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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Plant Polymer Research » Research » Research Project #428643

Research Project: Improved Utilization of Proteinaceous Crop Co-Products

Location: Plant Polymer Research

2018 Annual Report


Objectives
The long-term objective of this project is to develop novel products utilizing current and new co-products from the industrial processing of agricultural materials. As a result our research will reduce dependence on non-renewable materials and produce higher value products that will benefit a large segment of our economy. Objective 1: Enable, from a technological standpoint, the commercial production of marketable products from the proteins in crops such as pennycress, camelina, soybean, cottonseed or corn. Sub-objective 1A. Establish pilot-scale extraction and biorefining techniques that generate protein-rich industrial feedstocks from plant crops, such as pennycress, camelina, soybeans, or cottonseed. Sub-objective 1B. Determine ability to form solvent cast and melt processed films or articles, as well as surface and interfacial tension agents; determine if suitable chemical modifications of these proteins will provide products that can replace petroleum-based products.


Approach
Establish pilot-scale extraction and biorefining techniques that generate protein-rich industrial feedstocks from plant crops, such as pennycress, camelina, soybeans, or cottonseed. Determine ability to form solvent cast and melt processed films or articles, as well as surface and interfacial tension agents. Determine if suitable chemical modifications of these proteins will provide products that can replace petroleum-based products.


Progress Report
Significant progress was made on both objectives which fall under National Program 306: Quality and Utilization of Agricultural Products, specifically Component 2, addressing Objective 1.3 of the ARS Strategic Plan. Under Sub-Objective 1A of this research project, the effects of genetic modification for oil quality on camelina protein composition and extractability, compared with wild-type camelina was evaluated. Protein was extracted using the base solubilization/acid precipitation route. The genetically modified camelina provided twice as much protein isolate than the wild camelina and had greater purity. However, protein yield from both sources was very low. To increase yield and improve protein purity, the method was modified by using single-stage extraction with dilute base and replacing the step of re-dissolving the precipitate with simple water-washing. Using these changes, protein extraction from wild camelina increased 10-fold and protein purity increased from 67 to 80%. For genetically modified camelina, protein isolate yield increased 5-fold and protein purity increased from 78 to 86%. Higher amounts of protein was extracted from press cake of the modified camelina versus wild camelina. Protein isolate from the modified camelina had identical solubility as wild camelina protein isolate from pH 2 to pH 7 but was more soluble of alkaline pHs. Under Sub-Objective 1A of this research project, additional modifications and replicate runs were performed using the pilot scale saline extraction process of protein from pennycress press cake. The two modifications made to the saline-based pilot-scale (5 kg) process for producing pennycress protein isolate (PPI) were: (a) only single-stage saline extraction followed by ultrafiltration-diafiltration, and (b) water-washing the spent solids. Incorporating these changes allowed the production of 0.5 kg of high-purity (92.0% protein content) protein isolate (PPI-saline). The product purity and protein extraction efficiency (49%) are similar to that of the conventional method used in industry. The PPI-saline from the replicate extractions showed identical electrophoretic band patterns demonstrating good reproducibility. The PPI-saline was found to be more soluble in acidic and alkaline solutions than at neutral pH. The PPI-saline was also far more soluble than the acid-precipitated PPI (PPI-acid) at all tested pHs. Under Sub-Objective 1B of this research project, this high purity PPI-saline was used to prepare films. Colored transparent films were prepared using formic acid as solvent and glycerol as the preferred plasticizer. Surprisingly, the PPI-saline protein could produce solutions having high (> 30%) polymer solids; much higher than that obtained using the PPI-acid. The PPI-saline film surface was free from defects as determined using scanning electron microscopy, and were chemically homogenous when they were analyzed using microscopic infrared spectroscopy. The films produced had tensile strength similar to other oilseed proteins, but significantly lower than films made from PPI-acid. Quality films could be made from blends of PPI-saline and corn protein. However, as the amount of PPI-saline was increased, the strength of the resulting films decreased commensurate with the amount of PPI added. Chemical derivitization will be required in order to improve the properties of PPI based films. Preliminary electrospinning tests using PPI were begun. Fibers could be made from PPI-saline protein using formic acid as the spinning solvent. The fibers were predominantly ribbon fibers having diameters from 1 to 3 micrometers. The use of protein and starch derivatives have been studied to protect high value crops from various pathogenic insects and microbes. Antimicrobial polymers are known and are often naturally occurring amphiphilic and cationic antimicrobial peptides. When amylose-inclusion complexes (AIC) are formed using amine salts, such as N-hexadecylammonium chloride, as the ligand, the AIC becomes cationic, amphiphilic, and water soluble. These amine salt AIC’s were found to be effective antimicrobial agents against gram(+) bacteria (some effect against gram(-) bacteria), yeast, fungi, and mold at concentrations comparable to commercial products. The AIC were blended with polyvinyl alcohol (PVOH) and used as a treatment for potato dry rot (a pathogenic fungus). The treatment eliminated the fungal infection and formed a seal at the wound site until the potato could heal naturally. When AIC, PVOH and cedar oil is applied to wood, the wood not only resists dry rot but also kills termites. Preliminary research suggests the AIC may be relatively safe as they showed no apparent damage to plant tissues, and no lysis of mammalian blood cells or liposomes composed of zwitterionic phospholipids. With these results, a U.S. Patent Application was filed and outgoing Material Transfer Agreements have been executed. Amylose inclusion complexes (AIC) formed from readily available inexpensive food grade ingredients, fatty acid salts and corn starch, were found to be effective emulsifiers comparable to commercial emulsifiers. Modified starches, such as octenyl succinic anhydride (OSA) modified starch, are widely used as emulsifiers, but the amount used is limited by the Food and Drug Administration regulations. The AIC, which are physical mixtures and made from food grade constituents, were found to be water-soluble surface-active nanoparticles capable of forming emulsions with properties superior to those of the known emulsifiers such as OSA modified starch, soy protein or sodium lauryl sulfate. The emulsification performance of the AIC is impacted by the length of the fatty acid salt; longer chain length versions give improved performance. The fatty acid salt AIC exhibits excellent emulsion characteristics in neutral and alkaline pH solutions. Outgoing Material Transfer Agreement’s have been executed using these reagents. Corn samples provided by a commercial partner under a collaborative research agreement were tested using the protein extraction method developed in this lab. The extracts were highly colored and the proteins present were only sparingly soluble in water, saline, ethanol, and alkali, indicating protein loss or denaturation took place during corn processing. This finding is supported by molecular weight analysis using gel electrophoresis, which showed loss of some proteins and the presence of high molecular weight proteins. Properties of proteins in pulse seeds before and after sprouting was undertaken using navy beans, pinto beans, and lentils. Standard compositional and performance tests were carried out on these samples to define their value and how it may change as the plant grows. The proximate composition of the beans and lentils did not change with germination. The solubility of the proteins from sprouted flours increased significantly at pH 2 (navy beans) and pH 8.5 (pinto beans). Sprouting had no effect on foaming properties of proteins in pulse flours. Sprouting improved the emulsifying activity and emulsion stability of the pulse proteins. Sprouting had a detrimental effect on heat coagulability of the pulse proteins.


Accomplishments
1. Biobased safe pesticidal starch complexes. There is a constant need for new compounds that can control pests, and most of these compounds utilize petroleum-based hazardous chemicals. ARS scientists in Peoria, Illinois, have found that certain corn starch based compounds are very effective at controlling a variety of pests including: gram(+) bacteria, yeast, mold, fungi and insects. These starch compounds are complexes, where the starch wraps around the active agent. The active agent being used is a vegetable oil derivative. The complexes are fairly safe – they do not reduce plant yields nor do they break red blood cells. When incorporated into films or applied to cellulosic articles, the pesticidal activity remains. The resulting antimicrobial film may be of use in settings where infection (hospitals, homes, etc.) are a concern. Antimicrobial bandages may also use this technology; given that the agent is a polymer, it is less likely to cause allergic reactions. A U.S. Patent Application has been filed covering this technology. This technology will be able to replace techniques which utilize hazardous chemicals and processes, allowing for improved pesticidal/antimicrobial products which will be less expensive and have a smaller carbon footprint. These new products will result in new applications for corn starch, benefiting producers, processors, and, ultimately, the consumer.

2. Improved water resistant paper and cotton articles and wood. There are numerous routes to produce water resistant paper or cotton fabrics and wood, however, these approaches typically use petroleum-based hazardous chemicals. ARS scientists in Peoria, Illinois, have found that cellulosic articles, such as paper or cotton fabrics and wood, can have dramatically increased water resistance by applying certain starch complexes to them. The degree of improvement is such that an applied water droplet may evaporate before soaking into the article. A U.S. Patent Application has been filed covering this technology. This technology will be able to replace techniques which utilize hazardous chemicals and processes, allowing for water resistant paper or cotton articles and wood which will be less expensive and have a smaller carbon footprint. These new products will result in new applications for corn starch and allow new markets for paper and cotton articles, benefiting producers and processors.

3. Pilot-scale production of pennycress protein isolate. Pennycress is a winter annual crop whose seed oil is being developed as a biodiesel source, but other components in the seed must also be utilized to maximize the value of pennycress. ARS scientists in Peoria, Illinois, have determined the conditions for pilot-scale salt-based extraction and purification of protein pennycress. The process can be easily used by industry. The process produced 0.5 kg of high purity protein isolate (92% protein content) which can be used by internal scientists or researchers from other institutions. The extraction efficiency and protein purity are similar to that of conventional industrial processes. The isolated pennycress protein was highly soluble in water, expanding its use in various applications. Additionally, the pennycress protein was found to be effective at stabilizing vanilla extracts, particularly when combined with ARS produced gums. Producing larger amounts of high purity pennycress protein allows for conversion into more valuable products, and testing by external labs. These efforts will result in increased value of pennycress crop and generates additional revenue streams for farmers and downstream processors.


Review Publications
Hay, W.T., Fanta, G.F., Peterson, S.C., Thomas, A.J., Utt, K.D., Walsh, K.A., Boddu, V.M., Selling, G.W. 2018. Improved hydroxypropyl methylcellulose (HPMC) films through incorporation of amylose-sodium palmitate inclusion complexes. Carbohydrate Polymers. 188:76-84.
Selling, G.W., Hojilla-Evangelista, M.P., Hay, W.T., Utt, K.D., Grose, G.D. 2018. Preparation and properties of solution cast films from pennycress protein isolate. Journal of the American Oil Chemists' Society. 95:1091-1103. http://dx.doi.org/10.1002/aocs.12034.
Hay, W.T., Vaughn, S.F., Byars, J.A., Selling, G.W., Holthaus, D.M., Price, N.P. 2017. Physical, rheological, functional and film properties of a novel emulsifier: Frost grape polysaccharide (FGP) from Vitis riparia Michx. Journal of Agricultural and Food Chemistry. 65(39):8754-8762. https://doi.org/10.1002/aocs.12034.
Eller, F.J., Hay, W.T., Kirker, G.T., Mankowski, M.E., Selling, G.W. 2018. Hexadecyl ammonium chloride amylose inclusion complex to emulsify cedarwood oil and treat wood against termites and wood-decay fungi. International Biodeterioration and Biodegradation. 129:95-101.
Hojilla-Evangelista, M.P., Sutivisedsak, N., Evangelista, R.L., Cheng, H.N., Biswas, A. 2018. Composition and functional properties of saline-soluble protein concentrates prepared from four common dry beans (Phaseolus vulgaris L.). Journal of the American Oil Chemists' Society. 95:1001-1012. doi: 10.1002/aocs.12135.
Xu, J., Selling, G.W. 2017. A comparison of the viscoelastic properties of starch-polyacrylamide graft copolymers produced in dimethyl sulfoxide and water. Rheology. 1(2):1000109.