture Research Unit (Starkville, Mississippi). In discussing this work with the researchers involved, a few seed of their finished lines and their initial crosses were obtained for testing. Variable numbers of seeds of the finished lines showed levels of oleic acid greater than 30% compared with typical agronomic varieties having values about 17-18%. Although not as high as seed from the initial GB-713 plants (>40%), this represents an increase worth investigating. Additionally, a number of the plants of these seeds were positive for our gene mutation. This material may shorten the time needed to breed the higher oleic acid trait, and a series of field tests have been designed to further explore this possibility. Cottonseed oil contains three cyclopropyl fatty acids (or CPFAs) that may be less desirable for some whole seed feeding applications. Cotton contains several cyclopropane synthase (CPS) genes but it is currently unknown which of these contributes to CPFA production in seed oil. As part of Objective 1b, a series of gene promoters associated with CPS activity were identified. These regions were cloned and will be used for functional testing in cultured cotton roots and other model plant systems to identify which genes should be engineered to lower the level of seed CPFA acids. A relatively new gene knockout technique for making changes to specific sequences of DNA is being developed in pursuit of this objective and as part of other collaborative efforts. The Cotton Fiber Bioscience Research Unit (New Orleans, Louisiana) has produced one stable cotton transgenic plant using the components of this first-generation system. Ongoing collaborations between our two groups will continue to develop cotton lines that have stable, heritable gene knockout pedigrees that may lead to improved insect or drought resistance or seed property changes. As part of Objective 2, efforts were made to show that crude oils from glandless cottonseed would be easier to refine than crude oils from glanded seed. Using the traditional chemical refining technique, bleached oils from glandless seed were obtained with a Lovibond red value of 0.3, compared with a Lovibond red value of 2.5 that is normally acceptable for cottonseed oil. However, physically refining oils (without sodium hydroxide) resulted in higher red values (2.6) that would be borderline acceptable. Experiments with solvent-extracted oils are in progress but a similar trend is apparent. Deodorization of the oils is also planned and is part of a Cotton, Inc. reimbursable agreement that is discussed below. Work on the use of cottonseed protein as wood adhesives also continues as part of Objective 3. By adding small levels of additives to cottonseed protein isolate-based adhesives, improved adhesion was found. The addition of acidic amino acids or one of several phosphate compounds resulted in increased tensile strength and water resistance, suggesting that charge-charge interactions are important for adhesion. The addition of small amounts of protein denaturants or anionic polymers resulted in variable improvement. Washed cottonseed meal was also evaluated in some studies. Formulation pH, protein storage time, and the rheological properties of the formulations were evaluated. Electron micrographs were obtained to evaluate the structures of the wood-adhesive interface. As part of a recently developed CRADA agreement with Mississippi State University to develop bio-based boards with protein adhesives and termite inhibitory properties, our first plywood and particle boards were prepared with cottonseed protein isolate. While the properties of these boards are inferior to those prepared from petroleum-based adhesives, improvement is expected as many variables have not been optimized. Dispersion of the protein appears to become more difficult with increased scale of adhesion, i.e., getting a uniform covering of the wood particles with the adhesive formulation, and this problem will need further study. The properties of guayule plant resin, which is known to impede termite activity, are also being studied. Understanding how the viscosity is affected by temperature and its solubility in solvents is currently being determined to help formulate methods of application. Glandless cottonseed extracts were used to treat mouse macrophages and cancer cells and cytotoxicity and key gene expression of immunological responses were monitored as a component of Objective 4a. No toxicity was observed from the extracts, but cells treated with gossypol present in the glanded cottonseed extracts did show effects. Additionally, proteins were extracted and separated and the bioactivity of any novel proteins will be tested. Xylan, a natural polymer of xylose, has been isolated from cottonseed hulls and cotton burrs as part of Objective 5, and anionic and cationic derivatives of these polymers have been made. The properties of these derivatives have been determined, including rheological behavior and viscosity. When these preparations were applied to the surface of paper, paper strength was found to increase appreciably. In response to stakeholder issues, a few additional efforts were furthered. First, the Unified Methods Committee of the American Oil Chemists’ Society (AOCS) has approved an update of the gossypol-HPLC method (AOCS Ba 8-93) and has requested that we work with AOCS staff on an inter-laboratory study to upgrade the assay to an AOCS Official Method. Second, a three year survey of commercial cottonseed-fiber properties was completed this year. Seed-fiber ratio, seed index, the percentage of linter, hull, and kernel components and the oil, protein, and gossypol composition of the seed were studied to better understand the changes that have occurred to seed properties because of decades of intensive breeding for fiber yield. Seed-fiber ratio and seed index have decreased appreciably compared with early reports (before 1950), indicating that seed has become smaller with the breeding changes. Small differences in the percentage of linters were apparent, which may have occurred because of the combination of breeding and improved ginning. In contrast, the relative percentage of hulls, which might relate to weaker seeds that are currently an issue with ginners, does not appear to have been affected. Third, work on the use of cotton gin trash as a filler in polymer preparation, which is a collaborative effort with researchers at the National Center of Crops Utilization Research (Peoria, Illinois), is continuing. Progress report for Agreement 6054-41000-103-14R. The targeted gene knockout technology commonly known as “CRISPR” was incorporated into an existing gene overexpression/gene silencing toolbox. Promoter and terminator regulatory elements that drive activity of the two components of the system have been identified and shown to lead to reasonable rates of rapid, heritable gene knockouts, which is essential for breeding pure knockout lines within reasonable times. This system has been applied to model plant lines, and first-generation knockout lines targeting each of at least four different genes that might affect novel fatty acid production are being analyzed. Progress report for Agreement 6054-41000-103-11N. The opening and closing of rice florets takes 6 to 10 days. The regulation of this process is very important for hybrid rice seed production. However, little is known about the effects of plant hormones on this process. Previous studies have shown that the chemicals jasmonatic acid and methyl jasmonate promote rice floret opening. In this study, the effects of six plant hormones were investigated. Most of the treatments increased the percentages of open florets and increased the time that the floret were open. Progress report for Agreement 58-6054-7-0012. Construction of a laboratory-scale vegetable oil deodorizer has begun for studying cottonseed oil refining. An initial design has been prepared, and a glassblower from the Chemistry Department of the Louisiana State University in Baton Rouge, Louisiana, has been contracted to fabricate the three pieces of glassware needed. Two pieces are complete and the third piece is in preparation. The equipment for the refining and bleaching operation is in place and 10 gallons of pressed glandless cottonseed oil has been generated for the effort.

Accomplishments
1. Discovery of high oleic acid trait in the seed oil of previously breed cotton cultivars. ARS researchers in New Orleans, Louisiana, have seeds from recently identified and bred cotton varieties that have higher than normal levels of oleic acid in the seed oil. While higher levels of oil oleic acid are known in wild cotton germplasm, this finding should shorten the breeding time that is needed for developing cottonseed oils with elevated levels of oleic acid. This should help with the development of cottonseed oil to complete with other vegetable-based oils for some market applications. Oils with high levels of oleic acid are more stable in deep fat frying applications and are nutritionally more beneficial.

2. Use of carbohydrate polymers as promoters for cottonseed protein in wood adhesive application. There is ongoing interest in using agro-based materials (like proteins) in wood adhesives, but cost, adhesive strength, and water resistance are the major issues. ARS researchers in New Orleans, Louisiana, showed that the addition of a few specific carbohydrate polymers to cottonseed protein adhesive formulations increased the tensile strength of glued wood veneers by 50% and increased their water resistance by 30%. The improved performance should make cottonseed protein more competitive as an ingredient in wood adhesive formulations and it should lower the costs of formulations. The work will help develop cottonseed proteins as bio-based wood adhesives.

3. New interpenetrating polymer involving cottonseed protein. ARS researchers in New Orleans, Louisiana, have earlier shown cottonseed protein (CSP) to be a viable ingredient in wood adhesives. They now came up with a new approach to further improve the adhesive performance of CSP. This involved the in situ polymerization of a polyester in the presence of CSP. In an optimal case, the new CSP-derived polymer increased the dry tensile strength by 100% compared with CSP alone. The hot water resistance test was increased 50-70% increase over CSP alone. These new composite adhesive formulation may lead to new ideas and direction that further promote the use of agricultural products in value-added applications.

4. Xylan isolated and characterized from cotton byproducts. Hemicellulose, a common plant polymer, is an abundant material in nature but is underutilized. Xylan, a common form of hemicellulose was isolated from cotton burrs (outer boll husks) and cottonseed hulls. Anionic and cationic derivatives of these xylans were synthesized by ARS researchers in New Orleans, Louisiana. Whereas the viscous properties of anionic and cationic xylan by themselves were not interesting, when the cationic and anionic modified xylans were combined the solution the viscosity dropped indicating interaction between the two polymer forms. When these were applied to the surface of paper, the paper’s dry strength increased significantly. Thus, the combination of cationic/anionic xylan may have promising properties that should have industrial and product applications.

U.S. DEPARTMENT OF AGRICULTURE

Commodity Utilization Research: New Orleans, LA