aqueous enzymatic essential oil
TRANSCRIPT
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Development of a Green AqueousEnzymatic Process to Extract Corn Oil
from Corn germRobert A. Moreau, David B. Johnston,
Leland Dickey and Kevin B. Hicks
SUSTAINABLE BIOFUELS AND COPRODUCTS RESEARCH UNITEastern Regional Research Center, ARS, USDA
600 E. Mermaid Lane, Wyndmoor PA 19038
mailto:[email protected]:[email protected] -
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Problem: Corn oil isvaluable but cornkernels only containabout 4% oil so it isusually too costly toobtain oil from cornkernels.
Solution: Obtain cornoil from the part ofthe corn kernel thatcontains the most oiland that portion is
The germ (embryo).
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Structure of the Corn Kernel
Tip Cap
Germ
Pericarp(Bran)
Endosperm
(~90 % of the total oil)
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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 toextract pure corn starch.
Corn oil is obtained by hexane extractionand/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 (#1soybean oil 33.6 MMT and #2 palm oil 31.4MMT).
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Chemical Composition(crude, unrefined)
wt%
Triacylglycerols ~97Diacylglycerols ~0.5Free Fatty Acids 1-2
Phytosterols ~1Vitamin E* 0.1Carotenoids 0.001
Phospholipids ~1
Conventional Corn (germ) Oil
O
O
O
O
O
O
HO
O
O
O
O
OH
HO
O
Lutei n
http://upload.wikimedia.org/wikipedia/commons/2/20/Luteine_-_Lutein.svghttp://upload.wikimedia.org/wikipedia/commons/2/20/Luteine_-_Lutein.svg -
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Corn whole kernels
Protein 10%
Fat 4%
Starch 70%
Other 16%
Corn Germ from acommercial wet mill (source of 90% of corn oil)
Corn Germ from acommercial dry mill (source of 10% of corn oil)
Protein 16%
Fat 20%
Starch 20%
Other 44%
Protein 14%
Fat 40%
Starch 9%
Other 37%
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Methods for the Obtaining CornOil from Corn germ
Method Advantage Disadvantage
Hexane Extraction High oil yields Health and safety issuesand associated costs
Pressing Safe, simple Lower oil yields, highenergy use
Pre-Pressing + Hexane High oil yields Health and safety issuesand associated costs
Supercritical CO 2Extraction
Good oil yields, safe Higher cost thanhexane, high pressures
Propane/ LiquefiedPetroleum Extraction
Good oil yields, lowpressures
Higher cost thanhexane, safety issues
Ethanol Extraction Semi-green Higher cost to removesolvent from meal
Aqueous Extraction green Very low oil yields
Aqueous EnzymaticExtraction
green High cost of enzymes
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Aqueous Extraction with WetMilled Corn Germ (factory dried)
Shi, L., J. Lu, G. Jones, P.A. Loretan, and W.A. Hill, Characteristicsand Composition of Peanut Oil Prepared by an Aqueous ExtractionMethod, Life Support & Bioscience 5 :225 (1998).
Objective 1: Evaluate a published aqueous methodto extract corn oil from corn germ.
Results published in 1998,- with peanuts = 80% yield of peanut oil
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Bench Scale Process for Aqueous Oil ExtractionStudies (Modeled after Shi et al. , 1998)
Weigh duplicate 6 gram samples of dry germ into50 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 rotaryincubator/shaker
Cool tubes to room temperature Centrifuge at 2500 g (4000 rpm) for 60 min Remove top oil layer with a pipit
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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)
______________________________________________
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Oil Yields Using the Aqueous Oil Extraction Protocol(6 grams germ + 40 ml buffer)
Oil Yield = 36.6%compared tohexane extraction= 100%
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Bocevska, M., D. Karlovic, J. Turkulov, and D. Pericin,Quality of Corn Oil Obtained by Aqueous EnzymeExtraction, 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).
Objective 2: Evaluate a published aqueousenzymatic method to extract corn oil from corn germ.
Results - published in 1993,- with dry milled corn germ = 80% yield of corn oil
AND improve its oil yields.
Aqueous Enzymatic Extractionwith Wet Milled Corn Germ
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0.5 ml Multifect GC 0.5 ml GC 220
Oil Yield = 80%compared tohexane extraction= 100%
Oil Yields Using Aqueous Enzymatic Oil ExtractionProtocol (6 grams germ + 40 ml buffer + E)
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A Comparison of the Oil Yields Using the Aqueous Enzymatic OilExtraction Protocol, wi th Various Commercial Enzymes, Listed inOrder f rom Highest to Lowest Oil Yields
___________________________________________________________
Enzyme Company Brand Name Oil YieldRelative%
___________________________________________________________
Cellulase Genencor Multifect GC 81.7 0.7Cellulase Novozyme Celluclast 1.5L 81.5 0.9Cellulase Genencor CG220 78.8 0.7
Xylanase Genencor Multifect Xylanase 65.6 1.4Cellulase Sigma C 1794 64.6 3.4Xylanase Sigma X 2753 54.0 0.1Cellulase Calbiochem Cellulysin 43.5 2.7Cellulase Sigma C 1184 9.6 2.2Pectinase Calbiochem Macerase 34.9 1.3Protease Genencor GC 106 33.3 0.8Protease Novozyme Alcalase 32.3 1.4Cellulase Novozyme Carezyme 29.9 8.1No enzyme 27.3 7.3
____________________________________________________________
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Nonpolar lipid compos ition of corn oi l obtained by hexane extraction versusaqueous enzymatic extraction (0.5 ml MCG) of oven dr ied corn germ.
_________________________________________________________________Lipid Class Hexane Extracted Aqueous Enz Extracted
___________________________________Wt % of oil
________________________________________________________________Triacylglycerols 97.10 0.01 97.95 0.77Free Fatty Acids 1.52 0.11 1.01 0.07
Phytosterol fatty acyl esters 0.61 0.01 0.48 0.05Free phytosterols 0.61 0.03 0.24 0.01Hydroxycinnamate phytosterol esters 0.03 0.00 0.00 0.00
________________________________________________________________
Moreau, R.A., D.B. Johnston, M.J. Powell, and K.B. Hicks, A Comparison ofCommercial Enzymes for the Aqueous Enzymatic Extraction of Corn Oil from CornGerm, J. Am. Oil. Chem. Soc. 81;1071-1075 (2004).
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AEOE with Raw (Undried) WetMilled Corn Germ
_________________________________________________________________
Enzyme Oil Yield
(relative to hexane extraction) __________________________________________
Cellulase only 0
Cellulase + 80.9 3.5 Alkaline Protease (pH 8 with buffers)
__________________________________________
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Ultrastructure of oil-containingorganelles in corn germ
A model of the maize oil body. Notehow the oleosins cover the outersurface of the membrane, shieldingthe phospholipids. Drawing
courtesy of Dr. Anthony Huang
Hypothesis - proteasesmay release oil byhydrolyzing oleosins,structural proteins inthe oil body membrane
Oleosin proteins
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AEOE with Dry Milled Corn Germ Enzyme Oil Yield
__________________(relative to hexane extraction)Cellulase only 0
Boiled Germ + Cellulase 42.6 2.3
Microwaved Germ + Cellulase 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 theaqueous 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.
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Effect of Cooking on CornGerm Ultrastructure
Dickey, L., Cooke,P.H., Kurantz, M.J.,McAloon, A., Parris,
N, and Moreau, R.A.,Using microwaveheating andmicroscopy toestimate optimal corngerm oil yield with a
bench-scale press,JAOCS 84:489-495,2007.
Fig. 4 Effect of heating on the ultrastructure of dry-milledcorn germ A as studied with confocal (100 m), light(25 m) and transmission electron (2.5 m) microscopy.
a control,uncooked corngerm;
b corn germcooked in aconvectionoven at 180 Cfor 6.5 min;
c corn germcooked in a1,500-Wmicrowave ovenfor 4.5 min
h
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AEOE with New GenerationCorn Germ from a Dry Grind
Ethanol PlantDry-Fractionated Corn GermMarket Flex TM, CPC
BFRAC, PoetWet-Fractionated Corn Germ
Quick Germ, U of IL
E-Germ, ERRC and U of ILMOR-Frac Plus TM, Mor Technologies
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Evaporation
Cleaning
Hammer Mill
Slurry MixLiquefaction
Saccharification
Fermentors
CO2 Scrubber
WATERCO2
Beer Column
Rectifier
Stripping
Centrifuge
Thin Stillage
Wet DDG S Conveyor DDGS Dryer
Cook Retention Tanks
Degasser
Beer Degas Vent Condenser
Molecular Sieves
CORN
Weigh Tank
YEAST
ETHANO
ACID
G-AMYLASE
LIME
DDGS
AMMONIA
A-AMYLASE
DENATURAN
Dry Grind Process for Ethanol using Corn(single co-product = DDGS)
Fractionation =Front EndRemoval ofGermViaDryFractionationor
WetFractionation
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AEOE with Dry FractionatedCorn Germ from a Dry Grind
Ethanol PlantEnzyme 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)
__________________________________________
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AEOE with Wet FractionatedCorn Germ (E-Germ) from a Dry
Grind Ethanol PlantEnzyme Oil Yield (%) __________________(relative to hexane extraction)Cellulase only 0
Cellulase + 87.4 Alkaline Protease (pH 8 buffer)
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 theaqueous enzymatic extraction of corn oil from dry milled corn germ and enzymaticwet milled corn germ (E-germ), J. Am. Oil Chem. Soc. 86: 469-474, 2009.
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AEOE with oil enrichment viaa bubble column
Dickey, L.C., M.J. Kurantz, N. Parris, A. McAloon, R.A.Moreau, Foam separation of oil from enzymaticallytreated wet-milled corn germ dispersions, J. Am. Oil
Chem. Soc. 86, 927-932, 2009.
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AEOE with oil enrichment viaa bubble column
Dickey, L.C., M.J. Kurantz, P Cooke, N Parris, R.A. Moreau, Separation ofbuoyant particles from an aqueous dispersion of corn germ particles usinga bubble column, Chem Eng Sci 63:4555-4560, 2008.
Using this approach with wet milled corn germ,we have been able to enrich the oil in the foamabout 4-fold, so that only about 25% of the
original volume of AEOE solution then needs tobe centrifuged to float the free oil. It isanticipated that adding this bubble columnapproach will reduce the cost of AEOE from corn
germ by reducing the centrifugation costs.
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Experiments conducted at ERRC in May-June2010 by Dr. Vera Van Hoed from U of Ghent
Red Raspberry = 15% oil by hexane extractionCranberry = 21% oil by hexane extraction
Using AEOE we were able to extract 34% ofthe hexane-extractable oil from Red Raspberryseeds. We were unable to obtain any oil fromCranberry using AEOE.
AEOE with Cranberry andRaspberry Seeds
Van Hoed, N. De Clercq, C. Echim, M. Andjelkovic, E. Leber, K. Dewettinck& R. Verh. Berry seeds: a source of specialty oils with high nutritionalvalue. Journal of Food Lipids, 2009, 16(1), 33-49. (cold pressed berry oils)
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AEOE with Camelina Seeds
Experiments conducted at ERRC in June-July2010 by Mr. Christopher Senske, Ursinus U
Camelina = 28% oil by hexane extraction
Using AEOE we were able to extract 60% ofthe hexane-extractable oil.
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Evaporation
Cleaning
Hammer Mill
Slurry MixLiquefaction
Saccharification
Fermentors
CO2 Scrubber
WATERCO2
Beer Column
Rectifier
Stripping
Centrifuge
Thin Stillage
Wet DDG S Conveyor DDGS Dryer
Cook Retention Tanks
Degasser
Beer Degas Vent Condenser
Molecular Sieves
CORN
Weigh Tank
YEAST
ETHANO
ACID
G-AMYLASE
LIME
DDGS
AMMONIA
A-AMYLASE
DENATURAN
Dry Grind Process for Ethanol using Corn
Back End Removal
of Corn Oil
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Obtained from the back end of a dry grind ethanolplant
Proprietary process developed by GreenShiftCorporation
Obtained by centrifugation/condensation of the thinstillage
The oil contains 10-15% free fatty acids. Theirremoval is costly and eliminates edible applications butare OK for biodiesel and other industrial applications.
~1% of commercial corn oil in US Rich in Lutein and Zeaxanthin (valuable for feedapplications)
Lutein and zeaxanthin, 200x higher in postfermentation corn oil than in corn germ oil
Post Fermentation Corn Oil
C l i
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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 wetfractionated corn germ.
Since several cellulase preparations appear to result in high oil yields , weanticipate that the new generation of celluloytic enzymes that are beingdeveloped for biomass hydrolysis and fermentation (Accellerase 1500, andDUET 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 inadditional valuable protein and carbohydrate coproducts , the sales of whichcould lower the overall cost of the process.
A new aqueous enzymatic extraction process has been developed that
results in oil yields of greater than 90% from wet milled corn germ or wetfractionated corn germ (including E-Germ).
A new aqueous enzymatic extraction process has been developed thatresults in oil yields of greater than 65% from dry milled corn germ or dryfractionated corn germ.
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Acknowledgments
Corn Germ
BungeCargillCereal Process Technologies
EnzymesGenencorNovozymes
Corn Germ AEOE
Mike PowellMike DahlmerJhanel Wilson
Fruit Seed AEOE
Vera Van HoedCamelina AEOE
Chris Senske
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Thanks to for inviting us to present ourUSDA-ARS research results
Questions ?
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Chemical CompositionCrude RBDwt%
Triacylglycerols ~97 ~99Diacylglycerols ~0.5 ~0.5Free Fatty Acids 1-2 0
Phytosterols ~1 0.7Vitamin E* 0.1 0.07Carotenoids 0.0002 0.0001Phospholipids ~1 0
refining
bleaching
deodorization
deodorization
degumming
Conventional Corn (germ) Oil
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Applications:Edible corn oil, margarine, etc,
corn oil fatty acids, high linoleate, like, soy+peanutNonedible oleochemicals etc
Value: unrefined $/lbSoybean 0.34Corn Oil 0.39
Cottonseed 0.40Canola 0.40Sunflower 0.56Peanut 0.68Source, Economic Research Service, USDA, June 2010
Historically corn oil has been considered tobe a premium oil sold for a premium price
Today, it sells for a 5 centpremium over soybean oil
Possible reason for the relative loss in value -consumer preference for high monounsaturateoils (olive, canola, sunflower oil (NuSun) andoils with more omega 3 fatty acids
Conventional Corn (germ) Oil
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Bench Scale Process for Aqueous Enzymatic OilExtraction Studies (protocol Designed to CombineSome of the Features of Shi et al ., 1998 and Karlovicet al. , 1994)
Weigh duplicate 6 gram samples of dry germ into 50 mltubes
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 shakinghorizontally Cool tubes to room temperature Centrifuge at 2500 g (4000 rpm) for 10 min Remove top oil layer with a pipet
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Scale up of the aqueous enzymatic extraction to 24 grams of w et milledgerm, 160 ml buffer, and 2.0 ml enzyme.
_________________________________________________________________
Enzyme Oil Yield ________________________________
Wt% of germ Relative% 1
_________________________________________________________________
Multifect GC (82 GCU/gram) 38.2 0.9 93.2 2.2
Celluclast 1.5L (790 EGU/gram) 37.3 1.4 91.1 3.5
_________________________________________________________________1 Oil Yield relative to hexane extraction (see Table 1)
Oil Yields Using 4X Scale p of the Aq eo s En matic
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Oil Yields Using 4X Scale-up of the Aqueous EnzymaticOil Extraction Protocol (24 g germ + 160 ml buffer + E)
Oil Yield = 90%compared tohexane extraction= 100%
TABLE 2A
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TABLE 2A A Comparison of the Oil Yields Using the Aqueous Enzymatic OilExtraction Protocol, wi th Various Commercial Enzymes, Listed inOrder f rom Highest to Lowest Oil Yields
___________________________________________________________
Enzyme Company Brand Name Oil YieldRelative%
___________________________________________________________
Cellulase Genencor Multifect GC 81.7 0.7Cellulase 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 ofCommercial Enzymes for the Aqueous Enzymatic Extraction of Corn Oilfrom Corn Germ, J. Am. Oil. Chem. Soc. 81;1071-1075 (2004).