2008 Annual Report 1a.Objectives (from AD-416) Develop new environmentally safe aqueous/enzymatic processes to extract the edible oil from corn germ (obtained from new-generation dry-grind corn-to-ethanol plants) and develop processes to fractionate the de-oiled germ into value-added protein and carbohydrate coproducts, to improve the overall economics of making fuel ethanol in new-generation plants. 1b.Approach (from AD-416) Corn germ from several new generation processes will be treated with mechanical (various forms of milling, homogenization and pressing), chemical (e.g. pH adjustment) and various enzymatic treatments, with the goal of causing the oil to coalesce and float upon centrifugation or other means of separation. 3.Progress Report Developed a Column for Corn Oil Separation after Aqueous Enzymatic Extraction – described in accomplishment below. Developed an Aqueous Enzymatic Oil Extraction Process for Enzymatically Wet Milled Corn Germ - described in accomplishment below. Identified two unique peptides from corn germ - During the last year we identified and sequenced two peptides (one with eight amino acids and one with fourteen) from protease-treated corn germ that appear to have unique biological activities. Both peptides inhibit Angiotensin Converting Enzyme (ASE) and one or both may have antihypertensive and other applications. Collaborative studies with the ERRC Proteomics Unit have revealed that the amino acid sequence of both peptides are identical to regions in the sequences of two proteins that are localized in the corn embryo (germ), globulin 2 and oleosin. Further studies are being conducted to characterize these peptides and to evaluate possible applications for them. Research was conducted under a CRADA (58-3K95-5-1087) between ARS and the Illinois Corn Marketing Board which was initiated on 01/01/05 and will terminate on 12/31/08. During the last year researchers at ERRC continued to compare the composition of conventional corn oil (corn oil obtained by pressing and/or hexane extraction of corn germ) versus corn oil obtained by extracting ground corn with ethanol. The analyses focused on comparing the levels of xanthophylls (lutein and zeaxanthin) in the two oils. Various fractionation methods were evaluated by the CRADA partner and the samples generated were analyzed at ERRC. A research publication (Moreau et al, 2008, J Am Oil Chem Soc 84:1039-1044) describing some of the results was recently published. During the year numerous telephone and e-mail messages were exchanged to monitor progress of the collaboration. Samples of ethanol extracted corn oil and other fractions were produced by the CRADA partner and analyzed at ERRC. On July 29-31, 2007, the ARS cooperator traveled with the CRADA partner to the International Food Technology Annual Meeting in Chicago, IL, to meet with prospective companies to discuss technology transfer options. This research responds to Component 3 Biorefining (Problem Statement 3a4: Biorefinery co-products) from the National Program 213: Bioenergy Action Plan because it identifies new high value biorefinery coproducts. 4.Accomplishments 1. Developed a Column for Corn Oil Separation after Aqueous Enzymatic Extraction -There is a need for a new separation technology to reduce the cost of obtaining valuable corn oil from corn germ that is removed before fermentation of corn to make fuel ethanol. Aqueous extraction will be a relatively inexpensive method of separating oil from corn germ if the centrifugation volume can be minimized. A pilot column/heat bath/stirred pot system was developed and used to show that 70-80% of the oil in corn germ could be collected with a foam overflow. The foam overflow was only 1/4 the mass of the original dispersion and thus will require only 1/4 of the centrifugation to collect the oil compared to centrifugation of the entire germ dispersion. Adding this new corn oil separation technology to a typical 50 million gallon per year corn-to-ethanol plant could reduce the annual operating costs by about $1.2 million, compared to a process that employed only centrifugation. There are more than 100 of these plants now with another 100 or more in various stages of planning and this corn germ and corn oil separation technology could potentially be added to some or all of them. This research supports the ARS National Program 307, Bioenergy and Energy Alternatives, Component 1, Ethanol Coproduct Development. 2. Developed an Aqueous Enzymatic Oil Extraction Process for Enzymatically Wet Milled Corn Germ - There is a need for a new on-site enzymatic process to reduce the cost of obtaining valuable corn oil from corn germ that is removed before fermentation of corn to make fuel ethanol. One of the ways that corn germ can be removed from the corn kernel before fermentation is to remove it via a new process called enzymatic wet milling. During the last year we identified an appropriate combination of commercial enzymes (a combination of a cellulase and a protease) and developed a process which allowed us to achieve oil yields of 80-90% from several samples of enzymatically wet milled corn germ. This new aqueous enzymatic oil extraction process, when combined with enzymatic germ removal, may provide a way for ethanol plants to produce a new valuable coproduct on-site, without the use of hexane or other hazardous solvents. Edible corn oil is currently selling for record high prices (~$0.50/pound), so adding this enzymatic corn oil extraction technology to a typical 50 million gallon per year corn-to-ethanol plant could increase revenues by about 20 million dollars.During the last twelve months the price of a bushel of corn and the price of corn oil have both increased more than 100%, and the price of the current coproduct, DDGS, has not kept pace, so there is an urgent need to develop economical new processes to produce corn oil as a coproduct if dry grind plants are to retain their profitability. This research supports the ARS National Program 307, Bioenergy and Energy Alternatives, Component 1, Ethanol Coproduct Development. Goal “To develop higher-valued new coproducts or improve the existing ones from the non-starch portion of the grain, resulting in a reduced net cost of the starch used for producing ethanol.” 5.Significant Activities that Support Special Target Populations NONE 6.Technology Transfer Number of Active CRADAs 1 Number of Invention Disclosures Submitted 1 Number of New Commercial Licenses Executed 3 Review Publications Moreau, R.A., Johnston, D., Hicks, K.B. 2007. A comparison of the levels of lutein and zeaxanthin in corn germ oil, corn fiber oil and corn kernel oil. Journal of the American Oil Chemists' Society 84:1039-1044. Srinivasan, R., Singh, V., Moreau, R.A., Rausch, K.D.,Tumbleson, M.E., Singh, V. 2007. Phytosterol distribution in fractions obtained from processing of distillers dried grains with solubles using sieving and elutriation. Cereal Chemistry 84:626-639. Dickey, L.C., Kurantz, M.J., Parris, N. Oil separation from wet milled corn germ dispersions by aqueous oil extraction and aqueous enzymatic oil extraction. 2008. Industrial Crops and Products 27(3):303-307. Sharma, V., Moreau, R.A., Singh, V. 2008. Increasing the value of hominy feed as a coproduct by fermentation. Applied Biochemistry and Biotechnology. 149:145-153. Nystrom, L., Moreau, R.A., Lampi, A., Hicks, K.B., Piironen, V. 2008. Enzymatic hydrolysis of steryl ferulates and steryl glycosides. European Food Research and Technology. 227:727-733. Moreau, R.A., Doehlert, D.C., Welti, R., Isaac, G., Roth, M., Tamura, P., Nunez, A. 2008. The identification of mono-, di-, tri-, and tetragalactosyl-diacylglycerols and their natural estolides in oat kernels. Lipids. 43:533-548. Parris, N., Moreau, R.A., Johnston, D., Dickey, L.C., Rotimi, A.E. 2008. Angiotensin I Converting enzyme inhibitory peptides from commercial wet- and dry-milled corn germ. Journal of Agricultural and Food Chemistry. 56(8):2620-2623.