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Research Article pubs.acs.org/journal/ascecg

Monitoring Nanostructure Dynamics and Polymerization in Glycerol Plasticized Wheat Gliadin and Glutenin Films: Relation to Mechanical Properties Ramune Kuktaite,*,† William R. Newson,† Faiza Rasheed,† Tomás S. Plivelic,‡ Mikael S. Hedenqvist,§ Mikael Gal̈ lstedt,∥ and Eva Johansson† †

Department of Plant Breeding, Swedish University of Agricultural Sciences, Växtskyddsvägen 1, SE-230 53 Alnarp, Sweden MAX IV Laboratory, Lund University, Fotongatan 2, SE-225 92 Lund, Sweden § School of Chemical Science and Engineering, Fibre and Polymer Technology, Royal Institute of Technology (KTH), Teknikringen 56-58, SE-100 44 Stockholm, Sweden ∥ Innventia AB, Drottning Kristinas väg 61, SE-114 28 Stockholm, Sweden ‡

S Supporting Information *

ABSTRACT: Gliadin and glutenin proteins with 10, 20, 30 and 40% of glycerol were compression molded into films (130 °C) and evaluated for protein polymerization, β-sheet structure and nano-structural morphology. Here, for the first time we show how different amounts of glycerol impact the nano-structure and functional properties of the gliadin and glutenin films. Most polymerized protein was found in the gliadin films with 20 and 30% glycerol, and in all the glutenin films (except 10%), by RP-HPLC. A β-sheet-rich protein structure was found to be high in the 10 and 20% glycerol gliadin films, and in the 20 and 30% glycerol glutenin films by FT-IR. Glycerol content of 20, 30 and 40% impacted the nanostructural morphology of the gliadin glycerol films observed by SAXS, and to a limited extent for 10 and 20% glycerol gliadin films revealed by WAXS. No ordered nano-structure was found for the glutenin glycerol films. The 20%, 30% and 40% glycerol films were the most tunable for specific mechanical properties. For the highest stiffness and strength, the 10% glycerol protein films were the best choice. KEYWORDS: Protein morphology, Protein film, Glycerol content, Polymerization, SAXS, WAXS

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extensibility of films come from the amount and type of intermolecular disulfide bonds as well as, covalent and irreversible bonds that occur during film formation. For Glu, which is polymeric in nature, both inter- and intra-molecular disulfide bonds together with irreversible bonds play a role in the thermoformed materials.8 The Glia and Glu proteins are rich in molecular interactions dictating secondary, tertiary and quaternary structures. For Glia, conformation is mostly stable until about 30 °C (in acidic solution), whereas above this temperature conformation drastically changes toward a lower helical content.9 For wheat gluten films processed at 130 °C, the amount of β-sheets increased.10 In Glia−glycerol films8 and Glia−glycerol-chemical additive films,6 the formation of nanohierarchical structures is greatly affected by the use of low molecular weight polar compounds such as glycerol. Glycerol in WG proteins contributes to conformational changes, assists in

INTRODUCTION Wheat gluten (WG) protein is valued due to its unique viscoelastic, cohesive−adhesive and barrier properties, making it suitable for production of bioplastics, e.g., films, sheets and adhesives. For these applications, a plasticizer such as glycerol is generally used.1−4 WG and two its protein fractions, the monomeric gliadins (Glia) and the polymeric glutenins (Glu), are responsible for extensibility and strength in dough, respectively. Blends of Glia and Glu with glycerol have been used to produce bioplastics with diverse functional properties.5,6 The properties of these protein based materials are impacted to a large extent by the use of glycerol, e.g., the glass transition temperature of protein is reduced and the protein chain mobility is generally increased.7 Gliadin, isolated from WG using 70% of ethanol, was used in blend with glycerol for making translucent and flexible films.4,8 Whereas the residual of the 70% ethanol extraction, the insoluble Glu-rich fraction, when blended with glycerol, forms rather rigid thermo-molded plastics films.8 In films from Glia, occurring as monomers in their native state, flexibility and © XXXX American Chemical Society

Received: December 11, 2015 Revised: April 4, 2016

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DOI: 10.1021/acssuschemeng.5b01667 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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Size Exclusion-High Performance Liquid Chromatography (SE-HPLC). Protein molecular size distribution in Glia− and Glu− glycerol films was studied using the SE-HPLC method13 with some modifications.14 For obtaining a fine powder from the pressed proteins, small samples of films were cut by hand into approximately 0.5 mm size particles.15 For SE-HPLC analysis, proteins were extracted in a 3 step extraction procedure. During the first extraction, 16.5 mg (±0.05 mg) of each protein sample was mixed with 1.4 mL of buffer solution (0.05 M NaH2PO4, 0.5% sodium dodecyl sulfate (SDS), pH 6.9) in 1.5 mL Eppendorf tubes (in triplicate). Samples were then vortexed for 10 s (Labassco, Whirli VIB 2) at maximum speed, shaken for 5 min at 2000 rpm (IKA-VIBRAX VXR), centrifuged for 30 min at 16,000 g at RT (Sorvall, Legend Micro 17) and the supernatant collected for SE-HPLC analysis (Ex1). For the second protein extraction 1.4 mL of the same buffer solution was added to the pellet from Ex1 and sonicated (Sanyo Soniprep 150 Ultrasonic Disintegrator) for 30 s at an amplitude of 5 μm, centrifuged as Ex1 and the supernatant was collected for analysis (Ex2). The pellet of the second extraction was sonicated for 30, 60 and 60 s in 1.4 mL of buffer solution with sample cooling to RT between sonications, followed by centrifugation with the supernatant decanted for analysis (Ex3). Supernatants were analyzed using a Waters HPLC system (Waters 2690 Separation Module) with Waters 996 Photodiode Array Detector (Waters, USA).8 For SE-HPLC analyses, 20 μL of sample was injected onto a size exclusion column (Biosep-SEC-S 4000, Phenomenex, USA) at an isocratic flow of 0.2 mL/min (50% acetonitrile, 0.1% TFA; 50% H2O, 0.1% TFA). Three extractions for each of the samples were performed and a standard deviation was calculated. Chromatograms were recorded and extracted at 210 nm (Empower Pro, Waters, USA), and integrated into two arbitrary protein groups according to the time of elution, high molecular weight (HMw), 8 to 13 min, and low molecular weight (LMw), 13 to 28 min. The integrated data was corrected for initial protein content and normalized according to the total extractability for each of the raw protein types, e.g., Glia and Glu. Reversed Phase-High Performance Liquid Chromatography (RP-HPLC). Glia− and Glu−glycerol films were analyzed for the protein solubility in the selected number of solvents and further separated by RP-HPLC16 with modifications.8 For the analysis, 100 mg of each sample was used in triplicate. Glia− and Glu−glycerol films were chopped into small pieces by using a scalpel as above. The extraction was done by six serial extraction steps, in the following order: (1) 70% ethanol, shaking for 30 min, (RT); (2) 50% propanol, extraction (shaking for 30 min at RT); (3) 50% propanol, keeping in a water bath at 60 °C (30 min, and centrifuge for 30 min at RT); (4) 0.5% SDS, 50% propanol, extracted at 60 °C (30 min, and centrifuged for 30 min at RT); (5) 1% DTT, 50% propanol, extracted at 60 °C (30 min, and centrifuged for 30 min at RT); (6) 1% DTT, 0.5% SDS, 6 M urea solution, 100 °C (5 min, and centrifuged for 30 min at RT). One mL of extraction solvent was used for each extraction. The first three alcohol based extractions were aimed to solubilize the unbound monomeric and polymeric proteins, whereas the following fourth extraction was aimed to disrupt intra- and intermolecular hydrogen bonds and hydrophobic interactions. The fifth and sixth extractions are targeted for disruption of intra- and intermolecular disulfide crosslinks. Each of six extraction steps was followed by a centrifugation at 16,000 g (30 min) and the supernatant collected for RP-HPLC analysis. The RP-HPLC system (Waters 2690 Separation Module with Waters 996 Photodiode Array Detector) was equipped with a precolumn (5 μm, 2 cm × 4.0 mm, discovery bio wide, Supelco) and a main separation column (5 μm 25 cm × 4.6 mm, discovery bio wide, Supelco; C-8 column) were used for analysis. A 50 μL sample was injected on to the column and separated for 40 min followed by a 15 min cleaning procedure by eluent. A gradient flow (28−72%) (Acetonitrile-0.1% TFA, and H2O-0.1% TFA) was used to elute the proteins at a rate of 0.8 mL/min. The chromatograms were extracted at 210 nm and further integrated for the time interval of 6.5−35 min for each extraction. Small Angle X-ray Scattering. All thermoformed Glia− and Glu−glycerol samples were analyzed by small-angle X-ray scattering (SAXS). The SAXS experiments were carried out at the MAX IV

unfolding of the tightly packed aggregated gluten protein and decreases inter-molecular interactions. Plasticization of WG protein chains comes at a large cost of decreased strength and stiffness; therefore, it is of interest to explore the impact of variations in Glia and Glu structure in thermoformed films due to the amount of added glycerol. It is also of interest to investigate how these structures at the macromolecular scale are linked to tensile properties. To our knowledge, neither such studies have been performed before on systematical monitoring of the nano- and macromolecular structures upon the use of different amounts of glycerol in the Glia and Glu films. The novelty of this study is on how different glycerol amounts can impact the structure−functional properties of Glia and Glu materials. Therefore, the aim of this study was to monitor the nano- and macro- structural and cross-linking reactions of Glia and Glu films in the presence of different glycerol amounts, as well as to investigate how different glycerol amounts effect the Glia− and Glu−protein polymerization behavior. In this study, we are focusing on the relationships between Glia and Glu plasticization upon the use of different amounts of glycerol and how plasticization impact nano-structure and mechanical behavior of processed Glia and Glu films.

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EXPERIMENTAL SECTION

Materials and Methods. Materials. Wheat gluten powder was kindly supplied by Reppe AB, Lidköping, Sweden and contained 77.7% of protein (dry weight; according to NMKL Nr 6, Kjeltec, N × 5.7), 5.8% starch (dry weight; Ewers method) and 6.9% moisture (total weight; NMKL method 23, 1991). Glycerol (99.5%) was supplied by Karlshamns Tefac AB, Sweden. Extraction of Glia and Glu. Commercial WG powder was dispersed in an 70% ethanol in Millipore water using a magnetic stirrer and further using a mechanical shaker for 30 min at 300 rpm at room temperature (RT) (Hunkel Ika, Werk KS 500).8 The WG−ethanol mixture was then centrifuged for 10 min at 12,000 g (Beckman centrifuge J2.21) and the supernatant was collected. Ethanol was removed from the supernatant by rotary evaporation under vacuum (Buchi) and Glia protein was collected after freeze-drying (Edwards, Modulyo).8 Glu protein was separated as the residual after Glia extraction. Glu pellet was immediately washed with Millipore water after centrifugation and freeze-dried (Edwards, Modulyo).8 The protein contents in Glia and Glu proteins were determined according to the Dumas method11 (Thermo Scientific, Flash 2000 NC Analyzer) and were 91% for Glia and 75% for Glu.6 Protein Film Preparation. Glia− and Glu−glycerol films were pressed according to the previously published procedure.12 Protein powders were mixed with glycerol by hand, and the blend was placed in the center of a 10 cm × 10 cm × 0.5 mm aluminum frame between polyethylene terepthalate sheets and preheated aluminum plates. The mixture was thereafter placed into a hot press (Polystat 400 s, Servitech, Germany) for 10 min at a pressing force of 100 kN and temperature of 130 °C for all samples. The protein−glycerol films were removed after compression molding, and cooled to room temperature between aluminum plates.12 The Glia and Glu powders were blended with glycerol in ratios of 90:10, 80:20, 70:30, 60:40, (protein:glycerol, w:w). The samples were abbreviated as Glia for gliadin and Glu for glutenin following by the amount of glycerol added, e.g., Glia10 and Glu10 corresponded to samples with Glia− glycerol and Glu−glycerol with a weight ratio of 90:10, respectively. The samples without glycerol were abbreviated as Glia0 and Glu0. Protein concentration of the gliadin glycerol blends was as follows: 81.9% for Glia10, 72.8% for Glia20, 63.7% for Glia30 and 54.6% for Glia40. Protein concentration of the glutenin glycerol blends was 67.5% for Glu10, 60% for Glu20, 52.5% for Glu30 and 45% for Glu40. For HPLC analysis the absorbance was normalized according to each of the protein concentrations. B


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ACS Sustainable Chemistry & Engineering Laboratory, Lund, Sweden at beamline I911-417 using a wavelength of λ = 0.91 Å. The scattering vector range q was 0.0082−0.47 Å−1 (q = 4π/λ sin(θ), where 2θ is the scattering angle) with a sample to detector distance of 1904 mm. Exposure times of 30 s and 5 min were used. The detector employed was a bidimensional CCD of 165 mm (Marresearch). The SAXS data were analyzed using the software bli911417 and the data were normalized with respect to integrated intensity incident on the sample during the exposure time and corrected for sample absorption and background. To study the evolution of the protein structures in films, SAXS measurements at different temperatures were performed for the Glia10, Glia30 and Glia40 films. Heating of the films was done using a customized high-temperature furnace (Linkam, TS1500) for X-ray scattering experiments. The temperatures were 25, 35, 45, 55, 60, 70, 80 and 90 °C with an X-ray exposure time of 60 s. Temperature at each measuring point was kept constant for 5 min before the SAXS image was taken. Wide Angle X-ray Scattering. Wide angle X-ray scattering analysis (WAXS) was carried out at the MAX IV Laboratory, Lund, Sweden, using beamline I911-218 with a wavelength of λ = 1.04 Å and a sample to detector distance of 149.8 mm. An area-CCD detector was used for the measurements. Silicon powder was used as a standard for calibration. The data was analyzed using Fit2D software.19 WAXS patterns of the Glu−glycerol films were measured at the beamline I911-4 using a detection range of 1 ≤ q ≤ 18 nm−1 and a wavelength of 0.91 Å. Fourier Transform Infrared Spectroscopy (FT-IR). FT-IR spectroscopy analysis was carried out on all the Glia− and Glu− glycerol films using a Spectrum 2000 FTIR spectrometer (PerkinElmer inc., USA) equipped with single reflection ATR (Golden Gate, Speac Ltd.). Prior to analysis the samples were dried for at least 72 h in a desiccator over silica gel. IR absorbance spectra were first Fourier selfdeconvoluted (FSD) using Spectrum software with γ = 2 and smoothing of 70%. Peak fitting to FTIR data was carried out with peak positions indicated by second derivative analysis of the spectra (Savitzy-Golay filter, 5 points) followed by the fitting of 9 Gaussian curves to FSD spectra between 1700 and 1580 cm-1using Fityk v0.9.8 (Wojdyr 2010). Fits were of R2 = 0.9998 or better. Peak assignments were performed according to Cho et al. (2011)20 and Gällstedt et al. (2010).21 Tensile Testing. Compression molded Glia− and Glu−glycerol films were punched into dumbbell shaped samples (ISO 37 type 3, Elastocon, Sweden). Samples were conditioned in a climate controlled chamber for 48 h at 23 °C at 50% relative humidity before testing. The film thickness was averaged from five points using a Mitutoyo IDC 112B indicator and a stand. Tensile testing was carried out under the same controlled climate conditions as during conditioning using a crosshead speed of 100 mm/min and 1 kN load cell with 30 mm clamp separation on an Instron 5566 universal test machine. Data were collected using Bluehill software (Instron AB, Sweden).22 Statistical Analysis. Statistical analysis software (SAS, version 9.3) was used to perform general linear model (GLM), analysis of variance (ANOVA) and Tukey’s HSD (significance level α = 0.05) tests for evaluating significance of glycerol level on tensile properties of Glia− glycerol and Glu−glycerol films. Microsoft Excel was also used to calculate mean values and a standard deviation.

Figure 1. Protein size distribution referred to the solubility of proteins at three extraction steps by SE-HPLC: (a) Glia films, and (b) Glut films, compressed with different amounts of glycerol; extractability is normalized according to the protein level of total raw Glia (a) or Glut (b), respectively. HMw, high molecular weight proteins; LMw, low molecular weight proteins. Bars represent standard deviation. Values with the same letter are not significantly different according to Tukey’s posthoc test at level p < 0.05.

and Glia40 films showed a similar solubility for the HMw proteins, resulting into nearly no (or an extremely small amount) HMw proteins being soluble. For the LMw proteins, the greatest amount of extracted protein was observed for Glia0 and Glia40 films (Figure 1 a). Similar protein extraction behavior was observed for 20, 30 and 40% glycerol samples, i.e., where greater amounts of LMw were extracted during the extraction step 3 (longer sonication times). This finding suggests that specific cross-links have been formed between the LMw proteins (most likely gliadins) resulting into formation of polymeric Glia with intra- and inter-chain disulfide bonds. For the Glu films similarly to the Glia films, the greatest amounts of both HMw and LMw were extracted for the Glu0 film (Figure 1b). For all glycerol containing Glu films, nearly none of the HMw protein was extracted. For the LMw, similar amounts were extracted for Glu10, Glu20 and Glu30, and a slightly greater amount was observed for the Glu40 film. Different from the rest of the films, for Glu30 film the lowest amount of LMw protein was extracted during the third extraction, followed by the second lowest amount extracted for Glu20, respectively (Figure 1b). For all the Glia and Glu films plasticized with glycerol, it was observed that glycerol favored the formation of insoluble polymers from the HMw proteins (small extractable amounts observed) (Figure 1). Greater amounts of the LMw proteins were observed in the Glia20 film compared to the rest of the Glia films (especially during first and second extractions). From

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RESULTS AND DISCUSSION Glycerol Effect on Protein Polymerization Evaluated by SE-HPLC. Qualitative assessment of protein polymerization behavior of protein glycerol films has been studied by SEHPLC. The results for the Glia and Glu films with different amounts of glycerol showed that the solubility of the HMw polymeric proteins and LMw proteins differed (Figure 1). The Glia films with no glycerol had the highest solubility and the Glia10 film showed the second highest amount (although relatively small) of soluble polymeric proteins compared to the rest of the Glia−glycerol films (Figure 1a). The Glia20, Glia30 C


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Figure 2. Protein extractability in various solvents during six extraction steps analyzed by RP-HPLC: (a) Glia−glycerol films, (b) Glut−glycerol films. Solvents during extractions steps are referred as follows: (1) 70% ethanol, (2) 50% propanol, (3) 50% propanol, 60 °C, (4) 50% propanol + 0.5% SDS, 60 °C, (5) 50% propanol + 1% DTT, 60 °C, (6) 1% DTT + 1% SDS + 6 M urea solution, 100 °C.

the Glu films, the great amounts of polymerized LMw proteins were found in all the Glu with glycerol films, except the Glu40 film. This fact suggests, that lower amounts (20 and 30%) of glycerol are in somewhat “optimal” for cross-linking Glia and Glu protein. While greater amounts of glycerol (40% in our study) reduce intermolecular forces between the proteins.3 In fact, the least polymerized LMw proteins were found in the 40% glycerol Glia and Glu films (Figure 1). Protein Solubility in Different Solvents Studied by RPHPLC. In this study, we compared the solubility/extractability of Glia− and Glu−glycerol films in different solvents using the RP-HPLC method6 (Figure 2). High variations in protein solubility from Glia− and Glu−glycerol film were observed for the extraction steps 1−2 (70% ethanol and 50% propanol, respectively) and 5−6 (DTT and DTT−SDS−urea), respectively (Figure 2a). The smallest amounts of solubilized Glia were observed for Glia30 film through all the extraction steps, except the step 6, compared to the other glycerol contents. For 30% glycerol film, a high solubility was reached only with reducing and/reducing denaturing agents (0.5% SDS, 1% DTT,

6 M urea), indicating a strongly cross-linked protein network (Figure 2a). Particularly, during the sixth step, when DTT denaturant together with urea and SDS were used, a higher solubility was observed for the Glia30 film compared to the rest of the Glia films. This indicated that the disulfide cross-links, probably of intermolecular type, were responsible for the protein−protein interactions in the Glia−glycerol films. For the Glia10, Glia20 and Glia40 films, the impact of the glycerol amount on the protein−protein interactions was similar. Therefore, the unprocessed Glia powder was mostly dissolved in the first (ethanol) and second (propanol) extraction steps (Figure 2a). Regarding Glu−glycerol films, the highest protein solubility was observed for steps 5−6 (both with reducing conditions) for all the glycerol contents (Figure 2b). For step 5 (with DTT), a rather low protein solubility (around 35%) was observed for all the glycerol Glu films, except the 40% glycerol film (reaching 55%). For the Glu−glycerol films (10, 20 and 30% glycerol), a quite opposite protein solubility behavior was observed during step 6, with high solubility for the Glu10, Glu20 and Glu30 D


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ACS Sustainable Chemistry & Engineering films, and relatively lower solubility for the Glu40 film (Figure 2b). This behavior indicates that greater amounts of glycerol (40%) increased the distance between the Glu chains23 and made the Glu more soluble (at step 5). Protein Secondary Structure by FT-IR. The protein secondary structure in Glia− and Glu−glycerol films has been studied by FT-IR, and the results are shown in Figure 3. From

explained by the plasticization effect of glycerol, which can be attributed to glycerol’s ability to locate between Glu polymer molecules, bind water and disrupt or weaken inter-molecular associations.7 The relative intensities at 1666 (β-turns), 1650 (α-helix and random), 1630 (β-sheet) and 1620 cm−1 (inter-molecular βsheet) were used to estimate secondary structures24 of Glia and Glu proteins in the protein−glycerol films and to justify Figures S1 and S2. In Table 1, Glu20 and Glu30 films at 1626 cm−1 Table 1. WAXS Scattering Distances of Glia− and Glu− Glycerol Films

a

sample name

d1 (Å)

d2 (Å)

d3 (Å)

Glia10 Glia20 Glia30 Glia40 Glut10 Glut20 Glut30 Glut40

9.74 9.77 9.77 9.77 9.75 9.80 9.65 9.87

4.42 4.42 4.42 4.37 4.45 4.44 4.45 4.40

4.65 4.65 n.f.a n.f. n.f. n.f. n.f. n.f.

n.f.: not found.

showed an increase in β-sheet strong interactions of 5% for each film. This indicate that increased glycerol allows a higher degree of chain mobility resulting in protein rearrangement to a higher level of 1626 cm−1 β-sheet content compared to the 0 and 10% glycerol cases. The 1626 cm−1 content decreases again at 40% glycerol as there is enough glycerol in the system for it to interfer with hydrogen bonding of the those β-sheet structures, at 20 and 30% glycerol levels the available glycerol is associated with other structures. In the Glu40 film of this study, the polymeric network became less dense/cross-linked due to that the polymer chains becoming further apart due to the presence of glycerol (Figure 1b). Thus, the intermediate glycerol levels (20% and 30%) appear to be more in favor of generating a greater cross-linking of Glu, where glycerol behaved as a chaperone, which stabilizes protein folding.8 Nanostructural Morphology of Glia− and Glu− Glycerol Films by SAXS. Variation in the nano-structural morphology was observed by SAXS for the Glia and Glu films with varying glycerol amounts (Figure 4). The Glia20, Glia30 and Glia40 films had the three well-defined Bragg peaks, following the peak positional ratio 1:√3:√4, indicating a hexagonal structure (Figure 4a, peaks indicated by arrows). The Glia10 film showed a broad and rather intense peak, and a small shoulder (low intensity) (Figure 4a, broken line). No structural feature was observed for the glycerol sample (no protein; Figure 4a). For all the Glia−glycerol films, variation in the shape of the Bragg peaks reflected differences of the system morphology (Figure 4a). Comparing films with intermediate levels of glycerol, e.g., the Glia30 film, showed the three well-defined Bragg peaks (intense and sharp; indicating a small dispersion of the scattering elements in the average peak position) indicating a well-defined hexagonal structure. For the Glia20 film, the three Bragg peaks were rather broad (indicating large dispersion of the scattering elements in the average peak position) compared to the Glia30 film (Figure 4a). No such three high order peaks from the hexagonal structure were

Figure 3. Protein secondary structure measured by FT-IR in (a) Glia− glycerol films and Glia powder; (b) Glut−glycerol films and Glut powder.

the IR spectra, for the Glia−glycerol films a peak can be clearly seen, indicating an α-helix structure (peak position at 1652 cm−1) in all the samples studied (including Glia powder) (Figure 3a). For all the Glia−glycerol films, the α-helix peak was somewhat more developed compared to the Glia40 film. When the β-sheet region (1624−1632 cm−1) between the Glia−glycerol films was compared, the most dominant broad shoulder was observed for Glia10 and Glia20 films (Figure 3a; indicated by arrows; Figure S1a and Table S1). For Glia30 film, the rather broad shoulder for β-sheets was less developed. In the Glia40 film, no clear shoulder was present, indicating a low level of β-sheets, compared to the other glycerol films (Figure S1a; Table S1). For all the Glu−glycerol films, the IR spectra showed a clear peak in the α-helix structural/amorphous region8 (Figure 3b; Table S1). The peak intensity for α-helix structure was decreasing with an increasing concentration of glycerol in the Glu−glycerol films. From Glu−glycerol films a broad shoulder in the β-sheet region (1624−1632 cm−1)8 was clearly observed for Glu20 and Glu30 films (indicated by arrows), and this shoulder was found to be rather undeveloped for the Glu40 film (Figure 3b; Figure S1b; Table S1). This tendency can be E


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30% glycerol film (with NH4OH and salicylic acid).10 Although, no such Bragg peaks were observed for the Glu−glycerol films in this study. Regarding the absence of hexagonal structure in the Glu−glycerol films one of the explanation is the glutenin aggregated/agglomerated structure, which has been observed in the commercially separated gluten.25 For future studies, it would be of interest to extract Glu by mild extraction procedure25 and investigate nanostructure of proteins in Glu−glycerol films. From this study, it is quite evident that glycerol amounts higher than 10% contribute to the formation of well-defined hexagonal structures. In fact, 20% glycerol seems to be enough for plasticizing Glia (also due to the Glia nature), whereas 40% glycerol seems to lead to the formation of unbound glycerol and less interactions between proteins (Figure 3a) (slightly greater α-helix versus β-sheets ratio) (Figure S1). In addition, the Glu-rich fraction was found more sensitive to water plasticization compared to the Glia-rich fraction,26 suggesting that the secondary proteins interactions have played a role here. We have not measured glass transition temperature (Tg), but the mechanical data indicate that the Tg is somewhere close to room temperature or below for the 20−40% glycerol samples, as observed by the large sample extensibilities (from 100% to over 500%) (Figure 6). In addition, the broad peak at the lowest q found for the Glia40 film, suggesting an interbilayer distance, which is reflected by the observed phase separation at high glycerol content for example, as in glycerol/water and copolymer system.27 Morphological study at different temperatures e.g. from 25 to 90 °C for Glia30 film has been included in this study and showed a disappearance of the Bragg peaks, and thus the hexagonal structure from 55 to 90 °C (Figure S2). Appearance of a broad peak, which was shifted toward greater q values with increasing temperature was observed (Figure S2). The disappearance of hexagonal structure has been observed previously for wheat gluten−starch composites (at the temperature interval 50−60 °C).28 Similarly, in this study, the hexagonal structure disappeared above 55 °C, for the Glia10 and Glia40 films (data not shown). For the Glu−glycerol films, no Bragg peaks were observed, except a broad and undeveloped shoulder, indicating a disorganized nanostructure (no specific structural hierarchy) (Figure 4b). The broad peak indicated the characteristic scattering distance, d, at ql, calculated as follows: d = 2π/ql. The broad peak position shifted with increasing glycerol content toward the lower q values, and the distance d increased with the increasing amounts of glycerol: 59.8, 63.5, 69.5 and 71.2 Å for the Glu10, Glu20, Glu30 and Glu40 films, respectively. The Glu−glycerol films (10%, 20% and 30%) here were more cross-linked (less protein extractable with reducing agents) compared to the Glu40 (high extractability during step 5) (Figure 2b). This fact also correlates with the lower amounts of β-sheets observed for the Glu40 sample (Figures 3b and Table S1). To conclude, the largest impact of glycerol was the swelling of the hexagonally arranged structure of Glia in the Glia− glycerol films when 20% or higher amounts of glycerol were used. Protein Nano-structural Morphology by WAXS. Glia− and Glu−glycerol films morphology at the atomic scale has been studied using WAXS shown in Figure 5. The two broad peaks having d1 and d2 values (around 9.7 and 4.4. Å, respectively) were observed for all the Glia and Glu films

Figure 4. SAXS profiles of Glia− and Glu−glycerol films; (a) Glia− glycerol and (b) Glut−glycerol films.

observed for the Glia10 film, except the broad wide peak and the very small peak at lower q values (indicating very large dispersion of the scattering elements) (Figure 4a). For the Glia40 film, the first Bragg peak was observed together with two small peaks, which were well developed, similarly to Glia30. All the three Bragg peaks were shifted toward lower q values in Glia40 film compared to the lower glycerol content samples. Importantly, a broad peak was also observed close to the first Bragg peak (Figure 4a). This observation indicated, that the Glia protein chain mobility increased and the hexagonal structure increased (Figure 4a). From the position of the first Bragg peak, ql, the interdomain distance of the hexagonal structure, dl was extracted as follows: dl = 4π/(√3 ql) for the Glia−glycerol films. The interdomain distance dl, for the cell parameters increased with the increasing amount of glycerol, e.g., 50.5, 59.6, 58.4 and 67.4 Å for 10, 20, 30 and 40% glycerol Glia films, respectively. This observation clearly indicated, that higher glycerol amounts (above 20%) increased the interdomain distance, i.e., a swelling of the hexagonal structure. The findings in this study (for the Glia films) are comparable with the lattice parameters of 70 Å found for wheat gluten with F


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structure (Table 1). However, a clear decrease in the peak d1 amplitude and the peak width was observed with the increasing glycerol amount (Figure 5a). The d1 distance seemed to correspond to the average distance of α-helices,28 suggesting a decrease in α-helices with the increasing glycerol content. An additional small peak d3= 4.65 Å was observed in the Glia10 and Glia20 films (Figure 5b; indicated by arrows). This peak seemed to be correlated with the greater amounts of βsheets for the Glia10 and Glia20 films (Figure 3a). Glu−glycerol films showed similar morphology as Glia− glycerol film by WAXS (Figure 5c; Table 1). Two broad peaks indicating amorphous structure of Glu−glycerol films were observed for all the samples studied. A decrease in the peak d1 amplitude with increasing amount of glycerol was observed for the Glu−glycerol films. This finding suggests that the average dvalues for Glu−glycerol films did not change significantly due to the varying amount of glycerol. Mechanical Properties of Glia− and Glu−Glycerol Films. The mechanical properties were determined for Glia− and Glu−glycerol films (Figure 6). The highest modulus was found for Glia10 and Glia20 films, corresponding to 562 and 94 MPa, respectively (Figure 6a). For Glia30 and Glia40 films, the E-modulus was much lower and corresponded to 4.8 and 1.4 MPa, respectively. Similarly, the maximum stress for the Glia− glycerol films was the greatest for the Glia10 films (Figure 6a). The strain at maximum stress (elongation) was the greatest for Glia30 film (489%) and the second greatest was for Glia40 film (344%) compared to the other Glia−glycerol films (166% and 3.8% for Glia20 and Glia10, respectively; Figure 6). Less extensible Glia films were found for Glia10 or Glia20 samples (Figure 6a). The possible reason for larger elongation values for Glia30 film can be due to the glycerol ability to facilitate protein chain−chain secondary forces and the increase in the free volume of the material.29 For Glia40 film, additional 10% (compared to 30%gly) contributed to both plasticization and weakening of the protein−protein interaction. We observed the smallest protein extractability during extraction step 5 with reducing conditions (with DTT, propanol at 60 °C) by RPHPLC for the Glia30 film, followed by second lowest solubility for the Glia10 and Glia20 films (Figure 2a). This, suggests that the development of inter-molecular disulfide bonds between the Glia polypeptide chains has occurred.6 In addition, for the Glia30 film the inter-molecular interactions could also be supported by the greater amount of hydrogen-bonded β-sheets versus α-helixes (Figure 3; SI Figure 1). Importantly, is that the nano-organization for Glia30 indicated a well-defined hexagonal structure (with less distortion) (Figure 4a). Consequently, a specific amount of plasticizer, 30%, produced films with the greatest elongation/extensibility in Glia−glycerol blends. In the case of high stiffness (E-modulus) and strength, Glia10 film is the best choice. When comparing the tensile properties of Glia−glycerol films in this study with the solution casted Gliaglycerol films (at similar relative humidities (RH) 50 vs 46%, respectively),30 we observed large variation. In general, the tensile performance for Glia10 film from this study was better compared to the solution-casted Glia films with 10% glycerol. Both the E-modulus and strength decreased significantly with increasing glycerol contents in the Glu−glycerol films (Figure 6b). The highest E-modulus was observed for the Glu10 film, 553 MPa, whereas the lowest E-modulus was found for the Glu40 film, 4.8 MPa (Figure 6b). The lowest E-modulus value observed in this study is similar to the tensile strength of Glu with 33% glycerol film (5.9 MPa) observed in the study by

Figure 5. WAXS profiles of Glia− and Glut−glycerol films; (a and b) Glia−glycerol; (c) Glut−glycerol films.

(Figure 5a; Table 1). For all the Glia−glycerol films, d values were rather similar, indicating no glycerol effect on the atomic G


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Figure 6. Mechanical properties of Glia− and Glu−glycerol films: (a) elastic modulus, (b) maximum stress, (c) elongation at maximum stress. Error bars indicate standard deviation. Values with the same letter are not significantly different according to Tukey’s posthoc test at level p < 0.05.

Hernandez-Munoz et al.31 The elongation for the Glu30 film in this study was also similar to the above referred study (215% vs 260−+40%). The maximum stress was highest, 17.2 MPa, for the Glu10 film. Different from the Glia−glycerol films, the strain at maximum stress of the Glu−glycerol films was considerably lower, with no significant difference between the Glu20 and Glu30 samples. The elongation at break for these samples was 190−278% (Figure 5b). In fact, a possible explanation for the lower extensibility is the presence of a highly aggregated protein (DTT reagent, extraction step 5) (Figure 2b) in the Glu10, Glu20 and Glu30 films. Previous studies have shown that a glycerol content above 20% facilitates the formation of a microphase separation, which is essential for the significant decrease in tensile strength and Young’s modulus, and for the improvement of the strain at maximum break.3 Lower strain at break is also due to impurities in the wheat gluten (bran fibers

etc.). For WG materials (with 30% glycerol), the use of mildly produced wheat gluten improved the mechanical properties and extensibility of the gluten protein films.32,33 The extraction of Glia and Glu fractions from mildly produced WG would be of high interest to include in future studies.

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CONCLUSIONS To conclude, the tuning of the nanostructural and mechanical properties upon the use of different amounts of glycerol in Glia and Glu films is presented in Figure 7. For the first time, we show that 20, 30 and to some extent 40% of glycerol are among the most favorable levels to plasticize gliadins, inducing the formation of hierarchically ordered nano-structure. This novel knowledge is of high importance for designing the nanostructures with smaller lattice distances, e.g., around 59 Å, in the Glia−glycerol films. These hierarchical structures contribute H



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to the development of Glia−glycerol films, which are aimed for applications where maximum extension (both high and medium extensibility) properties are important. If a stiff and relatively strong Glia−glycerol film is desired, no specific nanostructure tuning to mechanical properties is possible, due to a too little plasticized protein at low glycerol levels (e.g., Glia10 film; E-modulus 562 MPa). In the Glu−glycerol films, which were highly aggregated, the highest E-modulus material (554 MPa) was obtained when 10% glycerol was blended with Glu. 40% glycerol was the most suitable for plasticizing glutenins. We conclude that this study shows new opportunities on the nano-structure and mechanical properties tuning of Glia proteins in Glia−glycerol films in a more precise manner. The Glu−glycerol films were less flexible for nano-structure and mechanical properties tuning when different amounts of glycerol were used.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.5b01667. Figure S1. Protein secondary structure α-helix versus βsheets ratio by FTIR, (a) Glia−glycerol and (b) Glu− glycerol, films. Figure S2. SAXS temperature profiles of Glia30 film measured at 25, 35, 45, 55, 60, 70, 80 and 90 °C (PDF).

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REFERENCES

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Figure 7. Schematic presentation of the nano-structure mechanical properties tuning in Glia−glycerol and Glut−glycerol films.

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Research Article

AUTHOR INFORMATION

Corresponding Author

*R. Kuktaite. E-mail: [email protected]. Tel.: +46-(0)40415337. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Authors acknowledge the MAX IV Laboratory Synchrotron, Lund for the provision of a beamtime at the beamlines I911-2 and I911-4, to the financing agencies VINNOVA (supporting through the research program Trees and Crops for the Future, TC4F), the Swedish Research Council (VR), Formas, PlantLink and The Nordic Council of Ministers. I


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