zein-fibrous clays biohybrid materials

Job/Unit: I20582 /KAP1 Date: 03-09-12 11:50:30 Pages: 10

FULL PAPER

DOI: 10.1002/ejic.201200582

Zein–Fibrous Clays Biohybrid Materials

Ana C. S. Alcântara,[a] Margarita Darder,[a] Pilar Aranda,*[a] and Eduardo Ruiz-Hitzky[a]

Keywords: Biohybrid materials / Zein / Sepiolite / Palygorskite / Bionanocomposites

The present work introduces a new type of biohybrid materi-als that is based on the combination of fibrous clays (sepioliteor palygorskite) with zein, a highly hydrophobic protein ex-tracted from corn. C, H, N, and S chemical analyses, FTIRspectroscopy, 13C solid-state NMR spectroscopy, field-emis-sion scanning electron microscopy (FESEM), thermal analy-sis, as well as dynamic vapor sorption were employed in thecharacterization of the resulting biohybrids to discern thetype of interaction between the protein and the clay fibersand to evaluate the reduced hydrophilic character of thenovel biohybrids in comparison to the pristine clays. With theaim of profiting from such a property, zein–fibrous clay bio-hybrids were tested as additives in the preparation of nano-

Introduction

The possibility of modifying clay minerals with diverseorganic compounds affords the preparation of a wide vari-ety of hybrid materials that are endowed with the desiredproperties.[1–4] Among clay-based hybrids, the term or-ganoclays has been mainly applied to those hybrids thatare based on the incorporation of alkylammonium organiccations through ion-exchange reactions and that showhydrophobic behavior.[4] Applications of such materials in-clude uses as rheological additives in the manufacture oflubricating greases and paints, adsorption of low hydrophi-lic pollutants, and more recently, nanofillers in polymer-based nanocomposites. One of the major concerns for theuse of this type of organoclays in certain applications�forinstance, as fillers in plastics for food packaging�is relatedto the presence of alkylammonium surfactants that mightshow toxicity. Thus, the preparation of bioorganoclays inwhich the organophilic counterpart is of biological origin isa new alternative for developing new clay hybrids for foodpackaging and other applications. Recently, it has been re-ported that the association of phospholipids to layered(smectites) and fibrous (sepiolite) clay minerals gives rise toa family of bioorganoclays that is able to be employed inthe removal of mycotoxins[5] or the immobilization of en-

[a] Instituto de Ciencia de Materiales de Madrid, CSIC,Cantoblanco, 28049 Madrid, SpainFax: +34-913720623E-mail: [email protected],Homepage: http://www.icmm.csic.es/Supporting information for this article is available on theWWW under http://dx.doi.org/10.1002/ejic.201200582.

Eur. J. Inorg. Chem. 0000, 0–0 © 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1

composites. For this purpose, alginate was chosen as a modelpolymer matrix, as it forms suitable self-supporting films andis a biocompatible and biodegradable material; but one ofits main drawbacks for applications is its high hydrophiliccharacter. In a similar way to conventional organoclays thatare based on alkylammonium compounds, the zein–clay bio-hybrids reported here were effective in improving severalfeatures of the biopolymer matrix, mainly those related tomechanical and barrier properties. In addition, the nontoxiccharacter of zein-based bioorganoclays is an advantageousfeature that would allow the application of these new eco-friendly materials in the food-packaging sector.

zymes.[6] In this way, the search for new clay-based bio-hybrids seems to be a promising line of research with a viewtoward replacing common organoclays for some of theirpresent uses and also in the search for new applications.

In this perspective, we propose in this work the develop-ment of new biohybrid materials that are based on the as-sembly of zein protein to sepiolite and palygorskite fibrousclays to reduce their hydrophilic character. Hence, analo-gously to organoclays based on layered silicates, clay min-erals of fibrous morphology such as sepiolite or palygorsk-ite (Figure 1) have also been employed to prepare or-ganoclays for different applications.[7] In this case, the or-ganic compounds interact directly with the external surfaceof the silicate as these clays do not exhibit intercalationproperties. Instead, they offer various interesting character-istics: a large specific surface area and microporosity(around 320 and 150 m2 g–1 for sepiolite and palygorskite,respectively),[8] the presence of OH groups at their externalsurface, and the possibility of functionalization for the in-troduction of new properties.[7] A remarkable characteristicof sepiolite and palygorskite is their well-known ability toadsorb a large variety of molecular and polymeric organiccompounds,[8] including macromolecules of biological ori-gin such as structural or functional proteins,[9–12] to affordthe denoted biohybrid materials in the latter cases. In thisway, the adsorption of the storage protein zein by both fi-brous silicates was also expected, and it was confirmed bya preliminary study.[13–15] Zein, the major storage proteinof corn, is classified as a globular protein that contains alarge fraction of nonpolar amino acid residues[16] andshows an average hydrophobicity that is 50 times larger


A. C. S. Alcântara, M. Darder, P. Aranda, E. Ruiz-HitzkyFULL PAPERthan albumin or fibrinogen. Zein protein is neither solublein pure water nor in alcohol since it contains both hydro-philic and hydrophobic character, and therefore it is solublein aqueous solutions of ethanol (60–95% v/v).[17] Zein has amolecular weight (Mr) between 23000 and 27000 daltons,[18]

and has been extensively used as a component in adhesives,fibers, chewing gum, cosmetic powders,[17] in the prepara-tion of drug-delivery matrices alone[19] or in combinationwith other systems,[20] and as an edible coating for foodproducts as well as biodegradable plastics that form biode-gradable films that act as a barrier to moisture and oxygendue to their strong hydrophobicity.[21–25] Therefore, theseinteresting properties of zein might prove beneficial in re-ducing the hydrophilic character of pristine clays, as hap-pens in the case of conventional organoclays that are basedon long-chain alkylammonium cations.

Figure 1. Schematic representation of the structures of (a) sepioliteand (b) palygorskite fibrous clay minerals.

In this work, the improved properties of water resistanceof zein–fibrous clay bioorganoclays when used as bioaddi-tive in biopolymer matrices have been tested in bionano-composites by selecting the polysaccharide alginate as poly-mer matrix. This application has been chosen because natu-ral polymers such as polysaccharides and proteins are thesubject of considerable attention for the development ofgreen plastics on account of their availability, low cost, highbiocompatibility and biodegradability, as well as their goodfilm-forming ability and flexibility in most cases.[26–28]

However, much work is still needed to improve the mechan-ical and physical properties as well as the low water resis-tance of this type of biopolymer films[29,30] to allow theiruse in wet environmental conditions.[31] In the present case,these novel bioorganoclays based on zein–fibrous clay sys-tems afford both the reinforcing role and the enhancementof water-barrier properties due to the presence of zein.

Results and Discussion

Zein–Clay Biohybrids

Zein–clay biohybrids have been prepared by direct ad-sorption of the protein on the two fibrous silicates, sepiolite(SEP) and palygorskite (PALY). Figure 2 shows the adsorp-tion isotherms (23 °C) of zein on sepiolite and palygorskitefrom ethanol/water solutions (80% v/v). In both cases, theadsorption isotherms show a sharp slope at low equilibriumconcentration values that fit an H-type isotherm, a special

www.eurjic.org © 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Inorg. Chem. 0000, 0–02

case for the L-type curve according to the Giles classifica-tion of isotherms.[32] This behavior is indicative of a highaffinity between the sepiolite and palygorskite substratesand the zein adsorbate. Both curves reach a plateau thatcorresponds to values of 25.0 and 14.7 g of zein per 100 gof sepiolite and palygorskite, respectively. According to theLangmuir model, these values are indicative of zein adsorp-tion as a monolayer providing complete coverage of the sili-cate surface. It has been observed that for equilibrium con-centrations higher than 3000 mgL–1, the adsorbed zeinamounts are greater than the values in the plateau. Thisbehavior might be related to the formation of molecularaggregates on the silicate surface, as will be discussed below.

Figure 2. Adsorption isotherm at 23 °C of zein from ethanol/water(80% v/v) solution on sepiolite and palygorskite. Adsorptionamounts were deduced from C, H, N, and S chemical analyses ofthe biohybrid solids.

Table 1 summarizes the zein–clay biohybrids prepared inthis work, including the initial amounts of zein employed intheir synthesis as well as the respective amount of adsorbedprotein on each one. The code number assigned to eachsample indicates the approximate content of zein (Z) ingrams per 100 g of inorganic solid. Comparing the valuesin Table 1, it is clearly observed that at equal initial concen-trations of zein, the amount of retained protein is greaterwhen the substrate is sepiolite. This result can be explainedby the higher specific surface area of this clay mineral thanpalygorskite, which in turn provides a larger specific areafor the protein adsorption. Samples prepared from solu-tions with very high zein content result in biohybrid materi-als with adsorbed zein that might exceed 50.0 and 28.0 gper 100 g of clay in sepiolite and palygorskite, respectively.These values are considerably higher than those that corre-spond to the plateau in the adsorption isotherms. This factcan be attributed to the surface coverage of the clays byseveral layers of protein or, most likely, to the formation ofmolecular aggregates in solution at high concentrations ofzein, which is a characteristic ability of this protein,[33] thatmight be directly adsorbed onto the silicate surface, or tothe existence of more complex processes related to forma-tion of zein microphases in ethanol/water media.[34]



Table 1. Zein–sepiolite (Z-SEP) and zein–palygorskite (Z-PALY) biohybrids prepared in this work by adsorption of zein from ethanol/water (80 % v/v) solutions that contained different initial amounts of zein.

Starting amounts Zein–sepiolite Zein coverage Zein–palygorskite Zein coverage(g zein/100 g clay) biohybrid codes (g zein/100 g SEP)[a] biohybrids codes (g of zein/100g PALY)[a]

10.0 Z-SEP10 9.85�0.08 Z-PALY9 9.30�0.1720.0 Z-SEP16 16.3�1.1 Z-PALY12 11.9�0.340.0 Z-SEP20 19.7�0.1 Z-PALY13 13.0�1.166.6 Z-SEP24 23.5 �1.5 Z-PALY14 14.0�0.9100.0 Z-SEP25 25.0�0.6 Z-PALY15 14.7�0.8166.0 Z-SEP29 29.2�1.4 Z-PALY18 17.8�1.4333.3 Z-SEP54 53.6 �1.3 Z-PALY21 20.8�1.7500.0 Z-SEP48 47.8�1.1 Z-PALY28 28.4�1.3

[a] Data are the average value from n = 3 C, H, N, and S elemental chemical microanalyses of the biohybrid solids.

The significant amounts of adsorbed zein, as shown inboth isotherms, suggest that a large part of the clay surfacemight be covered by the protein. As shown in Table 2, thespecific surface area [N2, Brunauer–Emmett–Teller (BET)]of the starting clays, 340 m2 g–1 in the case of sepiolite and186 m2 g–1 in palygorskite, was considerably reduced in thebiohybrid material on account of the protein adsorptionand reached values as low as 22 and 19 m2 g–1 for the bio-hybrids Z-SEP48 and Z-PALY28, respectively, which hadthe highest zein content. Given that both clays exhibitstructural micropores, the dimensions of which are only ac-cessible to small molecules (such as the N2 used in the spe-cific surface-area measurements), adsorption of the volumi-nous zein polypeptide chains inside the tunnels of theseclays is not possible and so it could take place only on theirexternal surface[9] (ca. 150 m2 g–1 in sepiolite and 120 m2 g–1

in palygorskite).[8,35] Table S1 in the Supporting Infor-mation presents the amount of adsorbed protein per m2 ofeach clay. In both cases the adsorbed amount is very sim-ilar, which suggests similar interaction sites in both clays.Although they are not able to penetrate the nanosized tun-nels, the important decrease in specific surface area of thebioorganoclays with respect to that of the starting clay min-erals confirms that the protein molecules block the accessof nitrogen to the nanopores during the BET measure-ments. The decrease in the specific surface-area values aszein content increases points to the agglomeration of par-ticles through the action of this biopolymer.

Table 2. Zein–sepiolite and zein–palygorskite biohybrids preparedin this work by adsorption of zein from ethanol/water (80% v/v)solutions that contain different initial amounts of zein.

Samples Specific surface area [m2 g–1]

Z-SEP16 147Z-SEP25 47Z-SEP48 22Z-PALY12 79Z-PALY15 36Z-PALY28 19

The FTIR technique provides information on the cover-age degree of the silicates by zein. The FTIR spectra in the4000–500 cm–1 wavenumber range of the starting compo-nents, zein, sepiolite, and palygorskite, together with thosecorresponding to two selected biohybrids are included inFigure 3. The spectrum of zein (Figure 3, a) presents the

Eur. J. Inorg. Chem. 0000, 0–0 © 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.eurjic.org 3

characteristic bands of proteins, amine (3310 cm–1), amide I(1658 cm–1), and amide II (1538 cm–1), which are associatedwith the presence of zein predominantly in the α-helixstructure.[18] In the same spectrum, the stretching vibrationbands in the 2950–2850 cm–1 range that are assigned to CHgroups can be also distinguished.[36] The spectrum of thepristine sepiolite (Figure 3, b) presents the characteristicbands of this silicate, including the broad band around3600 cm–1 that is assigned to the stretching vibrations (νOH)of zeolitic water, and a group of bands around 1626 to1615 cm–1 that is attributed to the bending vibrations(δHOH) of coordinated water molecules. Other characteristicbands are those that appear at 3720 cm–1, which are as-signed to the OH stretching vibrations of silanol groups(Si–OH) located on the external surface of the silicate, andthe band at 3680 cm–1 that is attributed to the stretchingOH vibrations of Mg–OH groups located inside the tunnelsof sepiolite.[9] Perturbations in the OH stretching vibrationsof silanol groups are often used to prove the existence ofinteractions between adsorbed species and the sepiolite sur-face;[37,38] they are also applicable in proving the interactionin diverse biohybrids.[6,39]

In the IR spectrum of the Z-SEP24 biohybrid sample(Figure 3, c), the amide I band of zein at 1658 cm–1 seemsto be overlapped with the characteristic bands of the bend-ing vibration modes of water in sepiolite from 1658 to1615 cm–1, which makes the interpretation of a possible in-teraction between these groups very difficult. In addition,the presence of other characteristic groups of the protein isclearly observed in the synthesized biohybrids, such as theamide II band at 1532 cm–1. An important fact observed inthe spectrum of this biohybrid (Figure 3, c) is the pertur-bation in the band at 3720 cm–1, which is associated withthe interaction of the silanol groups in the silicate with theadsorbed protein through hydrogen bonding;[39] it causesthis band to become barely visible. Similar results have beenobserved for the Z-PALY14 biohybrid (Figure 3, e), fromwhich it is possible to observe the presence of characteristicbands of the amide I and amide II at 1660 and 1530 cm–1

that correspond to zein. Even in this spectrum, a strongperturbation in the 3710 cm–1 band can be observed; it isassigned to the OH stretching vibration νSiOH of the silanolgroup in the natural palygorskite (Figure 3, d). The de-crease in the intensity of the bands at 3720 and 3710 cm–1


A. C. S. Alcântara, M. Darder, P. Aranda, E. Ruiz-HitzkyFULL PAPER

Figure 3. FTIR spectra in the 4000–500 cm–1 region of (a) zein, (b) sepiolite, (c) Z-SEP24 biohybrid, (d) palygorskite, and (e) Z-PALY14biohybrid. Inset (on the left) shows a magnification of the 3760 to 3650 cm–1 region.

in the sepiolite and palygorskite, respectively, can be corre-lated to the degree of coverage of the silicate surface byzein. In contrast, the bands that are characteristic of OHstretching vibrations of Mg–OH at 3680 cm–1 in sepiolite(Figure 3, b�) and at 3698 cm–1 in palygorskite (Figure 3,d�) remain unaltered in the respective biohybrids (Figure 3,c� and e�), even at high amounts of adsorbed zein. As dis-cussed above, these groups are located inside the talclikestructural blocks of both fibrous clays and therefore be-come inaccessible to the adsorbed species. These results canbe better observed in Figure S1 of the Supporting Infor-mation, which shows the variation of the relative intensityof these silanol bands with respect to those of Mg–OH asa function of the amount of adsorbed protein. Results inFigure S1 of the Supporting Information show a gradualcoverage of the silicate surface with the increase of ad-sorbed zein; complete coverage of the inorganic surface isachieved at approximately 23.5 g zein/100 g sepiolite and14.8 g of zein/100 g palygorskite, respectively.

Solid-state high-resolution NMR spectroscopy was alsoapplied to characterize the zein–clay biohybrids (Figure 4).The 13C NMR spectrum of zein (Figure 4, a) shows thetypical protein signals at δ = 174 ppm that are assigned tocarbonyl carbon atoms, from δ = 140 to 100 ppm thatcorrespond to the amino acid aromatic side-chain residues,from δ = 70 to 45 ppm on account of the α-carbon atoms,and signals from δ = 45 to 15 ppm that are assigned to theamino acid aliphatic side chains.[36,40] The chemical shift ofcarbonyl groups at δ = 174 ppm, which is sensitive to thesecondary structure, could indicate high α-helix content,thus corroborating the results from FTIR analysis.[36] In the13C NMR spectra of Z-SEP biohybrids, the characteristic


signals of zein increase with the degree of coverage of thesilicate surface by protein. However, the spectra of the hy-brid compounds Z-SEP16 (Figure 4, d) and Z-SEP24 (Fig-ure 4, c) show a very low signal-to-noise ratio on accountof the low carbon content, which is in accord with the re-sults of elemental chemical analysis of these compounds(7.4 and 9.9% of C, respectively). Because the spectra have

Figure 4. 13C NMR spectra of (a) pure zein, (b) Z-SEP48, (c) Z-SEP24, and (d) Z-SEP16.



a poor signal-to-noise ratio, it makes it very difficult to statethis point. Despite this difficulty, a small shift of the δ =174 ppm signal to lower ppm can be detected from the spec-tra of the biohybrids, which suggests an interaction pointof zein with the silanol groups (Figure S2 in the SupportingInformation). This effect is more pronounced for the bio-hybrids with lower zein content. Thus, from the spectrumof sample Z-SEP24 (Figure 4, c) it is possible to observe anew signal at δ = 163 ppm, which suggests a perturbationof the carbonyl groups. This fact could be related to theirinteraction with sepiolite Si–OH groups in an analogousway to that reported for zein–alginate systems.[20] This is incontrast to the Z-SEP48 material (Figure 4, b), which pres-ents a spectrum similar to starting zein, thus indicating thata large part of the macromolecule does not interact with thesilicate surface. This fact might be related to the existenceof adsorbed protein in multilayers or aggregates with slightinteraction with the sepiolite surface, since this sample wasprepared with very highly concentrated zein solutions.However, a new peak at δ = 29 ppm was observed; it wasrelated to the amino acid groups, thus corroborating theexistence of interactions between the protein and the Si–OH groups in the sepiolite surface. A similar effect cannotbe deduced for other samples due to the low resolution oftheir spectra in that region.

FESEM techniques allowed us to observe the differencesin the morphology of the biohybrids relative to the startingzein. This latter presents globular aggregates (Figure 5, a)that result from the strong zein–zein and zein–solvent inter-actions during the drying process, which leads to the forma-tion of thermodynamically stable aggregates, also reportedby Wang et al.[34,41,42] In contrast, it is not possible to dis-tinguish the presence of this type of protein agglomerate inthe biohybrid materials (Figure 5, b and c). In both cases,the silicate fibers appear to be well integrated into the struc-ture of the zein, thus making the protein–protein interac-tion that would cause the formation of aggregates difficult

Figure 5. FESEM images of (a) zein protein and the biohybrids (b)Z-PALY28 and (c) Z-SEP54. (d) TEM image of Z-SEP54.


due to a considerable interaction between the clay fibersand the protein, as already pointed out by FTIR and NMRspectroscopic results. However, a careful analysis of theimages of the sample with the highest zein content, Z-SEP54 (Figure 5, c), reveals the presence of small spheresthat could be attributed to this protein appearing as segre-gated particles. These small spheres justify the high value ofadsorbed zein found for this sample from chemical analysisresults (Table 1). TEM images of the same sample (Fig-ure 5, d) corroborate the presence of these small zein aggre-gates in the biohybrids that contain high amounts of theprotein.

The thermal properties of zein–clay biohybrids evaluatedby thermogravimetric/differential thermal analysis (TG-DTA) show good stability up to 300 °C, at which pointpolymer degradation starts (Figure S3 in the Supporting In-formation). The curves are quite similar for all the biohyb-rid samples and differ from that of pure zein, which is stableup to approximately 500 °C. This effect might be attributedto the different conformation of zein in interaction with se-piolite as discussed above. Concerning water-sorption prop-erties, it is remarkable that the assembly of zein into thepristine clay reduces the hydrophilic character of the silicate(Figure 6), although to a lesser extent than would be ex-pected due to the significant hydrophobicity of this protein.Figure S4 in the Supporting Information shows the fittingcurves by using the Park equation[43] [Equation (S1) in theSupporting Information], and the fitting parameters aresummarized in Table S2 in the Supporting Information. Interms of the Langmuir sorption region, the decrease in theLangmuir capacity constant [AL in Equation (S1)] as theamount of associated zein increases in the biohybrids pointsto a lower amount of adsorbed water with respect to pris-tine sepiolite, which indicates the reduction of sites forwater sorption. This fact is also supported by the analysisof the experimental kinetics data. As shown in Figure S5 inthe Supporting Information, sorption kinetics in the bio-

Figure 6. Moisture-sorption isotherms of pristine sepiolite and zeinprotein and different Z-SEP bioorganoclays.


A. C. S. Alcântara, M. Darder, P. Aranda, E. Ruiz-HitzkyFULL PAPERhybrids are slower across all the water-activity range thanthose observed for pristine sepiolite, and closer to that ofzein. Thus, it seems evident that the presence of zein in thebiohybrids affects the water sorption, which suggests theless hydrophilic character of these samples instead of amarked hydrophobic behavior. It is known that zein aggre-gates that are formed in aqueous ethanol have their hydro-phobic regions oriented towards the center of the aggregate,thus exposing the hydrophilic region.[44] This arrangementmight be responsible for the reduced hydrophilic characterof the biohybrids.

Zein–Clay Biohybrids as Additives in Bionanocomposites

Biohybrids could be advantageous for different applica-tions, particularly in the field of nanocomposites as an eco-logical alternative to alkylammonium-based organoclays.For this application, stability tests of these biohybrids werecarried out. The washing tests of the biohybrid Z-SEP48 inpure water provoked a 2.6 % weight loss. This result indi-cates that zein–clay hybrids show good stability in water,which is in agreement with the strong interaction betweenboth components. Thus, zein–clay bioorganoclays havebeen tested here as fillers or additives to polymer matricesin the preparation of nanocomposites, with the aim of prof-iting from their reduced hydrophilicity relative to pristineclays. This property might help improve the features ofpolymers of biological origin such as polysaccharides orproteins, which show high hydrophilicity and low stabilityin water, while at the same time the filler might modify themechanical properties of the polymer. Thus, the polysac-charide alginate was chosen as a model biopolymer matrixto test the efficiency of zein–clay bioorganoclays as addi-tives in the development of bionanocomposites. To this end,ternary bionanocomposite films were prepared by disper-sion of the zein–sepiolite (or zein–palygorskite) biohybridcompounds within an alginate matrix. For the sake of com-parison, alginate (ALG) bionanocomposites using neat se-piolite as filler as well as alginate films without incorpora-tion of biohybrid particles were also evaluated. The re-sulting self-standing films show considerable homogeneityand transparency, independent of the amount of bioorgan-oclays and the type of the latter incorporated in the alginatematrix.

Tensile modulus (E) and elongation at break (Eb) can beused to describe the mechanical properties of the films,which are studied in relation to the arrangement of the

Table 3. Tensile properties of the alginate films loaded with zein–sepiolite and zein–palygorskite bioorganoclays.

Biohybrid filler 1:1[a] 1:2[a] 1:3[a]

E [GPa] Eb [%] E [GPa] Eb [%] E [GPa] Eb [%]

Z-SEP16 3.79�0.95 8.84�2.03 3.02�0.98 9.62�2.03 2.58�1.04 9.24 �1.44Z-SEP24 2.76�0.87 15.43�1.01 2.33�0.84 15.21�1.89 2.06�0.95 14.94� 1.77Z-SEP48 2.40�0.89 20.09�1.11 2.00�1.04 19.30�2.44 1.95�0.97 19.90� 2.02Z-PALY12 1.86�0.24 5.78�1.58 1.47�0.98 6.90�1.56 1.33�1.00 6.06�1.52Z-PALY14 1.62 �0.75 10.39�2.04 1.35�1.02 10.06�2.14 1.22�0.78 10.36�2.66Z-PALY28 1.48 �1.00 12.98�1.53 1.25�0.99 12.59�2.12 1.00�0.83 12.98�0.09

[a] Alginate/[zein–fibrous clay] ratio.


components. Tensile modulus indicates the stiffness of thematerial, whereas elongation represents the capacity of thefilm for stretching. The pure alginate and pure zein films(2 % w/v) have a tensile modulus of approximately 3.9 and0.5 GPa, respectively. The incorporation of pure zein intoalginate diminishes both tensile modulus and elongation atbreak; it presents values of 1.12 GPa and 3.7%, respectively,for alginate film loaded with 25% of zein (w/w). Table 3shows the tensile modulus and elongation at break valuesof the obtained alginate films with and without the zein–clay biohybrid additive. In both ALG/Z-SEP and ALG/Z-PALY film systems, the increase in zein content in the algin-ate films by incorporating bioorganoclays with a higheramount of zein causes a slight decrease in the tensile modu-lus values, but at the same time, the values of elongation atbreak increase considerably. Similar results were observedwhen increasing the mass ratio of a given biohybrid in thealginate film. This effect indicates that the addition of bi-ohybrids increases the flexibility and stretchability of thebiopolymer matrix, thereby resulting in a plastic behaviorof the alginate-based bionanocomposite film. Sepiolitealone acts a filler to enhance the modulus (e.g., 5.1 GPa inalginate films that contain 50 % sepiolite) but it diminishesthe plastic behavior of the system by up to 3.3%.[45] Thisbehavior points out that zein might act as a certain plasti-cizer that significantly affects the mechanical properties andcauses a slight decrease in the tensile modulus and the rel-evant increase in the plastic properties in the final material.However, it is worth mentioning that the tensile modulusmeasured for ALG/Z-SEP and ALG/Z-PALY films yieldedminimum values of 1.95 and 1.00 GPa, respectively,whereas other reinforced biopolymer materials such asstarch[46] or cellulose[47] often present values below 1 GPa.

Given that one of the major drawbacks in the use ofpreviously studied alginate systems as bioplastics is theirwater-absorption tendency, any improvement in water resis-tance is highly important. In this way, the reduced hydro-philicity of the zein–clay bioorganoclays mentioned abovemight contribute to a decrease in the water uptake of algin-ate films. Figure 7 shows the weight increase due to wateruptake�expressed as grams of water incorporated per gramof dry sample�of the pure alginate film and its bionanoc-omposites with Z-SEP and Z-PALY as a function of theexposure time in bidistilled water (pH 5.5). For all thetested films, the amount of adsorbed water reaches a nearlyconstant content after around 1–2 h. In contact with bidis-tilled water, the pure alginate film reaches a maximum water



uptake of around 1.2 g of water per gram of film afterabout 3 h. After this time, the film disintegrates, thus mak-ing subsequent measurements impossible. The water-uptakeproperties of alginate films are influenced by the content ofzein in the Z-SEP or Z-PALY biohybrids that are used asbioadditives. In both systems, water uptake decreases whenthe zein content increases; it reaches values of 0.54 and0.80 gg–1 for films of alginate that contain Z-SEP48 and Z-PALY28 bioorganoclays, respectively, in a 1:1 ratio. The useof neat sepiolite as filler leads to water-uptake values closeto those of samples with the lowest zein content (ca.0.9 g g–1 film),[45] thus proving the effect of zein on the ob-served behavior. Similar results were reported for studieson the incorporation of zein protein in starch films, whichresulted in more water-resistant materials on account of thehydrophobic character of this protein.[24]

Figure 7. Effect of (a) zein–sepiolite and (b) zein–palygorskite bio-organoclays on the water uptake of alginate films with a 1:1 contentof nanofiller exposed to deionized water (pH 5.5).

Similar to water absorbency, the passage of water vaporthrough alginate films is altered by the presence of zein inthe bioorganoclay that is used as filler. The water-vaportransmission rate (WVTR) values of the pure alginate and


the bionanocomposite films are shown in Figure 8. The ob-tained values are in agreement with the observations in thewater-uptake study. Here again, the WVTR of bionano-composite films changed significantly depending on typeand concentration of incorporated biohybrid. The WVTRof bionanocomposite films decreases significantly de-pending on the type and the amount of the bioorganoclayused, and in all cases these values are lower than that whichcorresponds to the unmodified alginate film. Alginateloaded with neat sepiolite shows WVTR values of approxi-mately 0.88 mgh–1 cm–2,[45] which is close to those of analo-gous films that incorporate biohybrids with the lowest con-tent in zein (Figure 8, a). In this way, it seems that the zeincontent afforded by the biohybrid to the bionanocompositefilm is responsible for the barrier properties. Actually, theamount of zein in the bioorganoclay plays the major rolein the reduction of the passage of water vapor. It is clearthat Z-SEP biohybrids are more effective in reducing watervapor than Z-PALY biohybrids on account of their abilityto uptake a greater amount of zein.; the most effective sys-tem is the one that incorporates the Z-SEP48 biohybrid in a1:3 proportion of alginate/biohybrid. The increase in water-vapor barrier properties of alginate/zein–clay bionanoc-omposite films can be attributed not only to the tortuouspath of water-vapor diffusion due to homogeneous distribu-tion of the biohybrid particles within the biopolymer ma-trix, which consequently increases the effective diffusionpath length, but also to the hydrophobic character affordedby the presence of zein. The protein can exert a barrier atthe interface of the film and the medium, thereby reducingthe amount of incorporated water molecules, as previouslyobserved in drug-delivery beads prepared by the combina-tion of alginate and zein.[20] Thus, zein-based bioorgano-clays can be a promising alternative for increasing the bar-rier and water-resistance properties in hydrophilic matrices,with the possibility of preparing a wide variety of biohyb-rids with different zein content. This allows the develop-ment of bionanocomposites in which the properties can betuned as a function of the desired requirements.

Figure 8. Water-vapor transmission rate (WVTR) of alginate-basedbionanocomposite films incorporating (a) zein–sepiolite and (b)zein–palygorskite bioorganoclays at different alginate/biohybrid ra-tios.



Conclusion

The combination of the protein zein with sepiolite andpalygorskite fibrous clay minerals gives rise to new biohyb-rid materials in which the protein is assembled on the exter-nal surface of these clays. The amount of adsorbed zeincan be tuned to ensure the complete coverage of the fibers,although zein aggregates can be found when the biohybridsare formed at high equilibrium concentration of zein. Sepi-olite-based biohybrids show a larger amount of assembledprotein than those based on palygorskite due to the higherexternal surface area of the former silicate. The assemblingmechanism is related to the interaction of amide groups ofzein with silanol groups on the surface of the fibrous min-erals. The zein coverage reduces the hydrophilicity of theclays and confers new properties onto the resulting biohyb-rids, which might therefore be employed in diverse applica-tions. We have therefore evaluated these novel biohybrids asadditives in alginate films. The resulting alginate/zein–clayternary bionanocomposite films show interesting propertiessuch as improved flexibility with a slight detriment to theirmodulus as well as good water-vapor barrier properties andreduced water uptake in comparison to pristine alginateand alginate–sepiolite films. In conclusion, zein-based bio-organoclays are promising materials as bioadditives for dif-ferent polymeric matrices, because it is a green alternativeto currently employed synthetic fillers that are mainly usedfor food-packaging applications.

Experimental SectionStarting Materials and Reagents: Zein from maize (Z) and sodiumalginate (ALG) were purchased from Sigma–Aldrich. Sepiolitefrom Vicálvaro (Spain) commercialized as Pangel S9 (SEP) byTOLSA, S.A. and Brazilian palygorskite (PALY) kindly providedby Prof. L.S. Barreto (Universidade Federal de Sergipe) were usedas fibrous clay minerals. Absolute ethanol was supplied by Panreac.Deionized water (resistivity of 18.2 MΩcm) was obtained with aMaxima Ultrapure Water from Elga.

Preparation of Zein–Clay Biohybrids: Sepiolite and palygorskitesuspensions (6% w/v) were prepared in ethanol/water (80%, v/v),and a vigorous stirring was applied with a mixer (G2 model, Lomi)to properly disperse the clay. Different amounts of zein were dis-solved in an 80% (v/v) aqueous ethanol solution (50 mL) to pre-pare a set of alcoholic solutions with zein concentration rangingbetween 0.6 and 30.0 gL–1. Each zein solution was added to thesepiolite or palygorskite dispersion (50 mL), and the mixture wasstirred for 48 h at room temperature. The solid product was isolatedby centrifugation and dried overnight at 40 °C. The biohybrid ma-terials zein–sepiolite and zein–palygorskite were denoted as Z-SEPand Z-PALY, respectively.

Preparation of Alginate/Zein–Clay Bionanocomposite Films: The al-ginate films loaded with zein–clay bioorganoclays as additives wereprepared by using the following general procedure: A necessaryamount of biohybrid to achieve weight ratios of alginate/biohybrid1:0, 1:1, 1:2, and 1:3 were dispersed in deionized water by vigorousstirring for 24 h with a magnetic stirrer at room temperature. Next,the biohybrid dispersion was gradually added to the previously pre-pared alginate dispersion (2% w/v), thus forming a single batchthat was kept under constant stirring overnight. Finally, the re-


sulting bionanocomposite was placed on a glass plate and allowedto dry at room temperature. The films prepared from alginate thatincorporated zein–sepiolite and zein–palygorskite bioorganoclayadditives were denoted as ALG/Z-SEP and ALG/Z-PALY, respec-tively.

Characterization: FTIR spectra were recorded with a Bruker IFS66v/S FTIR spectrophotometer. The samples in film form or di-luted in KBr as pellets were placed in the sample holder andscanned from 4000 to 250 cm–1 with 2 cm–1 resolution. The amountof organic matter in the samples was determined by C, H, N, andS elemental chemical microanalysis with a Perkin–Elmer 2400 ana-lyzer. The thermal behavior of the different prepared materials wasanalyzed from the simultaneously recorded thermogravimetric(TG) and differential thermal analysis (DTA) curves with a SeikoSSC/5200 instrument in experiments carried out under air atmo-sphere (flux of 100 mLmin–1) from room temperature to 1000 °Cat a heating rate of 10 °C min–1. Solid-state 13C CP magic-anglespinning (MAS) NMR spectra were obtained with a Bruker Avance400 spectrometer by using a standard cross-polarization (CP) pulsesequence. Samples were spun at 10 kHz. A contact time of 2 msand a period between successive accumulations of 5 s were used.The number of scans was 800. Chemical shift values were refer-enced to tetramethylsilane (TMS). The specific surface areas of thebionanocomposites and starting clays were determined by applyingthe single-point BET method to the adsorption/desorption of N2

at 77 K (Micromeritics Flowsorb II 2300). Surface morphology wasobserved with a FEI-NOVA NanoSEM 230 FESEM instrument,which allowed the semiquantitative analysis of elements. Samplepreparation was performed by adhering the samples on a carbontap for direct observation without requirement of any conductivecoating on the surface. TEM was carried out with a LEO-910 in-strument operating at an accelerating voltage of 80 kV.

Moisture-Adsorption Isotherms: Moisture sorption was measuredby means of an Aquadyne DVS dynamic water-vapor sorption in-strument from Quantachrome. Moisture-sorption isotherms wererecorded at 25 °C in the range of relative humidity from 0 to 95%by using amounts of samples around 10 mg.

Mechanical Properties: Mechanical properties such as tensile mod-ulus (E) and percent elongation at break (Eb) of the film sampleswere evaluated with a Model 3345 Instron Universal Testing Ma-chine (Instron Engineering Corporation Canton, MA, USA) ac-cording to ASTM standard method D 882-88. The samples withrectangular shape (ca. 60 mm�15 mm) were mounted between thegrips with an initial separation of 50 mm, and the cross-head speedwas set at 5 mmmin–1. Three replicates were run for each film sam-ple.

Water-Uptake Determination: The alginate/zein–clay films wereplaced in a petri dish, immersed in distilled water, and shaken occa-sionally at room temperature. After a predetermined time interval,the films were withdrawn and, after removing the excess amount ofwater, weighed on an analytical balance, as indicated by Remuñán-López and Bodmeier.[48] The water uptake can be calculated ac-cording to Equation (1) in which Wt and W0 are the wet and initialmass of films, respectively.

(g/g water uptake) = (Wt – W0)/W0 (1)

Water-Vapor Transmission Rate: The water-vapor transmission rate(WVTR) of the film samples was measured gravimetrically by usingthe modified ASTM method E 96-80. Circular test cups made ofPVC and with a diameter of 45 mm (test area: 15.8 cm2) were used.The cups that contained desiccant (silica gel, 14 g) were placed in



a desiccator that contained a saturated aqueous NaCl solution(75% rel. humitity, 22 °C). The moisture transmitted through thecomposite films was determined gravimetrically by weighing thecups initially and over a 72 h period (2, 8, 12, 24, 48, and 72 h;n = 3). The rate of the water-vapor transmission was obtained fromthe slope of the line that resulted from plotting the weight of trans-mitted water vapor versus time. The effects of the concentrationand the type of biohybrid incorporated on the water-vapor trans-mission of alginate films was investigated.

Supporting Information (see footnote on the first page of this arti-cle): Details about 13C NMR spectroscopy, a discussion of moist-ure-adsorption data, additional results about zein adsorbed ontosepiolite and palygorskite from FTIR spectra and C, N, H, and Sanalysis, and thermal analysis curves of pristine clays and biohyb-rids.

Acknowledgments

This work was supported by the Spanish Comisión Interministerialde Científica y Tecnológica (CICYT) (MAT2006-03356,MAT2009-09960) and the Consejo Superior de Investigaciones Ci-entíficas (CSIC) (project numbers 201060I009 and 2010MA0003;CSIC-Academie Hassan II). A. C. S. A. acknowledges CSIC for aJAE-Predoc fellowship. The authors also thank Dr. I. Sobradosand Mr. A. Valera for fruitful discussions of the NMR data andtechnical assistance in the FESEM studies, respectively.

[1] E. Ruiz-Hitzky, P. Aranda, M. Serratosa, in: Handbook of Lay-ered Materials (Eds.: S. M. Auerbach, K. A. Carrado, P. K.Dutta), Marcel Dekker, New York, 2004, pp. 91–154.

[2] Functional Hybrid Materials (Eds.: P. Gómez-Romero, C. San-chez), Wiley-VCH, Weinheim, Germany, 2004.

[3] E. Ruiz-Hitzky, M. Darder, P. Aranda, in: Bio-Inorganic HybridMaterials: Strategies, Syntheses, Characterization and Applica-tions (Eds.: E. Ruiz-Hitzky, K. Ariga, Y. Lvov), Wiley-VCH,Weinheim, Germany, 2008, pp. 1–40.

[4] E. Ruiz-Hitzky, P. Aranda, M. Darder, G. Rytwo, J. Mater.Chem. 2010, 20, 9306–9321.

[5] B. Wickein, M. Darder, P. Aranda, E. Ruiz-Hitzky, Langmuir2010, 26, 5217–5225.

[6] B. Wicklein, M. Darder, P. Aranda, E. Ruiz-Hitzky, ACS Appl.Mater. Interfaces 2011, 3, 4339–4348.

[7] E. Ruiz-Hitzky, P. Aranda, A. Alvarez, J. Santaren, A. Esteban-Cubillo, in: Developments in Palygorskite-Sepiolite Research. ANew Outlook of these Nanomaterials (Eds.: E. Galán, A.Singer), Elsevier, Oxford, 2011, pp. 393–452.

[8] Developments in Palygorskite-Sepiolite Research. A New Out-look of these Nanomaterials (Eds.: E. Galán, A. Singer), Elsev-ier, Oxford, 2011.

[9] E. Ruiz-Hitzky, J. Mater. Chem. 2001, 11, 86–91.[10] N. Olmo, M. A. Lizarbe, J. G. Gavilanes, Biomaterials 1987, 8,

67–69.[11] F. M. Fernandes, A. I. Ruiz, M. Darder, P. Aranda, E. Ruiz-

Hitzky, J. Nanosci. Nanotechnol. 2009, 9, 221–229.[12] F. M. Fernandes, M. Darder, A. I. Ruiz, P. Aranda, E. Ruiz-

Hitzky, in: Nanocomposites with Degradable Properties: Synthe-sis Properties and Future Perspectives (Ed.: V. Mittal), OxfordUniversity Press, New York, 2011, pp. 209–226.

[13] V. Caballero, F. M. Bautista, J. M. Campelo, D. Luna, J. M.Marinas, A. A. Romero, J. M. Hidalgo, R. Luque, A. Macario,G. Giordano, Proc. Biochem. 2009, 44, 334–342.

[14] A. C. S. Alcântara, M. Darder, P. Aranda, E. Ruiz-Hitzky, Ma-cla (J. Spanish Miner. Soc.) 2008, 9, 25–26.

[15] E. Ruiz-Hitzky, P. Aranda, M. Darder, A. C. S. Alcântara,Spanish Pat. WO/2010/146216, 2010.


[16] A. C. S. Alcântara, P. Aranda, M. Darder, E. Ruiz-Hitzky, Ab-stracts of Papers, 241st National Meeting and Exposition of theAmerican Chemical Society, Anaheim, CA, March 27–31, 2011,American Chemical Society, Washington, DC, 2011, vol. 241,PMSE 299.

[17] R. Shukla, M. Cheryan, Indust. Crops Prod. 2001, 13, 171–192.[18] Y. Wang, F. L. Filho, P. Geil, G. W. Padua, Macromol. Biosci.

2005, 5, 1200–1208.[19] H. J. Wang, Z. X. Lin, X. M. Liu, S. Y. Sheng, J. Y. Wang, J.

Controlled Release 2005, 105, 120–131.[20] A. C. S. Alcântara, P. Aranda, M. Darder, E. Ruiz-Hitzky, J.

Mater. Chem. 2010, 20, 9495–9504.[21] H. Takahashi, K. Yamada, N. Yanai, US Pat. 5,585,060, 1996.[22] J. Bai, V. Alleyne, R. D. Hagenmaier, J. P. Mattheis, E. A. Bal-

dwin, Postharvest Biol. Technol. 2003, 28, 259–268.[23] G. W. Padua, A. M. Rakotonirainy, T. T. Ha, US Pat.

0056388A1, 2004.[24] M. Gáspár, Z. Benkó, G. Dogossy, K. Réczey, T. Czigány, Po-

lym. Degrad. Stab. 2005, 90, 563–569.[25] B. Ghanbarzadeh, A. R. Oromiehi, Int. J. Biol. Macromol.

2008, 43, 209–213.[26] R. N. Tharanathan, Trends Food Sci. Technol. 2003, 14, 71–78.[27] E. Ruiz-Hitzky, M. Darder, P. Aranda, in: Annual Review of

Nanoresearch (Eds.: G. Cao, Q. Zhang, C. J. Brinker), WorldScientific Publishing, Singapore, 2010, vol. 3, pp. 149–189.

[28] a) Nanocomposites with Biodegradable Polymers. SynthesisProperties, and Future Perspectives (Ed.: V. Mittal), OxfordUniversity Press, New York, 2011; b) Environmental SilicateNano-biocomposites (Eds.: L. Avérous, E. Pollet), Springer-Ver-lag, London, 2012.

[29] L. S. Zarate-Ramírez, I. Martínez, A. Romero, P. Partal, A.Guerrero, J. Sci. Food Agric. 2011, 91, 625–633.

[30] A. Jerez, P. Partal, I. Martinez, C. Gallegos, A. Guerrero,Rheol. Acta 2007, 46, 711–720.

[31] J. W. Rhim, Food Sci. Technol. 2007, 5, 691–709.[32] C. H. Giles, T. H. MacEwan, S. N. Nakhwa, D. Smith, J.

Chem. Soc. 1960, 3973–3993.[33] S. Kim, J. Xu, J. Cereal Sci. 2008, 47, 1–5.[34] Y. Wang, G. W. Padua, Langmuir 2010, 26, 12897–12901.[35] S. Xue, M. Reinholdt, T. J. Pinnavaia, Polymer 2006, 47, 3344–

3350.[36] L. A. Forato, T. C. Bicudo, L. A. Colnago, Biopolymers 2003,

72, 421–426.[37] P. Aranda, R. Kun, M. A. Martín-Luengo, S. Letaïef, I.

Dékány, E. Ruiz-Hitzky, Chem. Mater. 2008, 20, 84–89.[38] J. L. Ahlrichs, C. Serna, J. M. Serratosa, Clays Clay Miner.

1975, 23, 119–124.[39] M. Darder, M. López-Blanco, P. Aranda, A. J. Aznar, J. Bravo,

E. Ruiz-Hitzky, Chem. Mater. 2006, 18, 1602–1610.[40] T. C. Bicudo, L. A. Forato, L. A. R. Batista, L. A. Colnago,

Anal. Bioanal. Chem. 2005, 383, 291–296.[41] Q. Wang, A. R. Yin, G. W. Padua, Food Biophys. 2008, 3, 174–

181.[42] Q. Wang, A. R. Crofts, G. W. Padua, J. Agric. Food Chem.

2003, 51, 7439–7444.[43] A. Bessadok, D. Langevin, F. Gouanvé, C. Chappey, S. Rou-

desli, S. Marais, Carbohydr. Polym. 2009, 76, 74–85.[44] K. Yamada, H. Takahashi, A. Noguchi, Int. J. Food Sci. Tech-

nol. 1995, 30, 599–608.[45] A. C. S. Alcântara, M. Darder, P. Aranda, E. Ruiz-Hitzky, ma-

nuscript in preparation.[46] C. M. O. Muller, J. B. Laurindo, F. Yamashita, Food Hydrocol-

loids 2009, 23, 1328–1333.[47] A. B. Dias, C. M. O. Muller, F. D. S. Larotonda, J. B. Laur-

indo, LWT Food Sci. Techn. 2010, 44, 535–542.[48] C. Remuñán-López, R. Bodmeier, J. Controlled Release 1997,

44, 215–225.Received: May 28, 2012

Published Online: �



Biohybrid Materials

The assembly of the hydrophobic corn pro- A. C. S. Alcântara, M. Darder, P. Aranda,*tein zein with fibrous clay minerals (sepiol- E. Ruiz-Hitzky ................................ 1–10ite and palygorskite) affords new biohyb-rids with reduced hydrophilic character, Zein–Fibrous Clays Biohybrid Materialswhich are useful as bioorganoclays to im-prove the mechanical and barrier proper- Keywords: Biohybrid materials / Zein /ties in polysaccharide-based nanocompos- Sepiolite / Palygorskite / Bionano-ites. composites


zein-fibrous clays biohybrid materials

Documents