characterization of zein modified with a mild cross-linking agent

Industrial Crops and Products 20 (2004) 291–300

Characterization of zein modified witha mild cross-linking agent�

S. Kima,∗, D.J. Sessab, J.W. Lawtonb

a Cereal Products and Food Science Research Unit, National Center for Agricultural Utilization Research,Agricultural Research Service, USDA, 1815 North University Street, Peoria, IL 61604, USA

b Plant Polymer Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service,USDA, 1815 North University Street, Peoria, IL 61604, USA

Received 19 July 2003; accepted 29 October 2003

Abstract

Zein, a predominant corn protein, is an alcohol-soluble protein extracted from corn and is an excellent film former. The charac-teristic brittleness of zein diminishes its usefulness as a film. It is well known that zein has a propensity for forming aggregates insolution. When zein molecules were cross-linked with 1-[3-dimethylaminopropyl]-3-ethyl-carbodiimide hydrochloride (EDC)andN-hydroxysuccinimide (NHS), it was found that the film-forming property was improved and the aggregation phenomenonin solution was suppressed. At the air/water interface, native zein forms brittle film with rough surface, whereas cross-linkedzein forms rigid film with very smooth and even surface. Tensile strengths of the films were shown to be greatly increased bycross-linking. Objectives of this study are to determine the cross-linking mechanism of zein, the optimum reaction conditions,characteristics of the reaction product, and mechanical properties of cross-linked zein film. Through viscosity and dynamic lightscattering, the cross-linking reaction was monitored. Optimum amount of EDC and NHS was determined to be 30 mg each pergram of zein. The cross-linking of zein with EDC and NHS seemed to be self-terminating because the cross-linking reaction didnot proceed to precipitation.Published by Elsevier B.V.

Keywords: Zein; Cross-linking; Aggregation

1. Introduction

Zein is an alcohol-soluble protein and is an excel-lent film former. Its film-forming property attracted

� Names are necessary to report factually on available data:however, the USDA neither guarantees nor warrants the standardof the product, and the use of the name by USDA implies noapproval of the product to the exclusion of others that may alsobe suitable.

∗ Corresponding author. Tel.:+1-309-6816260;fax: +1-309-6816685.

E-mail address: [email protected] (S. Kim).

attention in the field of edible film and coating ma-terial (Gennadios and Weller, 1990; Herald et al.,1996; Krochta and De Mulder-Johnsto, 1997; Lai andPadua, 1997). However, zein films are brittle, andthus, some modifications are needed to improve theirflexibility. Cross-linking between zein molecules hadbeen induced by using reagents such as formalde-hyde, glutaraldehyde, epichlorohydrin, citric acid,1,2,3,4-butanetetracarboxylic acid, polymeric dialde-hyde starch, 1,2-epoxy-3-chloropropane and dialco-hols. As a result of cross-linking, a noticeable increasein tensile strength of zein films have been reported

0926-6690/$ – see front matter. Published by Elsevier B.V.doi:10.1016/j.indcrop.2003.10.013

292 S. Kim et al. / Industrial Crops and Products 20 (2004) 291–300

(Yamada et al., 1995; Yang et al., 1996; Parris andCoffin, 1997; Zhang et al., 1997). Despite the success-ful application of these reagents for cross-linking anddemonstration of improvements in mechanical prop-erties of cross-linked films, the reaction mechanismand characterization of reaction products have notbeen studied for these cross-linkers. Because basicinformation is essential to design films with specificmolecular structures and mechanical properties, a sys-tematic approach for understanding the cross-linkingprocess is required. In this investigation, we employed1-[3-dimethylaminopropyl]-3-ethyl-carbodiimide hy-drochloride (EDC) andN-hydroxysuccinimide (NHS)as cross-linking reagent for zein solution (Sehgal andVijay, 1994). Because these chemicals are known asmild reagents for cross-linking, denaturation of pro-teins is not expected. As a result of cross-linking,protein molecules should be interconnected, and alarger hydrodynamic radius for the reaction product isexpected. Objectives of this study are to determine thecross-linking mechanism of zein, the optimum reac-tion conditions, characteristics of the reaction product,and mechanical properties of cross-linked zein film.

2. Materials and methods

2.1. Materials

Zein was purchased from Freeman industries (Tuck-ahoe, NY). EDC and dimethyl sulfoxide (DMSO)were from Aldrich (Milwaukee, WI) and ethanolwas from Aaper Alcohol and Chemical Company(Shelbyville, KY). NHS was from Sigma (St. Louis,MO). All the chemicals were used without furtherpurification.

2.2. Viscosity measurement

Rheological properties of zein in 90% (w/w) aque-ous ethanol were measured with Brookfield Pro-grammable Rheometer (Model DV-III; Middleboro,MA) equipped with a coaxial cylinder cell. Timedependence of viscosity at 200 s−1 was measuredby using custom software written with QBASIC. Allmeasurements were performed at 23.0 ± 0.1◦C.

2.3. Dynamic light scattering experiment

DLS measurements were performed with BI-9000AT autocorrelator (Brookhaven InstrumentsCorp., Holtsville, NY) and a variable scattering angleBIC BI-200SM goniometer with a stepping motorcontroller, using theλ = 514.5 nm line of an argonlaser (Lexel Laser, Inc., Fremont, CA) to illuminatethe sample cell. The sample cell was placed in athermo-scattered glass vat with an optically flat win-dow. All experiments were carried out at 25.0±0.1◦C.The scattering intensities were determined by usingstandard photon counting method.

2.4. Transmittance measurement

Transmittance was measured with a custom-builtturbidometer that is composed of He–Ne laser, tem-perature controlled sample block, stirrer, neutraldensity filter, laser powermeter, A/D converter, andCRT display. The 633 nm beam from He–Ne laserpasses through sample solution in scintillation vialthat is surrounded by temperature-controlled copperblock. The diameter of scintillation vial is 2.5 cm.The turbidometer is sensitive to the existence of dustparticles, so sample solutions were filtered through5�m disposable Teflon filters prior to collecting data.During the transmittance measurement, the samplesolution is continuously stirred with magnetic spinbar with a digitally controlled stirrer. The intensityof transmitted laser beam was monitored with a laserpower meter, the output of which is interfaced with amicrocomputer equipped with an analog/digital con-verter. Time-dependent data could be collected witha program written with QBASIC.

2.5. Mechanical property measurement

Zein formulations for films were cast on Tefloncoated glass plates and then air-dried for 16 h. Filmswere then cut with a die conforming to the ASTMD-412-68 Type C Standard for testing. Three samplesfrom each film were then exposed to relative humid-ity of 50% for 7 days. Five thickness measurementsacross the gauge length of each sample (33 mm) weretaken using a Minitest 2500 (Electro Physik, Cologne,Germany) and averaged. Grip distance was 76.2 mm.Actual testing was then done using an Instron

S. Kim et al. / Industrial Crops and Products 20 (2004) 291–300 293

Universal Testing Machine (Model 4201, Canton,MA) in a constant relative humidity room (50%) at23◦C. Series IX software handled data recording andmanipulation. For these samples, a cross-head speed of50 mm/min was used. Elongation (%), tensile strength(MPa), and Young’s modulus (MPa) were comparedbetween modified films versus control films.

3. Results and discussion

3.1. Cross-linking reaction

To cross-link zein, we used EDC and NHS. The re-action mechanism for these chemicals have been wellstudied compared with the cross-linkers discussedin Introduction section (Sehgal and Vijay, 1994).EDC and NHS used for this reaction are classified aszero-length cross-linking reagent because during thecross-linking reaction, atoms are eliminated from thereactants, thus shortening the distance between thetwo linked moieties (Wong, 1991). In this reaction,cross-linking reagent connects carboxyl groups in oneprotein molecule to amine groups in the other proteinmolecules. The reaction scheme is shown inFig. 1.The water-soluble carbodiimide EDC is used to form

Fig. 1. Scheme for amide bond formation between zein molecules reacted with 1-[3-dimethylaminopropyl]-3-ethyl-carbodiimide hydrochlo-ride (EDC) andN-hydroxysuccinimide (NHS) where zein (A) and zein (B) represent zein molecules, and R1 and R2 are the two alkylgroups in cross-linking reagent, EDC.

covalent conjugates via amide bonds. The reactioninvolves the intermediary formation of the activatedO-acylurea derivative of the carbodiimide. A sub-sequent nucleophilic attack by the primary nitrogenof the amino compound brings about the formationof the amino linkage with the release of the solublesubstituted urea. NHS, which facilitates the reac-tion, reacts with carboxyl-containing compounds togive aminoacyl esters. The stable active esters, thusformed, hydrolyze slowly compared with their ratesof reaction with amino groups. Hence, NHS enhancesthe coupling efficiencies of carbodiimides for conju-gating carboxylated compounds with primary amines.

3.2. Concentration of cross-linking reagents

To optimize the reaction condition, i.e., the requiredrelative amounts of the EDC and NHS reactants, theconcentration effect of cross-linking reagents on theprogress of reaction was studied. When the solutionviscosities were compared both before and after thereactions, a noticeable increase was observed. Thisobservation offers direct evidence that the reactionscheme is successful for cross-linking of zein. Hence,the viscosity of the reacting solution was monitoredas a function of time and amount of cross-linking


Fig. 2. Viscosity used as a measure of the progress of the cross-linking reaction. Various amount of cross-linking reagents, EDC and NHSwere added to 20% zein in 90% aqueous ethanol.

reagents. For this series of experiments, 10 g of 20%zein solution was prepared in 90% (w/w) aqueousethanol. EDC and NHS were added to the solutions inequal amounts ranging from 10 to 200 mg each. Ac-cording to these results (Fig. 2), the optimum concen-tration of combined chemical reagents is 60 mg pergram of zein. Under this reaction condition, the mo-lar ratio of EDC:zein corresponds to 3.5:1. For thiscalculation, the molar mass of zein is assumed to be23,000 Da (Shukla and Cheryan, 2001). With this re-sult, we conclude that the number of binding site oneach zein molecule is very limited.

3.3. Hydrodynamic radius before and after thereaction

Dynamic light scattering technique was used to ob-tain the hydrodynamic radius of macromolecules insolvent medium. Correlation function is given as

g(1)(τ) = exp(−Γτ) (1)

whereΓ = Dtq2 (Johnson and Gabriel, 1994). From

the slope ofq2 versusΓ plot, we obtain diffusion co-efficient of solute molecule,Dt, where,q is the scat-tering wave vector. The hydrodynamic radius of solutemolecule can be calculated by Stokes–Einstein rela-tionship,

Rh = kT

6πηDt(2)

wherek is the Boltzmann constant,T the absolute tem-perature, andDt the diffusion coefficient. As the vis-cosity of the solvent medium,η, remains the same be-fore and after the reaction, we can relate the hydrody-namic radii before and after the reaction by comparingthe diffusion coefficients.

Theq2 versusΓ plot before and after the reaction isshown inFig. 3. This data set was obtained from 1%zein in 90% aqueous ethanol. For the cross-linkingreaction, 50 mg each of reagent was added to thesolution, and stirred for 2 days at room temperature.During the reaction, random cross-linking should


Fig. 3. q2 vs. Γ plot of dynamic light scattering data from before and after cross-linking zein with EDC and NHS. Hydrodynamic radiusis proportional to the slope of this plot.

have been induced. As a result, the solution willcontain cross-linked zeins with a broad range ofmolecular weights. Although this polydispersity havecaused nonlinearity in the data, the slowing-downof the diffusion coefficient is clearly observed. Thatis, diffusion coefficient of zein was decreased from4.8 × 10−7 to 8.8 × 10−8 cm2/s. This means the hy-drodynamic radius of zein increased by ca. 5.4 timeson the average, which corresponds to ca. 160 timesincrease in volume. In other words, the associationnumber of cross-linked zein molecules is about 160.

It seems that the cross-linking reaction stops afterforming multimers with a range of association num-bers. This observation is supported by the turbiditydata that will be further explained in the next sec-tion. Indefinite association of molecules is supposedto bring about precipitation because the solubilityof polymer decreases as molar mass of polymerincreases. Because no precipitation or aggregationwas observed, it is reasonable to conclude that thecross-linking reaction stops at certain point. We postu-

late that these observations indicate that a limited num-ber of reaction sites per molecule and steric hindranceprevent the reactants from further cross-linking.

3.4. Disintegration of aggregate with cross-linkingregents

When zein molecules are solubilized in solventmixtures, in most cases, they form aggregates overtime (Evans and Manley, 1943). As aggregates areformed, the whole solution becomes turbid. Thismeans that the aggregate formation can be studiedby measuring time-dependent turbidity of the so-lution medium. For that purpose, we employed acustom-built turbidometer. The relationship betweenaggregate formation and measured transmittance canbe explained as follows: when a light beam with in-tensity I0 passed through a sample cell, part of thebeam intensity is lost by absorption and/or scatter-ing by solute molecules. If incident laser beam doesnot interact with the sample solution, which is our


case, absorbance of light by sample material does nothave to be considered in the equation. DefiningIt asa transmitted beam intensity, the turbidity per unitlength of path through the sampleτ is,

τ = −ln

(It

I0

)(3)

The turbidity is viewed as the product of three factors,

τ = (πR2)QC (4)

whereR is the radius of opaque spheres,Q the scat-tering efficiency factor, andC is the concentration ofopaque spheres in the sample solution. Combiningthe above two equations, we obtain the followingrelationship:

It = I0 exp(−πR2QC) (5)

Assuming thatQ remains constant, the formation ofaggregates will result in the increase inC andR termsin Eq. (5) whereby aggregation of proteins can bemonitored by measuring turbidity of the solution.

Changes in the transmittance of the solution as ag-gregates are formed in the solution are presented inFig. 4. These data were obtained from 10% zein in90% aqueous ethanol. As soon as a clear solution wasprepared, vacuum was applied to the solution to re-move air bubbles, and transmittance was measured

Fig. 4. Stirring speed dependence of transmittance of 10% zein in 90% aqueous ethanol. Aggregate formation is irrelevant to stirring andstirring speed.

from that moment. The importance of this experimen-tal result is that stirring speed does not affect aggre-gate formation. Even without stirring, the same degreeof aggregate formation was observed. This means theformation of aggregates cannot be explained by col-lision theory, but by electrostatic interaction betweenmolecules.

When a solution full of aggregates was shaken, par-tial recovery of transmittance was observed. This par-tial recovery of transmittance is interpreted as disin-tegration of aggregates into smaller particles by shak-ing the solution because transmittance decreases againas a function of time. When the solution was shakenagain, transmittance was recovered to a lesser amountcompared with the first shaking (Fig. 5). This meansshaking motion disintegrates loosely bound aggregatesonly. It seems that repeated shaking helps in the for-mation of better-organized and more-densely-packedaggregates. Micrographs for shaking effect are shownin Fig. 6. As was previously discussed, shaking thesolution disintegrates aggregates in to smaller size.

We can better understand the mechanism of ag-gregate formation by usingEq. (4). There should betwo kinds of mechanism for the formation of ag-gregates. One is aggregate formation from individualmolecules, and the other is larger aggregate forma-tion from smaller ones. At the beginning of aggregate


Fig. 5. Formed aggregates of 10% zein in 90% aqueous ethanol can be disintegrated by shaking the solution. As this process is repeated,disintegration effect becomes less noticeable.

formation, the first mechanism will be a major one.However, at the later stage, the second mechanism willbe predominant because the concentration of solute islow, whereas the population of aggregates is high. Theexistence of second mechanism was illustrated by theshaking effect seen inFig. 6, which was also discussedpreviously. Therefore, among these factors contribut-ing to turbidity, we can conclude that the concentra-tion term is more important at the beginning and thesize term is more important at the later stage.

Fig. 6. Photographic images of aggregates formed from 10% zein in 90% aqueous ethanol for shaking effect: (A) before shaking, (B) aftershaking for 2 min.

Cross-linking effect was observed on 10% zein solu-tion in 90% aqueous ethanol. Upon preparation of zeinsolution, the solution was filtered and transmittancewas monitored. After aggregation was induced in thesolution, cross-linking reagents were added to the so-lution. As is seen inFig. 7, the transmittance recoveredto the initial condition. This means the cross-linkingreagents disintegrate the aggregates. Based on this ex-perimental result, the reaction time is estimated to bearound 1.5 days for this experimental condition.


Fig. 7. Cross-linking effect on aggregated zein formed in 10% zein in 90% aqueous ethanol: aggregation was induced for 20 h, after that,cross-linking reagents, EDC and NHS were added.

In order to make sure that the cross-linking reagentsprevent and disintegrate aggregates, they were addedjust after the preparation of the sample solution. Com-petition seems to be present between aggregate for-mation and disintegration (Fig. 8 ). At the beginning,aggregation appears to be faster than disintegration,but later, disintegration prevails, whereby, transmit-

Fig. 8. Transmittance data showing the competition between cross-linking and aggregation in 10% zein in 90% aqueous ethanol: cross-linkingreagents, EDC and NHS were added to zein solution from the beginning.

tance retains high value. The scatter of data is causedby floating small aggregates.

3.5. Mechanical property measurements

The tensile strength of the cast zein films wasgreatly increased by cross-linking with EDC (Table 1).


Fig. 9. Cross-linked zein films formed at the air/water interface are strong enough to be lifted with a glass rod (representations (1)–(4)).Without cross-linking, zein films are too weak to be lifted (not shown).

Flexibility of the films was also improved as shownby the decrease in Young’s modulus. The cross-linkedfilms showed a small, but significant increase in elon-gation. This is probably due to the high glass transi-tion temperature of zein in the absence of plasticizers(Lawton, 1992).

The effect of cross-linking was also shown by com-paring the strength of thin films with and withoutcross-linking. Because zein is hydrophobic, it easilyforms thin films at the air/water interface. In order toshow that cross-linking improves film-forming prop-erty of zein, the film was formed at the surface of

Table 1Tensile properties of zein films before and after cross-linking

Treatment Humidity(%)

Young’smodulus(%)

Elongation(%)

Tensilestrength(MPa)

Control 50 1710± 30 2.23± 0.04 33± 1Cross-linked 50 1620± 50 3.6± 0.5 43± 1

water by spreading zein using 90% aqueous ethanolas a spreading agent. The concentration of zein was1%. At the air/water interface, native zein formed abrittle film with a rough surface, whereas cross-linkedzein formed a rigid film with a very smooth and evensurface. Native zein formed too weak a film to belifted with a glass rod. In contrast, cross-linked zeinformed a very sturdy film that could be lifted with aglass rod (Fig. 9). Although the mechanical propertymeasurement data showed only 33% increase in ten-sile strength, prominent improvement in the strengthof thin film could be demonstrated.

4. Conclusion

Mild cross-linking reagents, EDC and NHS, wereemployed for the first time to form intermolecu-lar amide bonds in zein solutions. As a result ofcross-linking, aggregate formation was suppressedand the mechanical properties of films were improved.


The progress of the reaction as well as optimizationof the reactants could be monitored by measuringviscosity of reactant solution. The growth of zeinmolecules by cross-linking was shown by compar-ing hydrodynamic radii before and after the reaction.These experimental results show that even with par-tial cross-linking the mechanical properties of zeinfilms can be improved significantly.

Acknowledgements

We thank Mr. Gary Kuzniar for his assistance indetermining the mechanical properties of zein films.

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characterization of zein modified with a mild cross-linking agent

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