Development of a Green Aqueous Enzymatic Process to Extract Corn Oil from Corn germ

Robert A. Moreau, David B. Johnston, Leland Dickey and Kevin B. Hicks

SUSTAINABLE BIOFUELS AND COPRODUCTS RESEARCH UNIT Eastern Regional Research Center, ARS, USDA 600 E. Mermaid Lane, Wyndmoor PA 19038 Robert.moreau@ars.usda.gov

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).

Structure of the Corn Kernel

Endosperm Pericarp (Bran)
(~90 % of the total oil)

Germ

Tip Cap

Conventional Corn (germ) Oil

History The corn oil industry began >100 years ago Corn germ is one of three common coproducts of the corn wet milling industry developed to extract pure corn starch. Corn oil is obtained by hexane extraction and/or pressing of the oil-rich germ (embryo) portion of the corn kernel (95% from WMCG). Corn oil is currently the # 11 edible oil in the world with an annual production of 2.5 MT (#1 soybean oil 33.6 MMT and #2 palm oil 31.4 MMT).

Conventional Corn (germ) Oil

Chemical Composition (crude, unrefined) Triacylglycerols Diacylglycerols Free Fatty Acids Phytosterols Vitamin E* Carotenoids Phospholipids wt% ~97 ~0.5 1-2 ~1 0.1 0.001 Lutein ~1
HO
O O O O O O
O O

OH

HO

Protein Fat

10% 4% 70% 16%

Corn whole kernels

Starch Other

Protein Fat

14% 40% 9% 37%

Corn Germ from a commercial wet mill

(source of 90% of corn oil)

Starch Other

Protein

16% 20% 20% 44%

Corn Germ from a commercial dry mill

(source of 10% of corn oil)

Fat Starch Other

Methods for the Obtaining Corn Oil from Corn germ

Method Hexane Extraction Pressing Pre-Pressing + Hexane Supercritical CO2 Extraction Propane/ Liquefied Petroleum Extraction Ethanol Extraction Aqueous Extraction Aqueous Enzymatic Extraction Advantage High oil yields Safe, simple High oil yields Good oil yields, safe Good oil yields, low pressures Semi-green green green Disadvantage Health and safety issues and associated costs Lower oil yields, high energy use Health and safety issues and associated costs Higher cost than hexane, high pressures Higher cost than hexane, safety issues Higher cost to remove solvent from meal Very low oil yields High cost of enzymes

Aqueous Extraction with Wet Milled Corn Germ (factory dried)

Objective 1: Evaluate a published aqueous method to extract corn oil from corn germ.
Shi, L., J. Lu, G. Jones, P.A. Loretan, and W.A. Hill, Characteristics and Composition of Peanut Oil Prepared by an Aqueous Extraction Method, Life Support & Bioscience 5:225 (1998).

Results published in 1998, - with peanuts = 80% yield of peanut oil

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 Yield = 36.6% compared to hexane extraction = 100%

Aqueous Enzymatic Extraction with Wet Milled Corn Germ

Objective 2: Evaluate a published aqueous enzymatic method to extract corn oil from corn germ. AND improve its oil yields. Bocevska, M., D. Karlovic, J. Turkulov, and D. Pericin, Quality of Corn Oil Obtained by Aqueous Enzyme Extraction, JAOCS 70:1273 (1993). Karlovic, D.J., M. Bocevska, J. Jakolevic, and J. Turkulov, Corn Germ Oil Extraction by a new Enzymatic Process, Acta Alimentaria 23:389 (1994). Results - published in 1993, - with dry milled corn germ = 80% yield of corn oil

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).

AEOE with Raw (Undried) Wet Milled Corn Germ

_________________________________________________________________

Enzyme

Oil Yield (relative to hexane extraction) __________________________________________ Cellulase only 0

Cellulase + 80.9 3.5 Alkaline Protease (pH 8 with buffers) __________________________________________

Ultrastructure of oil-containing organelles in corn germ

Hypothesis - proteases may release oil by hydrolyzing oleosins, structural proteins in the oil body membrane Oleosin proteins

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

AEOE with Dry Milled Corn Germ

Enzyme Oil Yield __________________(relative to hexane extraction) Cellulase only 0 Boiled Germ + Cellulase Microwaved Germ + Cellulase 42.6 2.3 56.5 6.3

Cellulase + 63.6 4.1 Alkaline Protease (pH 8 with buffer)

Moreau, R.A., Dickey, L.C., Johnston, D.B., and Hicks, K.B., A process for the aqueous enzymatic extraction of corn oil from dry milled corn germ and enzymatic wet milled corn germ (E-germ), J. Am. Oil Chem. Soc. 86: 469-474, 2009.

Effect of Cooking on Corn Germ Ultrastructure

a control, uncooked corn germ; b corn germ cooked in a convection oven at 180 C for 6.5 min; c corn germ cooked in a 1,500-W microwave oven for 4.5 min
Fig. 4 Effect of heating on the ultrastructure of dry-milled corn germ A as studied with confocal (100 m), light (25 m) and transmission electron (2.5 m) microscopy.

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

CO2 W ATER DENATURANT

ETHANOL CO2 Scrubber Molecular Sieves

Fermentors

Rectifier Stripping Beer Column

Beer Degas Vent Condenser

Thin Stillage Centrifuge

Evaporation

DDGS W et DDGS Conveyor DDGS Dryer

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

Cellulase + 65.6 2.5 Alkaline Protease (pH 8, with buffer) __________________________________________

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

Cellulase + 79.5 Alkaline Protease (pH 8 w/o buffer)

Moreau, R.A., Dickey, L.C., Johnston, D.B., and Hicks, K.B., A process for the aqueous enzymatic extraction of corn oil from dry milled corn germ and enzymatic wet milled corn germ (E-germ), J. Am. Oil Chem. Soc. 86: 469-474, 2009.

AEOE with oil enrichment via a bubble column

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.

AEOE with oil enrichment via a bubble column

Using this approach with wet milled corn germ, we have been able to enrich the oil in the foam about 4-fold, so that only about 25% of the original volume of AEOE solution then needs to be centrifuged to float the free oil. It is anticipated that adding this bubble column approach will reduce the cost of AEOE from corn germ by reducing the centrifugation costs.
Dickey, L.C., M.J. Kurantz, P Cooke, N Parris, R.A. Moreau, Separation of buoyant particles from an aqueous dispersion of corn germ particles using a bubble column, Chem Eng Sci 63:4555-4560, 2008.

AEOE with Cranberry and Raspberry Seeds

Experiments conducted at ERRC in May-June 2010 by Dr. Vera Van Hoed from U of Ghent Red Raspberry = 15% oil by hexane extraction Cranberry = 21% oil by hexane extraction Using AEOE we were able to extract 34% of the hexane-extractable oil from Red Raspberry seeds. We were unable to obtain any oil from Cranberry using AEOE.
Van Hoed, N. De Clercq, C. Echim, M. Andjelkovic, E. Leber, K. Dewettinck & R. Verh. Berry seeds: a source of specialty oils with high nutritional value. Journal of Food Lipids, 2009, 16(1), 33-49. (cold pressed berry oils)

AEOE with Camelina Seeds

Experiments conducted at ERRC in June-July 2010 by Mr. Christopher Senske, Ursinus U Camelina = 28% oil by hexane extraction

Using AEOE we were able to extract 60% of the hexane-extractable oil.

Dry Grind Process for Ethanol using Corn

A-AMYLASE CORN Cleaning Hammer Mill Slurry Mix W eigh Tank Liquefaction Cook Retention Tanks Saccharification AMMONIA LIME ACID G-AMYLASE

CO2 W ATER YEAST DENATURANT

ETHANOL CO2 Scrubber Fermentors Molecular Sieves

Rectifier Stripping Beer Column Beer Degas Vent Condenser

Degasser

Back End Removal of Corn Oil

Thin Stillage Centrifuge

Evaporation

DDGS W et DDGS Conveyor DDGS Dryer

Post Fermentation Corn Oil

Obtained from the back end of a dry grind ethanol plant Proprietary process developed by GreenShift Corporation Obtained by centrifugation/condensation of the thin stillage The oil contains 10-15% free fatty acids. Their removal is costly and eliminates edible applications but are OK for biodiesel and other industrial applications. ~1% of commercial corn oil in US Rich in Lutein and Zeaxanthin (valuable for feed applications) Lutein and zeaxanthin, 200x higher in post fermentation corn oil than in corn germ oil

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

Thanks to for inviting us to present our USDA-ARS research results

Questions ?

Conventional Corn (germ) Oil

Chemical Composition Crude wt% ~97 ~0.5 1-2 ~1 0.1 0.0002 ~1 RBD ~99 ~0.5 0 0.7 0.07 0.0001 0

Triacylglycerols Diacylglycerols Free Fatty Acids Phytosterols Vitamin E* Carotenoids Phospholipids

refining deodorization deodorization bleaching degumming

Conventional Corn (germ) Oil

Applications: Edible corn oil, margarine, etc, corn oil fatty acids, high linoleate, like, soy+peanut Nonedible oleochemicals etc Value: unrefined $/lb Historically corn oil has been considered to Soybean 0.34 be a premium oil sold for a premium price Corn Oil 0.39 Today, it sells for a 5 cent Cottonseed 0.40 premium over soybean oil Canola 0.40 Possible reason for the relative loss in value consumer preference for high monounsaturate Sunflower 0.56 oils (olive, canola, sunflower oil (NuSun) and oils with more omega 3 fatty acids Peanut 0.68
Source, Economic Research Service, USDA, June 2010

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

_________________________________________________________________ 1 Oil Yield relative to hexane extraction (see Table 1)

Oil Yields Using 4X Scale-up of the Aqueous Enzymatic Oil Extraction Protocol (24 g germ + 160 ml buffer + E)

Oil Yield = 90% compared to hexane extraction = 100%

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).