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Aqueous Enzymatic Essential Oil
Aqueous Enzymatic Essential Oil
Problem: Corn oil is valuable but corn kernels only contain about 4% oil so it is usually too costly to obtain oil from corn kernels. Solution: Obtain corn oil from the part of the corn kernel that contains the most oil and that portion is The germ (embryo).
Germ
Tip Cap
OH
HO
Protein Fat
Starch Other
Protein Fat
Starch Other
Protein
Bench Scale Process for Aqueous Oil Extraction Studies (Modeled after Shi et al., 1998) Weigh duplicate 6 gram samples of dry germ into 50 ml polycarbonate centrifuge tubes Add 40 ml buffer, 0.05 M Na Acetate, pH 4.0 Grind mixture with a Polytron homogenizer Incubate in boiling water bath, 20 min Churn, at 65C for 20 hours, with tubes shaking horizontally at 160 rpm in a rotary incubator/shaker Cool tubes to room temperature Centrifuge at 2500 g (4000 rpm) for 60 min Remove top oil layer with a pipit
A Comparison of the Oil Yields Using the Aqueous Oil Extraction Protocol ______________________________________________ Oil Yield ______________________ Extraction Wt% Relative % ______________________________________________ Hexane extraction 42.7 2.0 100.0 (with homogenization) Aqueous extraction 15.3 0.4 36.6 1.1 (with homogenization) ______________________________________________
Oil Yields Using the Aqueous Oil Extraction Protocol (6 grams germ + 40 ml buffer)
Oil Yields Using Aqueous Enzymatic Oil Extraction Protocol (6 grams germ + 40 ml buffer + E)
Oil Yield = 80% compared to hexane extraction = 100%
0.5 ml Multifect GC
0.5 ml GC 220
A Comparison of the Oil Yields Using the Aqueous Enzymatic Oil Extraction Protocol, with Various Commercial Enzymes, Listed in Order from Highest to Lowest Oil Yields ___________________________________________________________ Enzyme Company Brand Name Oil Yield Relative% ___________________________________________________________ Cellulase Cellulase Cellulase Genencor Novozyme Genencor Multifect GC Celluclast 1.5L CG220 81.7 0.7 81.5 0.9 78.8 0.7
Xylanase Genencor Multifect Xylanase 65.6 1.4 Cellulase Sigma C 1794 64.6 3.4 Xylanase Sigma X 2753 54.0 0.1 Cellulase Calbiochem Cellulysin 43.5 2.7 Cellulase Sigma C 1184 9.6 2.2 Pectinase Calbiochem Macerase 34.9 1.3 Protease Genencor GC 106 33.3 0.8 Protease Novozyme Alcalase 32.3 1.4 Cellulase Novozyme Carezyme 29.9 8.1 No enzyme 27.3 7.3 ____________________________________________________________
Nonpolar lipid composition of corn oil obtained by hexane extraction versus aqueous enzymatic extraction (0.5 ml MCG) of oven dried corn germ. _________________________________________________________________ Lipid Class Hexane Extracted Aqueous Enz Extracted ___________________________________ Wt % of oil ________________________________________________________________ Triacylglycerols 97.100.01 97.950.77 Free Fatty Acids 1.520.11 1.010.07 Phytosterol fatty acyl esters 0.610.01 0.480.05 Free phytosterols 0.610.03 0.240.01 Hydroxycinnamate phytosterol esters 0.030.00 0.000.00 ________________________________________________________________
Moreau, R.A., D.B. Johnston, M.J. Powell, and K.B. Hicks, A Comparison of Commercial Enzymes for the Aqueous Enzymatic Extraction of Corn Oil from Corn Germ, J. Am. Oil. Chem. Soc. 81;1071-1075 (2004).
Enzyme
A model of the maize oil body. Note how the oleosins cover the outer surface of the membrane, shielding the phospholipids. Drawing courtesy of Dr. Anthony Huang
Dickey, L., Cooke, P.H., Kurantz, M.J., McAloon, A., Parris, N, and Moreau, R.A., Using microwave heating and microscopy to estimate optimal corn germ oil yield with a bench-scale press, JAOCS 84:489-495, 2007.
AEOE with New Generation Corn Germ from a Dry Grind Ethanol Plant
Dry-Fractionated Corn Germ Market FlexTM, CPC BFRAC, Poet Wet-Fractionated Corn Germ Quick Germ, U of IL E-Germ, ERRC and U of IL MOR-Frac PlusTM, Mor Technologies
Dry Grind Process for Ethanol using Corn (single co-product = DDGS)
A-AMYLASE CORN Cleaning Hammer Mill Slurry Mix W eigh Tank Liquefaction Cook Retention Tanks Saccharification AMMONIA LIME ACID G-AMYLASE
Fractionation = Front End Removal of Germ Via Dry Fractionation or Wet Fractionation
YEAST Degasser
Fermentors
Evaporation
AEOE with Dry Fractionated Corn Germ from a Dry Grind Ethanol Plant
Enzyme Oil Yield (%) (relative to hexane extraction) _________________________________________ Cellulase only 0 Micowaved Germ + Cellulase 52.3 3.2
AEOE with Wet Fractionated Corn Germ (E-Germ) from a Dry Grind Ethanol Plant
Enzyme Oil Yield (%) __________________(relative to hexane extraction) Cellulase only 0 Cellulase + Alkaline Protease (pH 8 buffer) 87.4
Dickey, L.C., M.J. Kurantz, N. Parris, A. McAloon, R.A. Moreau, Foam separation of oil from enzymatically treated wet-milled corn germ dispersions, J. Am. Oil Chem. Soc. 86, 927-932, 2009.
Degasser
Evaporation
Conclusions
A new aqueous enzymatic extraction process has been developed that results in oil yields of greater than 90% from wet milled corn germ or wet fractionated corn germ. germ (including E-Germ). A new aqueous enzymatic extraction process has been developed that results in oil yields of greater than 65% from dry milled corn germ or dry fractionated corn germ. Since several cellulase preparations appear to result in high oil yields, we anticipate that the new generation of celluloytic enzymes that are being developed for biomass hydrolysis and fermentation (Accellerase 1500, and DUET by Danisco/Genencor and Cellic CTec and CTec2 by Novozymes), will result in even higher oil yields and/or may be more economical to use than the current generation of cellulolytic enzymes. In addition to corn oil, it is envisioned that this process will also result in additional valuable protein and carbohydrate coproducts, the sales of which could lower the overall cost of the process.
Acknowledgments Corn Germ Bunge Cargill Cereal Process Technologies Enzymes Genencor Novozymes Corn Germ AEOE Mike Powell Mike Dahlmer Jhanel Wilson Fruit Seed AEOE Vera Van Hoed Camelina AEOE Chris Senske
Questions ?
Bench Scale Process for Aqueous Enzymatic Oil Extraction Studies (protocol Designed to Combine Some of the Features of Shi et al., 1998 and Karlovic et al., 1994) Weigh duplicate 6 gram samples of dry germ into 50 ml tubes Add 40 ml buffer, 0.05 M Na Acetate, pH 4.0 Grind mixture with a Polytron homogenizer Add enzyme Churn at 50C for 4 hours, with tubes shaking horizontally 160 rpm in a rotary incubator/ shaker Churn at 65C for an additional 16 hours shaking horizontally Cool tubes to room temperature Centrifuge at 2500 g (4000 rpm) for 10 min Remove top oil layer with a pipet
Scale up of the aqueous enzymatic extraction to 24 grams of wet milled germ, 160 ml buffer, and 2.0 ml enzyme. _________________________________________________________________ Enzyme Oil Yield ________________________________
Wt% of germ Relative%1 _________________________________________________________________ Multifect GC (82 GCU/gram) Celluclast 1.5L (790 EGU/gram) 38.2 0.9 37.3 1.4 93.2 2.2 91.1 3.5
Oil Yields Using 4X Scale-up of the Aqueous Enzymatic Oil Extraction Protocol (24 g germ + 160 ml buffer + E)
TABLE 2A A Comparison of the Oil Yields Using the Aqueous Enzymatic Oil Extraction Protocol, with Various Commercial Enzymes, Listed in Order from Highest to Lowest Oil Yields ___________________________________________________________ Enzyme Company Brand Name Oil Yield Relative% ___________________________________________________________ Cellulase Genencor Multifect GC 81.7 0.7 Cellulase Novozyme Celluclast 1.5L 81.5 0.9 Cellulase Genencor CG220 78.8 0.7 ____________________________________________________________
Moreau, R.A., D.B. Johnston, M.J. Powell, and K.B. Hicks, A Comparison of Commercial Enzymes for the Aqueous Enzymatic Extraction of Corn Oil from Corn Germ, J. Am. Oil. Chem. Soc. 81;1071-1075 (2004).