Industrial Crops and Products 26 (2007) 44–53

Separation of high-value products from ethanol extractsof corn by chromatography

Aniket Kale ∗, Fangyi Zhu, Munir CheryanAgricultural Bioprocess Laboratory, University of Illinois at Urbana-Champaign, 1302W. Pennsylvania Avenue,

Urbana, IL 61801, USA

Received 7 October 2006; received in revised form 6 January 2007; accepted 8 January 2007

Abstract

This research details the development of a new one-step process for producing high-value products such as zein and xanthophyllsfrom corn to enhance the economic viability of the dry-grind ethanol industry. This process was designed for incorporation ina dry-grind ethanol plant, and thus in-house ethanol was the processing solvent for the entire processing chain, from the initialextraction of the whole ground corn to the final purification steps. Dry-ground whole corn was extracted with 70% (v/v) ethanol andfiltered. The extract was processed by preparative-scale size exclusion chromatography with aqueous ethanol as the mobile phaseand an ethanol-stable resin as the stationary phase. The largest component (zein) was totally excluded in the void volume of thecolumn while the impurities and the xanthophylls interacted with or passed through the pores of the resin and separated accordingto their mobilities. Starting with 17 g of corn or 50 mL corn extract, one can obtain about 320 mg of zein with 90+% yield andpurity in this method. Thus, a typical dry-grind ethanol plant processing 50 million gallons of ethanol per year, can produce about

13 million kg of zein and 7.5 tonnes of xanthophylls and this implies an additional revenue of $95million (conservative valuation:zein ∼$4.4 kg−1 and xanthophylls ∼$5 g−1) without bringing in any additional raw materials. This is about 1.5–2 times the presentrevenues of such an operation.© 2007 Elsevier B.V. All rights reserved.

s; LH 2

Keywords: Size exclusion chromatography; Corn; Zein; Xanthophyll

1. Introduction

Corn is one of the major crops in the United States.About 25% of the corn was converted to food, feed andindustrial products (NCGA, 2006). Ethanol productionfrom corn by the dry-grind process has increased expo-

nentially since 2003 and will reach 7.5 billion gallonsby 2012 (RFA, 2006). At present, a dry-grind ethanolplant typically produces three products: ethanol, carbon

∗ Corresponding author. Tel.: +1 217 3339332; fax: +1 217 3339592.E-mail address: akale@uiuc.edu (A. Kale).

0926-6690/$ – see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.indcrop.2007.01.006

0

dioxide and distillers dried grains with solubles (DDGS).Since operating costs are almost equal to the revenuefrom ethanol, any profit is derived from the coproductsand the subsidies and tax waivers. The dry-grind ethanolprocess is a low-profit operation and there is a need fornew technologies and additional coproducts to improvetheir profitability.

This paper reports on two high-value coproductspresently not being produced by dry-grind ethanol

plants: zein and xanthophylls. Zein is a protein withseveral existing and potential applications in the food,pharmaceutical and biotechnology industries (Shuklaand Cheryan, 2001). It is commercially produced from

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orn gluten meal by extraction with aqueous alcohols.he extract is purified using various methods such asrecipitation, carbon adsorption, filtration and solventxtraction. Zein sells for $10–40 kg−1 depending on theurity and color (whiter zein commands higher prices).urrent purification methods usually do not provide highields and purity simultaneously and none produce zeinhat is completely devoid of xanthophylls.

Xanthophylls such as lutein and zeaxanthin are oxy-enated carotenoids that are found in high concentrationsn the macular region of the eye. Epidemiological studiesave shown that low concentrations of xanthophylls inhe macular region can increase the risk of age-related

acular degeneration (AMD) (Seddon et al., 1994).anthophylls cannot be synthesized by the human body

nd must be obtained from foods such as fruits, veg-tables, eggs or from dietary supplements containinghemical- or fermentation-derived compounds. Amongatural sources, egg yolk and corn contain the highestolar levels of lutein and zeaxanthin (more than 85%

f the total carotenoids). Most sources of xanthophyllsther than corn are rich in either lutein or zeaxanthinut not both (Sommerburg et al., 1998). In corn, xantho-hylls are preferentially bound to zein and as a result,xtraction and purification of xanthophylls from cornlso results in the simultaneous extraction and purifica-ion of zein, and vice versa.

Previous work in our laboratory (Moros et al., 2002;hukla et al., 2000; Kwiatkowski and Cheryan, 2005)stablished that zein and xanthophylls could be extractedith 70–85% aqueous ethanol with little or no co-

xtraction of corn oil. The zein and xanthophylls are thenurified using membrane technology (Cheryan, 2001,002). Several stages of ultrafiltration and nanofiltra-ion membranes are needed, together with substantialmounts of diafiltration, to obtain the desired (>90%)urity of zein. In addition, because most of the co-xtracted impurities have similar molecular sizes as theanthophylls, membranes alone cannot produce a highlyure xanthophylls stream. These factors prompted us toeek alternate methods for the production of pure zeinnd xanthophylls, especially a method of purificationhat can produce pure (white) zein.

Chromatography is one such technique of producingure zein and xanthophylls. It has the inherent advantagef being able to achieve high resolution (i.e., high degreef separation between zein, xanthophylls and the impu-ities) leading to high purity while not requiring harsh

rocessing conditions such as extremes of temperature,ressure or shear which could otherwise lower productuality. Zein has been separated using cation exchangehromatography by Craine et al. (1961). Ground corn

Products 26 (2007) 44–53 45

was defatted and then extracted with 70% ethanol. Thecorn extract was processed with an Amberlite IRC-50column using a salt (NaCl) gradient. The purity of theproduct was 85% and the yield was 86%. The remainingzein was irreversibly bound to the resin.

Since there is a large difference in molecular weightsof xanthophylls (∼560) and zeins (>17 kDa), an obvi-ous choice would be size exclusion chromatography(SEC). This technique is simple to operate and scaleup, provided ethanol-stable resins are available. Landryet al. (1967) and Landry and Guyon (1984) proposed aprotocol that used a complicated series of eight liquidextractions that included organic solvents such as etherand acetone, followed by two size exclusion chromatog-raphy steps with the Sephadex LH-20 resin utilizing adifferent mobile phase in each step. Danzer and Rees(1971) used a similar size exclusion resin followedby treatment with charcoal to separate zein from cornextracts to obtain zein of 90% purity but yield data hasnot been presented. Sessa et al. (2003) decolorized zein inseveral ways, such as supercritical fluid extraction, ultra-filtration, activated carbon, and column chromatographywith Sephadex LH-60. The pore size and fractionationrange of the LH-60 is too large for effective purificationof zein, since the molecular weight exclusion limit of theLH-60 is 18,000–20,000 (Amersham, 1995a,b; Coocniet al., 1980; Janshekar et al., 1982). Mosse and Landry(1980) used Sephadex G-100 and Sephadex G-200 forseparating zein monomers and dimers, but these resinsalso have exclusion limits that are too large for efficientpurification of whole zein.

Xanthophylls have traditionally been producedmainly from marigold flowers. Khachik (2005a,b)described extraction of lutein using an alkaline organicsolvent of tetrahydrofuran containing ethanol and KOHmaintained at pH 12 and chemical conversion of luteinto anyhydroluteins. Lutein was purified by column chro-matography using n-silica gel and mobile phases suchas C5–C7 hydrocarbons or petroleum ether in combina-tion with acetone or methyl ethyl ketone or ethyl acetateor tetrahydrofuran or C4–C6 ethers. However, the xan-thophylls in marigolds are in an ester form and requireadditional steps to obtain the pure form. The product isalso quite unstable and cannot be processed into veryhigh concentrations of the xanthophylls. In addition,marigolds produce mainly lutein and not zeaxanthin,while corn contains both isomers.

The main focus of this work was to develop a process

for production and purification of xanthophylls and zeinsimultaneously by chromatography and using only rawmaterials available in a dry-grind ethanol plant, such asethanol and whole ground corn.

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46 A. Kale et al. / Industrial Cr

2. Materials and methods

2.1. Extraction of zein and xanthophylls from corn

The extraction method was adapted from Shukla et al.(2000). Raw whole corn (yellow dent #2) was obtainedfrom Anderson Grain Co. (Champaign, IL). The cornwas ground using a bench top hammer mill, IKA MF10.2 (IKA Works Inc., Wilmington, NC) with a 1 mmmesh. Ethanol (200 proof; USP grade) was procuredfrom Aaper Alcohol and Chemical Co. (Shelbyville,KY). The water was distilled, deionized and microfil-tered using a 0.2 �m Maxi Capsule Filter (Pall GelmanLab, Ann Arbor, MI). Extraction was done in an Erlen-meyer flask using a solvent: solids ratio of 4:1 (4 L of70% ethanol per kg of corn) at 40 ◦C for 30 min. A NuovaII stirring hot plate (Thermolyne, Dubuque, IA) was usedfor temperature control and mixing. The slurry was thenfiltered with Whatman paper #1 (11 �m average porediameter; Whatman Inc., Clifton, NJ). The extract wasthen stored at 40 ◦C in an Oakton stable temperatureoven (Oakton, USA) until use to avoid any precipitation.The extract was microfiltered with a 0.45 �m hydrophiliclow protein binding PTFE filter (LCR, Millipore Corp.,Bedford, MA).

2.2. Chromatography

A semi-prep Waters (Waters Corporation, Milford,MA, USA) system consisting of a Waters 600E mul-tisolvent delivery system (600E pump with flow range0–20 mL/min and a 600E pump controller) and a Waters2996 PDA detector (equipped with of a light source ofDeuterium lamp with a flow cell capacity of 10 �L) wasused. The system was connected to a personal computerwith Waters Empower software for the data analysis.Manual injections were carried out directly through theinlet manifold valve. The injection volume was set to50 mL by controlling the sample injection time at a con-stant flow rate of 6.25 mL/min to 8 min.

An Omnifit column (2.5 cm diameter, 100 cm length,491 mL column volume) was purchased from West-ern Analytical Inc. (Murrieta, CA). It was packed withLH-20 resin according to the manufacturer’s instruc-tions. The cross-linked hydroxypropylated dextran basedresin Sephadex LH-20 (GE Healthcare-Biosciences, Pis-cataway, NJ) was used since it is one of the fewcommercially available SEC resins that are stable in

aqueous ethanol concentrations over 70% ethanol andhave a molecular weight exclusion of less than 10 kDa.It was equilibrated with 70% aqueous ethanol solutionat room temperature. Fifty milliliters of the corn extract

Products 26 (2007) 44–53

was injected into the column. The mobile phase waspumped into the column using a Waters 600E pump atroom temperature and a flow rate of 375 mL/h. Sam-ples were collected every 3 min for 1.5 column volumeselution.

2.3. Materials

Freeman zein with a protein content of about90% (F4000) was obtained from Freeman Industries,Tuckahoe, NY. Showa zein with a protein content ofover 90% was obtained from Showa Sangyo Corp,Tokyo, Japan. Xanthophylls were obtained from DSMNutritional Products (formerly Roche Vitamins Ltd.),Basel, Switzerland. The eluents used in the study werefiltered using a 0.2 �m filter (FGLP, Millipore Corp.,Bedford, MA).

2.4. Analysis

2.4.1. ChromatogramsThe absorbance of the samples was measured with the

PDA detector at three wavelengths: 280, 310 and 450 nm.The chromatograms obtained at 280 nm represent theprotein, 310 nm represent the impurities and 450 nm rep-resents the xanthophylls. The samples were sufficientlydilute and did not require further dilution for analysis.

2.4.2. Total solidsThe total solids in the sample were analyzed using

gravimetric method. The liquid samples were placed ina hood for 6 h to evaporate the ethanol in the sample. Thesamples were then dried at 103 ◦C for 6 h and weighedto determine the solids content of the original sample.

2.4.3. Protein analysisCrude protein was analyzed using a Leco FP-528

Nitrogen Combustion Analyzer (Leco, St. Joseph, MI)by the AOAC method (AOAC, 2000). Protein was cal-culated as nitrogen × 6.25.

2.4.4. Molecular weight distributionProtein molecular weight distribution was obtained

using an automated electrophoresis system (Agilent2100 Bioanalyzer, Agilent Technologies, Palo Alto,CA). This system is based on the proteins movingthrough microfluidic channels of the gel on a chip atdifferent velocities based on their size under an electric

current. Theoretically, all proteins have the same shapeand charge due to SDS or LDS micellization and have afluorescent marker attached. The fluorescence detectiongives the distance traveled by each protein based on its

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olecular size (assumed to be directly proportional tohe weight). This system provides the gel representationf the results as well as the electropherograms. The sam-le preparation is done as described by the manufacturer.o mercaptoethanol was used in our sample preparation.

.4.5. XanthophyllsRoutine determination of xanthophylls concentration

as done by spectroscopy at 450 nm. Individual iso-ers were analyzed using HPLC (Kale and Cheryan,

007). The HPLC column was a 4.6 mm × 250 mm C-0 carotenoid column (Waters Inc., Wilmington, NC).

guard column (4 mm × 23 mm) containing the sameacking material as the C-30 column was installed aheadf the carotenoid column. The solvents were HPLC gradeethanol and methyl tert-butyl ether (MTBE, Fishercientific, Pittsburgh, PA). A gradient system was used

nvolving two mobile phases. Mobile phase A was pureethanol and mobile phase B pure MTBE. The ini-

ial values were 90% of A and 10% B, to 50% A and0% B in 16 min, followed by a cleaning with 9%And 91% B for 5 min. The flow rate was 1.0 mL/minnd the temperature was 25 ◦C during the entire run. Allamples were injected via a 100-�L loop using a 1-mLyringe.

. Results and discussion

The separation capabilities of the chromatographyystem were evaluated using the LH-20 resin packed inn Omnifit column. Fifty milliliters of commercial zeinsShowa and Freeman Yellow ∼ F4000) prepared in 70%thanol to a concentration of 1.5% (w/v) and the cornxtract (containing about 7 g/L of zein) were injectednto the column. The chromatograms obtained for eachf the samples are shown in Fig. 1. Zein is representedy the chromatograms at 280 nm (solid line). In allases, it is completely excluded from the column andassed through it with minimal interaction with theydrophilic media. In addition, due to its size, it couldot pass through the pores of the LH-20 resin, which are1 nm in size (Kale, 2006). Zein is apparently the only

ompound in the extract that is larger than the moleculareight cut-off of the resin and hence the purity of the

ein is high, as will be seen later.On the other hand, the xanthophylls (represented by

he dotted line) eluted until after one column volumef elution. This could be because LH-20 has lipophilic

“L”) propyl groups and hydrophilic (“H”) hydroxylroups (Henke, 1994). The LH-20 resin separatesrganic compounds of low molecular weight by adsorp-ion using its inherent hydrophobicity (Melchert et al.,

Products 26 (2007) 44–53 47

2002). Thus, xanthophylls were adsorbed by the resinand eluted slowly after one half column volume. Fig. 1also shows the relative amounts of xanthophylls in thethree products. Showa zein is manufactured with a seriesof solvent washings to remove the color and non-zeinimpurities. It has the lowest level of xanthophylls and isthe whitest in color.

The fractions eluting after the zein (termed the “impu-rities” fraction in this paper) showed a high absorbance at280 nm, especially with the corn extract and to a lesserextent with the commercial zeins. However, the nitro-gen content was negligible in the impurities fractions, aswill be seen later. The peak spectra maximum at 310 nmfor this fraction. The impurities could be polyamineputrescine compounds, which show a very high UVabsorbance around 320 nm (Moreau et al., 2001). How-ever, these compounds amount to about 115 ppm in cornand are found mostly in the pericarp with a small amount(∼7%) in the endosperm (Sen et al., 1994). Thus, thesecompounds will be in the ppm or even ppb levels in ourextract. The oil content in the extract is negligible asobserved by Kwiatkowski and Cheryan (2005). Thus,the impurities may be either fine fiber (since it passedthrough the 11-�m pores of the Whatman filter paper#1) or sugar and/or soluble starch with a strong affinityfor the polyamines.

All three feed streams in Fig. 1 showed similar elutionpatterns with the LH-20 resin: zein eluted first followedby the “impurities” and finally the xanthophylls. Allchromatograms showed a small xanthophylls peak inthe void volume implying some binding of zein andxanthophylls. The Showa zein had a negligible amountof xanthophylls and low molecular weight impurities(MW < 4 kDa). Freeman yellow zein showed a promi-nent xanthophylls peak and negligible impurities peak.The corn extract showed abundant xanthophylls, impu-rities and zein.

The individual fractions from the corn extract werefurther analyzed for protein and total solids content andthe results are shown in Fig. 2 together with photographsof the samples. The void volume fractions (termed Frac-tion A) are abundant in zein. The large peak of solidsfollowing the zein protein peak, which eluted before thexanthophylls contained a significant amount of non-zein(from protein and total solids data) and non-xanthophylls(from chromatogram a color of the fraction collected)solids. This “impurities” fraction is called Fraction B.Fractions beyond 50% column volume elution are rich

in xanthophylls and are termed as Fraction C. The cornextract, which was the feed to the column, is shown onthe left of the picture and is a typical yellow color. Thezein fraction was substantially de-colored and was white

48 A. Kale et al. / Industrial Crops and Products 26 (2007) 44–53

Fig. 1. Chromatograms of (a) Showa zein at a concentration of 1.5% (w/v), (b) Freeman zein at a concentration of 1.5% (w/v), and (c) Corn extractcontaining zein at a concentration of 0.7% (w/v). Eluent was 70% aqueous ethanol at room temperature at a flow rate of 1 mL/min. Feed injectionvolume was 50 mL. The black solid line is absorbance at 280 nm (zein), the red dotted line is absorbance at 450 nm (xanthophylls) and the bluedashed line represents the impurities at 310 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the webversion of the article.)

A. Kale et al. / Industrial Crops and Products 26 (2007) 44–53 49

Fig. 2. Chromatogram and fractions from chromatography of corn extract at 25 ◦C. Eluent was 70% ethanol at 6.25 mL/min. Feed injected was5 hophylll , the re

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0 mL corn extract. Fraction sizes were 3 min or 18.75 mL each. Xantine). (For interpretation of the references to color in this figure legend

n color when dried, while the xanthophylls fraction (theample on the right in the picture) had a distinct yel-ow color characteristic of xanthophylls and appearedark red at higher concentrations obtained by membraneoncentration.

The operating conditions of this run resulted in base-ine separation of the zein and impurities. The totalolids and protein peaks almost completely overlappedn the zein fraction indicating a high purity of zein.here was a small xanthophylls peak appearing in theein fraction, which may be due to strong hydrophobicnteractions between zein and xanthophylls. This maye eliminated by modifying the mobile phase. Thereas also a small overlap of the impurities and xan-

hophylls. This can be eliminated by increasing theolumn length but column length is limited by resinnstability and hence a series of small columns may beequired.

s (dashed red line), impurities (dotted blue line) and zein (solid blackader is referred to the web version of the article.)

3.1. Zein yield and purity

Table 1 summarizes the material balance of theexperiment shown in Fig. 2. Zein and xanthophylls areextracted from 100 g of corn with 400 mL of 70% aque-ous ethanol at 50 ◦C in 30 min. Filtering the corn slurryresulted in about 325 mL of the corn extract with a totalsolids (TS) of 14.2 g/L (zein purity of the extract was∼49%). Fifty milliliters of this extract was injected intothe column, and eluted with 460 mL of mobile phase(70% ethanol). Fraction A was 80 mL in volume andhad TS of 4.4 g/L, of which 91% was zein, resultingin a yield of over 90% of the zein in the extract. Therest of the zein appeared in Fractions B and C. Frac-

tion C was 200 mL in volume, contained about 0.1 g/LTS and 88% of the xanthophylls in the extract. It may benoted that the total solids material balance does not closeand this could be attributed to the accuracy of measur-

50 A. Kale et al. / Industrial Crops and Products 26 (2007) 44–53

Table 1Material balance on the fractions collected with the 100 cm Omnifit column.

Corn extract Zein fraction Impurities fraction Xanthophylls fraction

Volume (mL) 50 80 180 200Total solids (g/L) 14.2 4.4 1.9 0.095Zein purity (%) 49 91 7.5 9.1Yield of zein (%) 100 92 7.3 0.5

Yield of xanthophylls (%) 100 6.5

Chromatogram is shown in Fig. 3.

ing the total solids in Fraction C. There is still evidenceof zein–xanthophylls binding, since 6% of the xantho-phylls appeared in Fraction A and 7.3% and 0.5% of thezein appeared in Fractions B and C, respectively. Thesubstantial removal of the xanthophylls from the zeinresulted in a white-colored zein, which is highly desir-able for pharmaceutical and other selected applications.The impurities elute before the xanthophylls resultingin pure (∼90%) zein and xanthophylls fractions. Thus,starting with 17 g of corn or 50 mL corn extract, one canobtain 320 mg of zein with 90+% yield and purity in this

method.

To confirm these results, the fractions from theOmnifit column were subjected to electrophoresis usingthe Agilent 2100 Bioanalyzer with the Protein 50 kit. A

Fig. 3. Gel electrophoresis of fractions and commercial zeins as described in thproteins; SZ = Showa zein; FZ = Freeman zein; CE = corn extract; C = Fraction

5.9 87

comparison of the corn extract and the three fractionsfrom the column along with the commercial zeins isshown in Fig. 3. Commercial zeins, the corn extract andFraction A show a set of bands at a molecular weightof 21,000–22,000, which represents alpha-zein. There isalso a set of minor bands visible in the 44,000–48,000region, which could be dimers of alpha-zein. Lowermolecular weight zeins are barely visible but it couldbe because of their low concentrations. The impuritiesand the xanthophylls fractions showed no zein bands.This implies that the reduction in zein yield as observed

in Table 1 may be due to accounting of non-proteinnitrogen, which are generally low molecular weightcompounds that may be expected to elute in FractionsB and C.

e figure. Abbreviations: MW = molecular weight (kDa); Std = StandardC (xanthophylls); B = Fraction B (impurities); A = Fraction A (zein).

A. Kale et al. / Industrial Crops and Products 26 (2007) 44–53 51

F t obtainee s to coloa

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carotenoids.The reproducibility of the separation was tested using

different batches of corn extracts. The chromatograms offive of those trials are shown in Fig. 7. The substantial

ig. 4. Electropherograms of zein fraction (Fraction A) and corn extracxtract; solid red line = Fraction A. (For interpretation of the referencerticle.)

Fig. 4 shows the electropherograms of the Fraction And the corn extract on the same plot. There is almost aerfect overlap of the two electropherograms confirminghat Fraction A contains essentially all of the zeins in thextract. This confirms the zein purity and yield data inable 1.

.2. Xanthophylls

The purity of the xanthophylls in Fraction C was eval-ated by HPLC as shown in Fig. 5. Fraction C showedour peaks. Peaks 1 and 2 match the spectra of stan-

ard lutein and zeaxanthin respectively and the othereaks could be beta-cryptoxanthin (Moros et al., 2002)r isomers of xanthophylls. Since peak maxima of eachompound also vary with the solvent, it is difficult to

ig. 5. HPLC of the xanthophylls Fraction C obtained in Fig. 2. Theetection is at 450 nm. The four peaks obtained have been identifiedased on the spectra shown in Fig. 6.

d from the gel electrophoresis shown in Fig. 3. Dashed blue line = cornr in this figure legend, the reader is referred to the web version of the

exactly identify the compounds eluting in Peaks 3 and4. However, the spectrum seems to indicate they are

Fig. 6. Peak spectra of the four peaks obtained in the HPLC analysisof xanthophylls Fraction C shown in Fig. 5.

52 A. Kale et al. / Industrial Crops and Products 26 (2007) 44–53

repeate

Fig. 7. Reproducibility of the chromatography process as shown byresin.

overlap in the chromatograms are a good indication ofthe consistency of the method for producing pure zeinand xanthophylls.

4. Conclusions

The objective of developing a process for producinghigh-value products from corn without bringing in anyextra raw materials to supplement the dry-grind ethanolprocess has been successfully accomplished. Existingmethods of purifying zein, such as precipitation usingthe CPC/Nutrilite technology or its variants (Shuklaand Cheryan, 2001), or liquid extraction (Morris et al.,1956; Takahashi and Yanai, 1996), or carbon adsorp-tion (McInnis and Tang, 2003; Cook et al., 1996) ormembrane technology (Cheryan, 2001, 2002) do notgive both high yields and high purity of zein. In mostcases, purity and yield are inversely related, with zeinyields of 20–40% being common. In addition, in mostother methods, the protein is exposed to enzymes, highpH, organic solvents other than ethanol, chemisorptionor shear, which could change the natural conformationand composition of the protein which may limit the sub-sequent utilization of the zein. On the other hand, the

chromatography method described here results in bothhigh purity (>90%) and high yield (90%) of zein, aswell as the simultaneous production of another extremelyhigh-value coproduct (xanthophylls) with minimal expo-

d injections of corn extract into the Omnifit column with the LH-20

sure to denaturation conditions. If the solvent constraintswere removed, better separations may be achieved withsize exclusion or other forms of chromatography.

Acknowledgements

The project was supported by the United StatesDepartment of Agriculture through the NationalResearch Initiative of the USDA Cooperative StateResearch, Education and Extension Service, grant num-ber 2004-35503-14116 and Illinois Corn MarketingBoard, Bloomington, IL. The Department of Food Sci-ence & Human Nutrition, University of Illinois providedtwo department scholarships to AK. AACC Interna-tional, St. Paul, MN awarded a fellowship to AKsponsored by National Starch & Chemical Company.The University of Illinois Research Board provided anequipment research grant. We thank Dr. Mark Band,W. M. Keck Center for Comparative and FunctionalGenomics, University of Illinois at Urbana-Champaignfor the use of the Agilent Bioanalyzer.

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