Structure and Morphology of Wheat Gluten Films: From Polymeric

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Structure and Morphology of Wheat Gluten Films: From Polymeric Protein Aggregates toward Superstructure Arrangements Ramune Kuktaite,†,* Tom
as S. Plivelic,‡ Yngve Cerenius,‡ Mikael S. Hedenqvist,§ Mikael G€allstedt,|| Salla Marttila,^ Rickard Ignell,^ Yves Popineau,# Oliver Tranquet,# Peter R. Shewry,3 and Eva Johansson† †

)

Department of Agriculture - Farming Systems, Technology and Product Quality, The Swedish University of Agricultural Sciences, SE-230 53 Alnarp, Sweden ‡ MAX-lab, Lund University, SE-221 00 Lund, Sweden § School of Chemical Science and Engineering, Fibre and Polymer Technology, Royal Institute of Technology (KTH), SE-100 44 Stockholm, Sweden Innventia, Box 5604, SE-114 86 Stockholm, Sweden ^ Department of Plant Protection Biology, The Swedish University of Agricultural Sciences, SE-230 53 Alnarp, Sweden # INRA, UR 1268 Biopolymeres Interactions Assemblages, Rue de la G
eraudiere, BP 71627, 44316 Nantes Cedex 3, France 3 Plant Science Department, Rothamsted Research, Harpenden, United Kingdom

bS Supporting Information ABSTRACT: Evaluation of structure and morphology of extruded wheat gluten (WG) films showed WG protein assemblies elucidated on a range of length scales from nano (4.4 Å and 9 to 10 Å, up to 70 Å) to micro (10 μm). The presence of NaOH in WG films induced a tetragonal structure with unit cell parameters, a = 51.85 Å and c = 40.65 Å, whereas NH4OH resulted in a bidimensional hexagonal close-packed (HCP) structure with a lattice parameter of 70 Å. In the WG films with NH4OH, a highly polymerized protein pattern with intimately mixed glutenins and gliadins bounded through SH/SS interchange reactions was found. A large content of βsheet structures was also found in these films, and the film structure was oriented in the extrusion direction. In conclusion, this study highlights complexities of the supramolecular structures and conformations of wheat gluten polymeric proteins in biofilms not previously reported for biobased materials.

’ INTRODUCTION Wheat gluten (WG) proteins show a number of advantages to be used for biobased materials, that is, their abundance as a byproduct from the bioethanol fuel industry in Europe, low cost, good biodegradability, and ability to polymerize.1
3 WG bioplastic films from gluten/glycerol blends produced by extrusion reveal that temperature and mechanical energy input,4 components of the blend,5 and the pH of the blend6 strongly affect the oxygen permeability and mechanical behavior of the biobased materials. Furthermore, the processing methods used to produce biobased materials decisively cause structural variation in the physical and chemical properties of the polymer, that is, chain orientation, molecular weight, and crystallinity.7,8 Molecular interactions and polymerization behavior of the WG proteins stand out among the factors affecting the end-use quality of WG materials.6 The structure of different WG proteins has been evaluated by a number of approaches.9
12 The central repetitive domains of the high-molecular-weight subunits of glutenin (HMW-gs) and r 2011 American Chemical Society

possibly also the repetitive domains of other gluten proteins have been found to form β-sheets (strands connected laterally by hydrogen bonds into a twisted sheet), whereas the nonrepetitive domains composed of these proteins are globular with R-helices.13 The β-sheet assemblies are highly stable, implicating the formation of gluten protein aggregates in the processed solid material with high strength.14,15 Structural WG models supported by small-angle X-ray scattering (SAXS)9 and scanning tunnelling16 and atomic force microscopy techniques17 indicated that the HMW-gs have a rod-like β-spiral structure when in solution and as hydrated solid, with a length of about 50
69 nm and a diameter of about 2
6.4 nm.10,11,18
21 The gliadins, R-, γ-, and ω-, have been modeled as prolate ellipsoids with a diameter of ∼3.2 nm.10 Film manufacture is likely to result in a more complex microstructure of proteins and protein polymers. Received: October 26, 2010 Revised: March 24, 2011 Published: March 24, 2011 1438

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Biomacromolecules During extrusion and heat processing, the proteins denature. The final molecular network is formed through disulfide cross-linking of the WG proteins, that is, monomeric gliadins and glutenins, into a macropolymer.4,11 Therefore, the WG extrudate structure results from a complete restructuring of protein molecules into an oriented protein peptide pattern (secondary structure). Despite the number of studies on individual WG protein types,9
12 no clear molecular structure model of the WG protein polymer has been established, particularly not for the WG materials. In this study, we have investigated WG protein polymerization behavior and polymer structure and morphology in a selection of WG-based films with varying end-use properties, differing in chemical composition, processing method temperature, and time (aging). We have also studied the interactions among specific WG protein components such as HMW-gs, low-molecularweight glutenin subunits (LMW-gs), and gliadins in WG films by a multianalytical approach, that is, small- (SAXS) and wideangle (WAXS) X-ray scattering, infrared spectroscopy (IR), confocal laser scanning electron microscopy (CLSM), and reversed-phase high-performance liquid chromatography (RPHPLC). Here we present, for the first time to our knowledge, a 3D structure modular approach to explain the structure of WG polymeric proteins in WG films.

’ EXPERIMENTAL SECTION Materials. The WG films selected for the present study were those that have been shown by Ullsten et al.6,14 having interesting characteristics in terms of tensile, gas barrier, and protein solubility properties. The strength was close to 6 MPa14 for a glycerol-plasticized film, and the oxygen permeability at dry conditions was as low as 0.18 mL mm/(m2 day atm).14 The protein solubility was low6 or almost zero.14 The WG films contained WG powder, glycerol (purity of 99.5%), sodium hydroxide (NaOH), and ammonium hydroxide (NH4OH) and samples were extruded as described in Ullsten et al.14 (the extruder temperature profile was 120
65
60
40 C, and the screw speed was 200 rpm) and/or salicylic acid (SA) (99%) and were extruded by a single screw extruder (screw speed: 265 rpm, the set “target” die temperatures were 90, 105, or 120 C; indicated in Table 1, Supporting Information) into WG films. The NaOH pellets were ground to powder in a Retsch ZM 1 laboratory centrifugal grinding mill (Haan, Germany) and then dry-mixed with the WG powder. To obtain a homogeneous distribution of SA in the material, we first ground 10 g of SA in a mortar with an equal amount of gluten powder before blending it with the remaining WG powder. The powder and glycerol were subsequently blended using a food processor (WATT; DUKA AB, Sweden) at the lowest speed, “speed 1: about 95 rpm”, for 20 s and thereafter at “speed 3: about 200 rpm” for 1 min. The ammonia solution was added in a similar way as the grounded NaOH to the WG powder. The weight percent of SA, NaOH, and NH4OH was based on the total mass of gluten and glycerol. Three selected films containing the base material (70 wt % WG and 30 wt % glycerol) and 5 wt % of NaOH were aged 120 days at 23 C and 50% RH.6 Also, one sample, WGG, which was selected as a reference, contained 70 wt % WG and 30 wt % glycerol in the present study. The composition, processing, and aging of the selected films are shown in Table S1 (Supporting Information). Methods. Small-Angle X-ray Scattering. The SAXS experiments were carried out at the I711 beamline of the MAX-lab Synchrotron, Sweden.27 A monochromatic beam of 1.1 Å wavelength was used, and the sample to detector distance was 1403 mm for all studied samples. Two-dimensional pictures were obtained using an area-CCD detector (165 mm in diameter active area, from Marresearch, GmbH) with 10 min of exposure time. WG films of ca. 3  3 mm2 and 1 mm thickness

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Figure 1. Orientation of the WG films for the X-ray scattering experiments: (a) normal direction (ND), (b) machine direction (MD); T, the third principal sample axis; X, direction of the X-ray beam. were placed in a multiple position sample holder and measured in an evacuated chamber. Samples were analyzed with the incoming X-ray beam perpendicular to the film plane surface (normal direction, ND) (Figure 1a). When clear anisotropic SAXS patterns were observed, complementary measurements were done with the X-ray beam parallel to the extrusion machine direction (MD) (see Figure 1b). X-ray scattering data were analyzed with the program FIT2D.28 Average radial intensity profiles, as a function of the scattering vector q (q = 4π/λ sin(θ), where 2θ is the scattering angle, and λ is the wavelength), were obtained by integrating the data in the complete image for isotropic scattering patterns or in a 20 angular sector around the maximum in the oriented WG images. The anisotropic character of some samples was discussed using azimuthal plots, I(j) versus j, where j = 0 when the scattering vector was at the equator and j = 90 at the meridian. For comparison of the samples, the intensities were normalized by the integrated intensity incident on the sample during the exposure, and corrected for sample absorption, parasitic scattering, and detector background. Wide-Angle X-ray Scattering. Wide-angle X-ray scattering (WAXS) measurements were carried out at the 911-5 beamline of the MAX-lab Synchrotron, Sweden.29 The wavelength was 0.907 Å, and the sampleto-detector distance was 150.6 mm. Silicon powder was used as a calibration standard for peak positions. Two-dimensional images were registered using an area-CCD detector (165 mm in diameter active area, from Marresearch, GmbH) with 3 min of data acquisition. Parasitic scattering was subtracted from all diffractograms. WG films of ca. 15  15 mm2 size and 0.4 to 1 mm thickness were placed on the top of a goniometric head with the beam perpendicular to the film surface. The WG films WGG, 5-NaOH (120) 120, 7.5- NH4OH-1.5-SA (120), and 10-NH4OH-1.5-SA (120) were selected for WAXS analysis. Infrared Spectroscopy (IR). Infrared spectra were recorded using a Perkin-Elmer Spectrum 2000 FTIR spectrometer, Perkin-Elmer, USA, equipped with a single reflection ATR accessory, Golden Gate from Specac (Kent, England). The samples were dried for at least 72 h in a desiccator containing silica gel before being tested. Confocal Laser Scanning Microscopy (CLSM) Immunostaining. WG films perpendicular to the film plane sections were blocked for 5
8 h and incubated with the following antibodies: (1) 1439

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Figure 2. (a) SAXS profiles of 5-NaOH wheat gluten (WG) films, extruded at 90, 105, and 120 C and aged for 120 days and WGG (reference) extruded at 105 C. (b) SAXS pattern of the 5-NaOH WG film, extruded at 120 C, measured at extended q values. Final fitted peak positions (red lines) corresponding to a tetragonal unit cell. (c) WAXS profiles of 5-NaOH WG films, extruded at 90 and 120 C, and WGG film extruded at 105 C. (d) CLSM image of a 5-NaOH WG film extruded at 120 C and immunostained with two antibodies, one directed to monoclonal HMW-gs and the other directed to polyclonal gliadins (Sigma). HMW-gs are green, and gliadins are red; arrows indicate three different regions, that is, gliadins (red, arrow 1), glutenins (green, arrow 2), and these intimately mixed (red and green, arrow 3); scale 10 μm. A mixture of two primary antibodies, a monoclonal cell line 1601, for the high-molecular-weight glutenin subunits (HMW-gs) 2, 5, 10, and 12 (courtesy to C. Mills, Institute of Food Research, U.K.; 1601 is essentially identical to 1602 described in Mills et al.22) and a polyclonal gliadin antibody (Sigma-Aldrich, St. Louis, MO, USA); antibody for the HMW-gs subunits 2, 5, 10, and 12 was used because of the fact that these subunits are rather common and present in all commercial wheats. (2) A monoclonal low-molecular-weight glutenin subunits (LMW-gs) antibody (IFRN 006722, provided by Rothamsted Research, U.K.) and a polyclonal gliadin antibody and (3) LMW-gs antibody (monoclonal, mouse, clone F98 14, peptide from N terminal sequence of LMW-gs SHIPGLERPSGC, provided by INRA, Nantes, France) and a polyclonal gliadin antibody were used. Specimens were incubated with two primary antibodies on the rotator for about

16
18 h at room temperature, washed, secondly incubated with two secondary antibodies, Alexa 488 and Alexa 546 (Molecular Probes, Eugene, Oregon, USA), washed, and viewed by CLSM according to Wretfors et al.24 with some incubation time modifications. Reversed-Phase High-Performance Liquid Chromatography (RPHPLC). Forty mg of each of two selected WG films, WGG and 7.5NH4OH-1.5-SA, was homogenized using a scalpel. The protein extraction was carried out in three steps. The homogenized WG films were mixed with each of three extraction buffers, and after each extraction, the supernatant was collected and a new portion of buffer was added to the pellet. The two samples were subjected to WG protein fractionation, separating the samples into two fractions: gliadins and glutenins. The following extractions were performed: (1) 0.5 mL of 50% 1-propanol, (2) 0.5 mL of 6 M urea, 0.5% sodium dodecyl sulfate (SDS), and 1% 1440

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Biomacromolecules dithiotreithol (DTT), and (3) 0.5 of mL 6 M urea, 0.5% SDS, and 1% DTT. For extraction 1, the samples were placed on a vibromix (IKAVibrax, VXR, Staufen, Germany) for 30 min at 2000 rpm at RT and centrifuged for 30 min at 10 000g. For extractions 2 and 3, samples were heated to 100 C for 5 min. Extracted protein fractionation was carried out using RP-HPLC according to Kuktaite et al.,25 and determination of proteins was done according to retention times, as was described in Wieser et al.26 Three replicates of each protein extraction were analyzed, and two-way ANOVA using KyPlot and Univariate Analysis of Variance (Tukey HSD, LSD, and Duncan tests) using R were calculated.

’ RESULTS AND DISCUSSION Structure Characterization of 5-NaOH Containing WG Films. The selected WG films containing 5-NaOH showed

specific polymer morphologies by SAXS, WAXS, and CLSM (Figure 2). The X-ray scattering measurements were performed using the incoming beam perpendicular to the WG film plane, and the following results were observed: (1) The 5-NaOH film extruded at 90 C and the WGG film extruded at 105 C showed SAXS profiles containing an undeveloped peak with average correlation distances d = 55 and 69 Å, correspondingly, calculated from the maximum peak position qmax and according to the relationship d = 2π/qmax (Figure 2a). (2) The 5-NaOH films extruded at 105 and 120 C showed a series of well-defined peaks, indicating the formation of a new more organized WG film structure compared with the film extruded at 90 C (Figure 2a). The characteristic spacing from the strongest and sharpest peak was 40 Å. (3) X-ray scattering measurement at higher q values (until q = 9 nm
1) enabled the fitting of all sharp peak positions with a unique set of parameters for the unit cell. A tetragonal structure with cell parameters a = 51.85 ( 0.06 Å and c = 40.65 ( 0.1 Å was obtained using the program Dicvol (from the package CRYSFIRE)30 (Figure 2b). (4) WAXS results of the 5-NaOH films extruded at 90 and 120 C and the WGG extruded at 105 C are shown in Figure 2c. Two strong diffuse peaks corresponding to the characteristic distances of 4.4 Å and 9.5
10.1 Å were observed for all three. No significant differences were observed between the diffractograms, which indicate that they had a similar degree of protein organization at the molecular level. (5) A representative CLSM image of the 5-NaOH (120) 120 film (Figure 2d) showed three dominating protein structure regions corresponding to solely gliadins (stained red; arrow 1), solely high-molecular-weight glutenins (HMW-gs) (stained green; arrow 2), and intimately mixed gliadins-HMW-gs (a mixture of red-green; arrow 3). To conclude, our results showed a transition of the WG protein molecular structure from a “simple” protein arrangement such as poorly organized protein aggregates in the reference film (WGG) toward tetragonal ordered supramolecular structures in the 5-NaOH (120) 120 film. This difference in structure agrees with previous results, which have shown that chemical additives, such as NaOH, and both specific concentration6 and higher pH,5 influence the WG films by contributing to protein polymerization/formation of β-sheet structures. Temperatures between 108 and 116 C activate thermosetting reactions, whereas higher temperatures, such as, 120 C, lead to a highly cross-linked WG network.31 The elevated extrusion temperature appears to activate shear mediated rearrangements. These are, for instance, attributable for the superstructure formation of the amyloid fibrils32 (note: at significantly higher shear rates than in extrusion and at only mildly elevated temperature), with most likely

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parallel similarities in the WG polymer in the film. There are, however, a number of issues to address such as: What is the change in superstructure between the sheared and nonsheared materials? What exactly is the mechanism responsible for the superstructure formation? In addition, an importance of the glycerol concentration on protein structure formation in the WG films, as was observed for the silk-glycerol films,33 should be noted. Therefore, the differences in structure may be the cause of the differences in mechanical properties observed between films produced at different die temperatures.6,14,15 WAXS diffraction patterns of the 5-NaOH WG film showed two strong diffuse rings in the region of 4.4 and 9.5 to 10.1 Å, which is in a good agreement with the diffraction patterns reported by Traub et al.34 and Lai et al.35 The observed peaks may originate from the R-helix conformations of the WG proteins. Specifically, the larger d spacing most likely is related to the average distance of neighboring helices (i.e., interhelix packing) and the shorter ones are related to the average backbone distance within the helices.35 Morphology Characterization of NH4OH Containing WG Films. SAXS Patterns of Films When X-ray Beam Is Perpendicular to Film Plane. Two-dimensional SAXS results of the 7.5NH4OH-1.5-SA film are shown in Figure 3a,b. Anisotropic intensity rings were clearly observed in the scattering pattern, which indicate a partially oriented structure of this film. Furthermore, the coexistence of both a broad inner ring and a sharp more intense external ring (Figure 3b) indicates the presence of a complex morphology (i.e., a mixture of morphologies) in the 7.5-NH4OH-1.5-SA film. The 1D intensity SAXS curve obtained after averaging the data for the maxima of the scattering intensities presented √ √ three well-defined Bragg peaks in the positional ratio 1: 3: 4 (Figure 3c, peaks indicated by arrows). This ratio indicates a 2D hexagonal close packed (HCP) arrangement36 of the scattering objects in the WG film. For this HCP symmetry, the distance between the centers of the objects (interdomain distances), a, can be calculated from the position of the first peak, q1, through the equation 4π a ¼ pffiffiffi 3 q1

ð1Þ

The obtained distance a was 70 Å for the 7.5-NH4OH-1.5-SA film. In addition, the broad peak in the scattering curve (at q values lower than q1) of the 7.5-NH4OH-1.5-SA film can be associated with larger correlation distances (ca. 80 Å) in the system and is probably related to other morphologies, that is, a “higher hierarchy” structures with distances >80 Å between the objects. The anisotropic scattering pattern of the 7.5-NH4OH-1.5SA film is shown in the plot of the intensity I(j) versus the azimuthal angle j in Figure 3d. The two observed scattering peaks, the inner broad peak and the first Bragg peak (Figure 3c), presented two maxima centered at the same angles, 163 and 343 (Figure 3d). The wider full width at half-maximum (fwhm) of the inner peak compared with the first Bragg peak might be an indication of the lower degree of orientation of this morphology. A complementary measurement on the 7.5-NH4OH-1.5-SA film choosing the equatorial direction as the extrusion MD and keeping the X-ray beam perpendicular to the film plane showed that the maxima in the azimuthal plots were perpendicular to the MD (at j = 90) (results shown in the Supporting Information, Figure S1). 1441

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Figure 3. SAXS patterns of the 7.5-NH4OH-1.5-SA film, extruded at 120 C when the X-ray beam was perpendicular to the film plane: (a) a 2D SAXS pattern and (b) the amplified image at the lowest q-values√of the √ scattering pattern; (c) 1D intensity profile of 7.5-NH4OH-1.5-SA. Three well-defined Bragg peaks (shown by arrows) in the positional ratio 1: 3: 4 indicate a bidimensional hexagonal close-packed (HCP) structure. WGG (reference) curve is shown for comparison. (d) Azimuthal plot I(j) versus j for 7.5-NH4OH-1.5-SA showing the anisotropic character of the scattering pattern.

This fits into a model where the objects are oriented mainly in the extrusion direction. Similar SAXS profiles and characteristic distances were observed for the 10-NH4OH-1.5-SA film (results shown in the Supporting Information, Figure S2) as for the 7.5-

NH4OH-1.5-SA film, indicating that both NH4OH-containing films had similar supramolecular organization. SAXS Patterns of Films When X-ray Beam Parallel to Film Plane. Because of the existence of anisotropic scattering patterns, 1442

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Figure 5. WAXS profiles of the 7.5-NH4OH-1.5-SA and 10-NH4OH1.5-SA WG films extruded at 120 C and the WGG (reference) extruded at 105 C.

Figure 4. SAXS patterns of the 10-NH4OH-1.5-SA WG film when the X-ray beam is parallel to the extrusion direction (MD): (a) 2D image (positions of the “inner” broad ring and 1st Bragg reflection are indicated by arrows); (b) azimuthal plot for both rings. The graph for the 1st Bragg reflection (continuous line) presented six peaks spaced j = 60 from each other. The plot for the “inner” ring was also anisotropic (dotted line).

complementary SAXS measurements on the NH4OH containing WG films were made with the X-ray beam parallel to the film plane and coincident with the MD. A 2D SAXS profile and the azimuthal plot of the 10-NH4OH-1.5-SA film are shown in Figure 4. We observed the same distinctive characteristics of the scattering pattern, as observed for those measured with a perpendicular beam: a broad inner ring and the external asymmetric ring, related to the first Bragg reflection, although six “spots” were identified on the outer ring (Figure 4a). The azimuthal intensity plot of the first Bragg ring confirmed the six-fold symmetry with peaks distanced 60 (Figure 4b), indicating a bidimentional HCP structure.36 The diffuse scattering at low angles characterized by a broad inner ring which was also oriented (shearing effect)

and indicated differences in intensity of the six maxima in the first Bragg ring (note: the maximum anisotropy of the broad inner ring corresponded well with the two reflections of higher intensities shown in Figure 4b). The six-fold symmetry provides new information on the main morphology of NH4OH containing WG films, indicating that they are composed of elongated objects packed in a hexagonal lattice and oriented by the extrusion process. However, the higher orders of the Bragg reflections were not observed in this particular geometry of the NH4OH WG films. This might be due to the fact that the samples obtained after extrusion are not a monodomain hexagonal lattice but polydomains separated by grain boundaries with some orientation distribution along the extrusion direction. Further studies of the NH4OH WG films including analysis of SAXS patterns at different WG film tilt angles would bring more details on the superstructure. WAXS Patterns of Films. WAXS diffractograms of the 7.5NH4OH-1.5-SA, 10-NH4OH-1.5-SA, and WGG (reference) films are presented in Figure 5. The diffraction pattern of the NH4OH WG films was similar to the diffraction patterns previously observed for the 5-NaOH WG films (Figure 2c), but the following differences were observed: (1) The first diffuse peak at q = 6.35 nm
1 was wider and less well-defined for the NH4OH WG films (Figure 5) compared with the 5-NaOH WG films (Figure 2c), indicating a less ordered structure at this characteristic distance, which can be related to more distorted interhelix packing in the NH4OH WG films.35 (2) The second peak of the diffraction curves of the NH4OH WG film showed the same characteristic distance, as observed for the NaOH films, 4.44 Å, although it appeared to be distorted by a new shoulder at lower q values, which indicated possible β-sheet formation. The specific d value of the new peak is 4.64 Å (Figure 5). It is also important to note that some authors, for example, Traub et al.,34 have suggested that this peak is related to the structural transformation from R-helix to β-sheet. In addition, a similar characteristic distance for the well-crystallized β-sheet structures was 4.7 Å.37 IR Spectrum of NH4OH Containing Films. The IR spectrum of the amide II region of undenatured dried vital WG powder is peaking somewhere between 1640 and 1650 cm
1 (Figure 6). This is consonant with a protein rich in R-helices and unordered material.34 The NH4OH samples show a prominent peak 1443

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Biomacromolecules between ca. 1618 and 1635 cm
1, peaking at ca. 1625 cm
1 (Figure 6). The high intensity in this region corresponds to a large content of β-sheets, in turn a consequence of a highly aggregated structure.38 Interestingly, the NaOH-containing samples (undried specimens6) did not show this prominent peak and were consequently less aggregated than the NH4OH samples. NH4OH WG Film Hexagonal-Closed-Packed (HCP) Structure Model and Orientation. Characterization of the nanoscale organization of the NH4OH-1.5-SA WG protein-based films showed scattering objects arranged in HCP structures similar

Figure 6. IR spectra of vital wheat gluten powder (dotted line) and the 7.5-NH4OH-1.5-SA (thick line), and 10-NH4OH-1.5-SA (thin line) films. Note that only the curve shapes and not the absolute intensities can be compared between the samples.

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to a model for peptide
hybrid block copolymers39 and the crystallization of self-assembled peptide filaments.40 The observed interdomain distance of the HCP structure was 70 Å. In addition, SAXS data for both NH4OH-1.5-SA films indicated the presence of a supramolecular orientation. We have evidence of the HCP structure and structure orientation are very similar for both of the NH4OH WG films and assume that these structures result from thermal, composition, and processing effects. Evidence of chemical composition of the blend affected functional properties as well as the protein extraction behavior of the film has been reported by Ullsten et al.14 A possible reason for the structural change and improvement in end-use properties may be the presence of the NH4OH, which may confer additional plasticization to the gluten protein system and increases the interactions between the protein matrix and the plasticizer (glycerol), although by a different mechanism than that reported for the quaternary ammonium ion in WG nanoclay composites.41 A beneficial effect of NH4OH (compared with NaOH) seems to be related to differences in protein
additive interaction mechanisms and changes in protein conformations that are often associated with changes in surface hydrophobicity, which was observed in pea protein films.42 The reasons can only be speculated regarding possible deamidation or other types of chemical reactions occurring under high temperature and basic conditions6 (i.e., Maillard reactions or lysinoalanine and isopeptide formation phenomena, which may contribute to cross-linking of the protein and reduced protein solubility43). A possible explanation for the beneficial effect of NH4OH might be greater like-charge repulsions that could be responsible for the emergent crystalline domains. Therefore, NH4þ ions would help to more effectively order those domains

Figure 7. CLSM images of the (a,b) WGG extruded at 105 C and (c,d) 7.5-NH4OH-1.5-SA extruded at 120 C; monoclonal HMW-gs was stained green and polyclonal gliadins (Sigma) were stained red; in parts a and b, arrows indicate areas rich in the HMW-gs aggregates; in parts c and d, arrows show intimately mixed areas of gliadins and HMW-gs. 1444

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Figure 8. CLSM images of (a
c) WGG extruded at 105 C and (d) 7.5-NH4OH-1.5-SA extruded at 120 C; WG proteins immunostained with antibodies directed to the LMW-gs (IFRN 0067) B group23
green and polyclonal gliadins (Sigma)
red (a,b); WG proteins immunostained with two antibodies directed to LMW-gs peptide (SHIPGLERPSGC, clone F98 14, INRA)
green and polyclonal gliadins (Sigma)
red (c,d). (a) Overview of protein structure showing individual areas of gliadins and HMW-gs. (b) Arrow indicates an area with more concentrated HMW-gs and to a smaller extent distributed gliadins. (c) Overview of overlapping binding sites of antibodies directed to gliadins and LMW-gs (yellow color); arrows show three different regions, gliadin rich (reddish, arrow 1), overlapping gliadin and LMW-gs (yellow, arrow 2) and LMW-gs rich (greenish, arrow 3). (d) Overview of intimately mixed gliadins and LMW-gs; scale 10 μm.

than Naþ would do through a charge screening alone. The reactive NH4OH together with mechanical deformation44,45 may induce the formation of a greater number of stable β-sheet structures (β-turns being converted to β-sheets). All gluten proteins contain long arrays of repeated sequences, which appear to adopt similar secondary structures, loose spirals, which comprise equilibrium between β-reverse turns and polyproline II-like structures.46,47 The equilibrium between those structures depends on temperature and chemical components,48 which may explain why greater cross-linking densities and specific arrangements of secondary protein structures occurred in the WG films with NH4OH. Apparent structure orientation of the WG protein in our study is clearly influenced by mechanical processing, that is, extrusion changed the balance of protein secondary structures in the WG polymeric protein system in the film. During extrusion, polymers are heated and stretched and form structures with oriented populations of macromolecules and microphase separation.7 Protein Structure Characterization of WGG and NH4OH Containing Films by CLSM. The structures of the WGG and 7.5-NH4OH-1.5-SA films observed by CLSM are shown in Figures 7 and 8 and can be summarized as follows: (1) In the WGG films, various protein groups, that is, the HMW-gs (subunits 2, 5, 10, and 12), the B group of the LMW-gs (recognized by the SHIPGLERPSGC antibody) and the gliadins, were distributed with varying degrees of homogeneity within the

films, which also contained a large number of voids of several micrometers in size (Figures 7a,b and 8a
c). (2) The WGG film showed areas where the HMW-gs seemed to be aggregated, and some gliadins were also present (Figure 7 a, b; glutenin aggregates indicated by arrows). (3) In addition, immunostaining of the WGG film with an antibody for the B group of LMW-gs (Figure 8a
c) showed gliadins dominating with some LMW-gs present (Figure 8a,b). An antibody for the LMW-gs peptide (SHIPGLERPSGC) (Figure 8c) indicated rather many binding regions for glutenins (green fluorescence; arrow 3), although the overall binding regions for both antibodies (glutenins and gliadins) were very similar (yellow fluorescence, arrow 2) through the entire film. (4) Unlike WGG and other WG films without NH4OH, the NH4OH-1.5-SA film appeared to have a rather uniform structure with the presence of intimately mixed areas of gliadins and glutenins. The polymer structure contained large regions rich in HMW-gs (Figure 7c,d) and LMW-gs (Figure 8d), which were very closely mixed with gliadins. An important observation was that a clearly uniaxial oriented protein structure pattern was observed (Figures 7c,d and 8d). It should be noted that the fluorescence intensity of immunolabeled gliadins and glutenins was uneven across most of the samples being considerably stronger at the surface than inside of the sample and also differed between the different antibodies. This is most likely related to the ability of the antibodies to “penetrate” into the sample because a penetration barrier was 1445

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Figure 9. RP-HPLC chromatograms of protein extractability of two WG films, the WGG and 7.5-NH4OH-1.5-SA, exposed to extracting solutions as follows: 50% 1-propanol, first extraction - gliadins; chromatogram is divided into three areas presenting ω-, R-, and γ-gliadins (a,b); 6 M urea, 0.5% SDS, and 1% DTT, second and third extractions - glutenins; chromatogram is divided into three areas presenting ω- gliadins, HMW-gs, and LMW-gs (c
f).

higher, especially for the more polymerized films such as the 7.5NH4OH-1.5-SA. The affinity of antibodies varies as well, making qualitative and highest semiquantitative comparisons possible.49 Comparison of the glutenin
gliadin structures in the WGG and NH4OH-1.5-SA films (Figures 7 and 8) indicates differences in protein patterns, which are related to the different protein conformations. At high extrusion temperatures, such as 120 C, the denaturation of proteins through dissociation and unfolding facilitates sulphydryl
disulfide interchange reactions and allows the gliadins and glutenins to repolymerize (forming intermolecular covalent bonds), which yields high tensile strength/ Young’s modulus.14 Indeed, the WG polymer structure of the NH4OH film was dominated by areas of intimately interacting gliadins
glutenins as well as a high content of HMW-gs regions with some gliadins present.

A significant difference in the WG protein structure in this study was clearly related to the distance between neighboring helices forming the β-sheet structure. Extrusion caused an increase in the formation of β-sheet structures in relation to βturns and R-helices, similar to the changes observed during biaxial extension of WG.44 In particular, the content of intermolecular β-sheets increased to various extents (from WGG to NH4OH-SA WG films), whereas the contents of β-turns and Rhelices decreased. The results indicate that interactions occurring between the HMW-gs and LMW-gs, and the incorporated gliadins along the direction of flow, quite clearly favored the formation of intermolecular β-sheets, whereas the content of intramolecular β-sheets might increase by changes in the globular nonrepetitive domains of the gluten proteins.44 The model for the WG structure proposed by Grosskreutz,50,51 which 1446

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of ω-gliadins with the glutenin polymer (some of ω-gliadins seemed to be “trapped”, whereas others were extracted with 6 M urea, SDS, and DTT during the first glutenin extraction (Figure 9b)).

Figure 10. Schematic drawing for WG polymer aggregates arranged in the HCP structure in NH4OH-containing WG films; a is the interdomain distance. The scattering objects were preferentially oriented with the long side along the extrusion direction.

suggested that β-sheets orientated mainly parallel to the WG film surface and stabilized by hydrogen bonding through an aqueous phase under the action of hydration and mechanical processing, therefore describes the WG polymer secondary structure complex very well. Protein Solubility by RP-HPLC. Figure 9 shows RP-HPLC profiles of protein fractions extracted from the WGG and 7.5NH4OH-1.5-SA films with 50% 1-propanol and in 6 M urea, SDS, and DTT. The following observations were made: (1) The amounts of extracted gliadins differed greatly between the samples with a larger amount of all types of gliadins, excluding the ω-type gliadins, extracted from the WGG, compared with the 7.5-NH4OH-1.5-SA film (Figure 9a,b; first extraction). However, a large peak in the ω-type gliadin region has eluted at 7.5 min. (2) The first extraction of the WGG film with 6 M urea, SDS, and DTT removed only small relative amounts of HMW-gs and large relative amounts of LMW-gs. In contrast, some ωgliadins, HMW-gs, and more polymerized/aggregated LMW-gs with some HMW glutenins were extracted from the 7.5NH4OH-1.5-SA film (Figure 9c,d; second extraction). (3) Relatively small amounts of proteins, mainly the LMW glutenins, were extracted by the second extraction using the same 6 M urea, SDS, and DTT buffer from both films (Figure 9e,f; third extraction). The LMW-gs extracted from the 7.5-NH4OH-1.5SA film were found to be more aggregated than those extracted from the WGG film. The previously discussed thermal, composition, and processing effects resulted in a high degree of polymerization for the NH4OH-1.5-SA film, whereas the polymerization level of the WGG film seemed to be extremely low because nearly all types of gliadins were extracted with 50% 1-propanol (Figure 9a). It should be noted here that in an intimately linked gliadin
glutenin matrix, such as the NH4OH-1.5-SA film, the gliadins seemed to appear “tightly” packed into the polymeric structure, as shown by CLSM. (See Figures 7c,d and 8d.) In NH4OH film, the incorporation of gliadins into glutenins structure seemed to proceed toward greater covalent cross-linkings, that is, through SH/SS interchange reactions43 and a more polymerized protein structure compared with the WGG film, whereas hydrophobic interactions and hydrogen bonds were easily broken with 50% 1-propanol. Other molecular interactions, such as ionic bonds with NH4OH, must be also present which favored the interaction

’ CONCLUSIONS Chemical components (i.e., NaOH) affect protein polymerization in the WG film and influence the film morphology and the functional properties at extrusion temperatures higher than 90 C. Components, NaOH and NH4OH, had different effects on WG protein aggregation, although both appeared to influence the equilibrium between secondary structures formed by the WG proteins. The presence of NaOH induced the formation of the tetragonal structures in WG materials at extrusion temperature >105 C. The more reactive NH4OH seemed to induce the formation of a greater amount of stable β-sheet structures compared with NaOH in the WG films. Also, NH4OH appeared to play an important role in the formation of the HCP structures in WG materials and to be responsible for the enhanced strength of material. The conclusions from our study are summarized in the HCP structure model of the WG films containing NH4OH, which is presented in Figure 10. The SAXS results showed that the extrusion affected the film morphology by orienting preferentially the HCP structures in the direction of extrusion. The ω-gliadins are probably “trapped” in the WG polymer, whereas the other types of gliadins seemed to interact with glutenins through SH/SS interchange reactions. The tetragonal and HCP supramolecular structures of WG proteins have not been previously observed in so-called “natural” cereal plant protein systems. Further studies are evolving quantification of the relationships between the protein secondary structures, supramolecular structures, and functional properties. Further work is also required to determine how the protein structures can be fine-tuned to modulate the functional of the WG biobased materials for specific applications. ’ ASSOCIATED CONTENT

bS

Supporting Information. Composition, processing temperature, aging time and maximum stress of the studied WG films, and SAXS patterns of the 7.5-NH4OH-1.5-SA with the equatorial direction parallel to the extrusion MD and the 10NH4OH-1.5-SA films when X-ray beam was perpendicular to the film plane. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Phone: þ46 40 415337. Fax: þ46 40 415119.

’ ACKNOWLEDGMENT Henrik Ullsten, Karlstad University, is thanked for providing the extruded films. We thank Dr. Christer Svensson for useful discussion and help with Dicvol and Sandra Denery for development of antibodies. Hasan T€ure and Thomas Blomfeldt are thanked for performing the IR measurements. FORMAS is gratefully acknowledged for financial support. Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the U.K. MAX-lab 1447

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Biomacromolecules Synchrotron beamline stations, the I711 and 911-5, are acknowledged for the beam time.

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