2009 Annual Report 1a.Objectives (from AD-416) To demonstrate the technical feasibility of using isolated enzymes in nonaqueous media to functionally modify vegetable oils on a scale suitable for commercial deployment. Optimize a pilot-scale system for non-aqueous enzymatic transformation of soybean oil to high value products with novel functional groups and properties, such as feruloylated monoacyl- and diacylglycerides, for food and non-food applications. Create a flexible, durable and environmentally benign platform technology for the bioelectrocatalytic transformation of plant triglycerides and phospholipids to higher-value products. 1b.Approach (from AD-416) Processes for producing modified plant lipids with novel and valuable functional properties are essential for developing new markets for commodity vegetable oils. Furthermore, these processes and products should have minimal adverse impact on the environment and pose no threat to plant operators and the surrounding community. It is the premise of this proposal that using isolated enzymes in selected nonaqueous media can meet these design goals. Two distinctly different approaches are considered. One approach, for enzymes not having complex cofactor requirements, employs soybean oil as a solvent as well as a reactant, while the other approach, for enzymes having more substantial structural and cofactor needs, places the enzyme in a highly structured environ and specifically tailored media. In the first approach, synthesis of a patented product is examined to optimize reaction variables for pre-commercial production. The second approach addresses several fundamental issues regarding ex vivo use of membrane-associated enzymes for the bioelectrocatalytic transformation of phospholipids and triglycerides. Taken together, new knowledge will be gained and useful processes will be developed that can deliver value-added vegetable oil derivatives for consumers and industry. 3.Progress Report Processes for improved production of a new skincare product were developed at the National Center for Agricultural Utilization Research (Peoria, IL). This work was conducted with the Biotechnology Research and Development Corporation (BRDC) under a Cooperative Research and Development Agreement (CRADA) in support of Project No. 3620-41000-126-00D, Biocatalytic Functionalization of Plant Lipids. Full pilot-scale production of one commercial product (Feruloyl Soy Glycerides) was successfully demonstrated. Several reactor configurations were examined. A reactor design capable of metric ton production with high efficacy was identified. Quality control measures were implemented for ongoing production and an end-user product validation certificate (Certificate of Analysis) was developed. Written Standard Operating Procedures were developed for operations in the pilot facility in collaboration with our commercialization partner (iSoyTechnologies Inc.). Third party clinical testing of the product for safety and efficacy was concluded. Project progress was discussed with CRADA partner representatives in frequent (daily or weekly) phone conversations and periodic on-site visits. Future products for commercial development were identified through discussions with BRDC, iSoyTechnologies and several end-user companies. All of these products will incorporate vegetable oils. 4.Accomplishments 1. LARGE SCALE PRODUCTION OF A POLYPHENOLIC, GLYCEROL-BASED SKIN CARE ACTIVE INGREDIENT. In response to the health and beauty-aid industry’s insatiable demand for natural and bio-based ingredients, ARS scientists at the National Center for Agricultural Utilization Research in Peoria, Illinois, developed a large scale, environmentally friendly process for making an all-natural, polyphenolic, glycerol-based compound suitable for use in consumer products. This compound possesses ultraviolet absorbing and antioxidative properties, and occurs naturally in wheat and rice bran in very small quantities. The ARS scientists, in collaboration with patent licensee and industrial collaborators, conducted metric ton scale productions of their patented, lipid-based cosmeceutical ingredient and discovered that, under specific process conditions, the polyphenolic glycerol-based compound is made as a byproduct. Research was conducted to optimize conditions to either suppress or maximize the glycerol-based product as desired. Analytical testing confirmed that the ultraviolet absorbing and antioxidant properties of the glycerol-based compound were comparable to petroleum-based active ingredients currently used in consumer products. The all natural, glycerol-based byproduct offers a water soluble alternative to the scientists’ patented lipid-based cosmeceutical ingredient currently used by the skin care industry. Developing such novel value-added uses for commodity crops and oils expands domestic market opportunities, which enhances the competitiveness and sustainability of rural and farm economies. 2. LIPIDS FORM ORGANIZED STRUCTURES IN ROOM-TEMPERATURE LIQUID SALTS. There is a need to process plant lipids in media that is more environmentally friendly than organic solvents. Room-temperature liquid salts are potentially such media. Typically, lipids do not organize into structures in organic solvents used to process the lipids. However, it was not clear what, if any, structures lipids would form in room-temperature liquid salts. To address this question, a study was conducted to determine how lipids organized in a room-temperature ionic liquid as one component of the liquid salt was varied. Results demonstrated that lipids formed several distinct structures in low concentration liquid salts. These fundamental results will be used by us and other scientists as a foundation to better understand how to process plant lipids in room-temperature liquid salt. 3. IMPROVED ENZYME IMMOBILIZATION PROCESS. The enzymatic conversion of vegetable oils to various new products, from biodiesel to sunscreens, is most readily conducted on a commercial scale with the employed enzyme affixed to an inert matrix (solid support). Lipases, which are often used to make these transformations, are usually attached to supports having a hydrophobic (water heating) surface. ARS scientists at the National Center for Agricultural Utilization Research in Peoria, Illinois, developed an improved procedure for allowing a lipase to optimally affix itself to its target hydrophobic support surface. The procedure ensures the enzyme establishes its proper, orderly orientation on the surface so that it may be in its most active and stable form. This accomplishment will provide a cost savings to manufacturers of vegetable oil derivatives. 6.Technology Transfer Number of Active CRADAs 1 Review Publications Laszlo, J.A., Evans, K.O. 2009. Influence of cosolvents on the hydrophobic surface immobilization topography of Candida antarctica lipase B. Journal of Molecular Catalysis B: Enzymatic. 58:169-174. Compton, D.L., Laszlo, J.A. 2009. 1,3-Diferuloyl-sn-glycerol from the biocatalytic transesterification of ethyl 4-hydroxy-3-methoxy cinnamic acid (ethyl ferulate) and soybean oil. Biotechnology Letters. 31(6):889-896.