Glutenin and Modified Potato Starch Green

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Innovative gliadin/glutenin and modified potato starch green composites: Chemistry, structure and functionality induced by processing Faraz Muneer, Mariette Andersson, Kristine Koch, Mikael S. Hedenqvist, Mikael Gällstedt, Tomás S. Plivelic, Carolin Menzel, Larbi RHAZI, and Ramune Kuktaite ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b00892 • Publication Date (Web): 12 Sep 2016 Downloaded from http://pubs.acs.org on September 14, 2016

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ACS Sustainable Chemistry & Engineering

Innovative gliadin/glutenin and modified potato starch green composites: Chemistry, structure and functionality induced by processing Faraz Muneer,*,† Mariette Andersson,† Kristine Koch,‡ Mikael S. Hedenqvist,§ Mikael Gällstedt,║ Tomas S. Plivelic,┴ Carolin Menzel,‡ Larbi Rhazi,┼ Ramune Kuktaite† †

Department of Plant Breeding, The Swedish University of Agricultural Sciences, Box 101,

SE-23053 Alnarp, Sweden ‡

Department of Food Science, The Swedish University of Agricultural Sciences, Box 7051,

SE-750 07 Uppsala, Sweden §

KTH Royal Institute of Technology, School of Chemical Science and Engineering, Fiber and

Polymer Technology, SE-10044 Stockholm, Sweden ║

Innventia AB, Box 5604, SE-11486 Stockholm, Sweden

┴MAX-IV Laboratory, Lund University, Box 118, SE-221 00 Lund, Sweden ┼

Institut Polytechnique, LaSalle Beauvais, 19 rue Pierre Waguet, 60026 Beauvais, France

Corresponding author’s e-mail: [email protected] Keywords: biopolymer, protein, materials, macromolecular structure, protein-starch interactions

ACS Paragon Plus Environment 1


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Abstract In this study, we combined two wheat proteins, gliadin (Gli)/glutenin (GT), and modified potato starch (MPS) into composites using extrusion. In the Gli/GT-MPS composites, we studied the structural dynamics of proteins and starch, protein-starch interactions, protein properties and composite morphology in relation to mechanical and barrier properties. Materials with different ratios of Gli/GT and MPS were extruded using either glycerol, or glycerol-water at 110 and 130 °C. For the first time, a hierarchical hexagonal structure of Gli proteins was observed in Gli-MPS composite at both extrusion temperatures. The higher temperature (130 °C) induced a higher degree of protein cross-links, an increase in the polymer size and β-sheets formation compared to 110 °C. The combination of plasticizers (glycerol and water), favored a macro-structural morphology with improved gelatinization of starch, processability, as well as strength, stiffness and extensibility of GT-MPS composites. The highest amount of the oxidized proteins was observed in the samples with the highest protein content and at high extrusion temperature. The Gli- and GT-MPS (30/70) samples showed promising oxygen barrier properties under ambient testing conditions. These findings provide in-depth information for tailoring the structural-functional relationship of the Gli/GTpotato starch composites for their promising use in designing various green materials.


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Introduction Naturally occurring plant based feedstocks have gained interest as “green” alternatives for replacing petroleum based materials. Wheat gluten (WG) protein and potato starch are two natural polymers known for their complex macromolecular structures in both un-processed and heat-treated products.1,2 Both polymers have shown attractive film forming and thermoplastic properties resulting from chemical reactions when processed with plasticizers (e.g. water or glycerol) and temperature into a green material.3 For example, WG films have demonstrated high viscoelasticity, extensibility and reasonable strength, while starch films have shown good processability and properties suitable for making biodegradable products.4-9 Wheat gluten proteins are available as a co-product from the bio-ethanol industry. Two major types of proteins from WG, namely the high molecular weight glutenin and low molecular weight gliadin are main determinants of the functional properties in films, foams and adhesives.6,10-12 In several studies on the thermo-processed gliadin/glutenin materials, the importance of the protein structure in a close relation to the mechanical properties of the final product has been observed.13,14 Although, the Gli molecular and nano-structure was considered as an indicator for predicting processing behavior of the protein film, still more knowledge is needed to fully understand and steer this process. In particular, a better control of the process chemistry and tuning of gliadin/glutenin protein structure in a blend with other components, like starch, still remain unclear. Native potato starch consists of 20-30% of amylose, a long chain linear glucose polymer and 70-80% of amylopectin, with a highly branched molecular structure. Potato starch has been chemically modified to change the amylose vs. amylopectin ratio, which led to improvement of the functional properties of starch materials due to increased entanglements between glucose molecules.15-17 Alteration in the ratio led to changes in the starch processing



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behavior, e.g. high amylose starches were more viscous and required high moisture content.8 Therefore, more knowledge about the macromolecular structure and processing behavior is needed in order to define the morphology of the potato starch polymer and its blends. In our previous study, we have shown that it is possible to extrude WG and modified potato starch composites with promising functional properties (mechanical and gas permeability) suitable for packaging materials.18 The macromolecular structure of WG-starch composites was developed under the influence of high processing temperature (130 °C) and plasticizer (45% glycerol or 30% glycerol and 20% water). The observed hexagonal structure of WG proteins in the composites having 50% or more of protein favored increased extensibility of the materials. In WG based materials, proteins undergo irreversible aggregation with the formation of disulphide cross-links, hydrogen and isopeptide bonds during thermomechanical and chemical treatment.4,19 The aggregation of proteins and the formation of an increased amount of β-sheets could be one of the reasons that commercial WG protein arranges into hexagonal structure.20,21 For gliadin materials, plasticization with glycerol during thermal processing was sufficient to achieve the hexagonal structural arrangement.14 However, more understanding towards the better control of protein/and starch structure during processing and in the final product is needed, and this still remains as a challenge when developing new plant-based polymer materials with desired functionalities. In this study, we have produced composites from the modified potato starch and gliadin/glutenin proteins using extrusion processing to study the chemistry, structure and macromolecular properties of protein-starch composites in relation to process-dependent modifications. The aim of this study was to monitor in detail the structural organization, polymerization and cross-linking of protein-starch composites induced by various processing conditions. The macromolecular structure in relation to the functional properties (mechanical and barrier) of the composite was also investigated.


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Experimental Section Materials Wheat gluten powder (protein 77.7%, starch 5.8%, moisture 6.9%, fat 1.2%) was provided by Lantmännen Reppe AB, Lidköping, Sweden. Modified potato starch (MPS) with long chain amylopectin, and an amylose content of 27% was used.18 Starch was processed at Lyckeby Starch AB, Kristianstad, Sweden. The glycerol (99.5%) was supplied by Karlshamn Tefac AB, Karlshamn, Sweden. Methods Separation of glutenin and gliadin from wheat gluten Glutenin (GT) and gliadin (Gli) were extracted from WG following the method described by Blomfeldt et al.6 WG powder (16 g) was dispersed through a metal sieve in 200 mL ethanol (70%) and constantly stirred until the mixture was homogenized. The mixture was placed on a shaker, IKA-KS 500 (IKA, Germany) (30 min, 300 rpm) and centrifuged for 10 min at 10,000 rpm in Sorvall RC 6+ centrifuge (Thermo Scientific, USA). The ethanol soluble Gli was extracted once and collected in a rotary evaporator at 70±5 °C (Buchi, Switzerland). The ethanol in-soluble GT fraction was collected as the remaining solids. Gli- and GT-protein rich fractions were lyophilized and grounded into powder using IKA A10 grinder (IKA, Germany). For Gli and GT rich protein fractions, the protein contents were 91% (Gli) and 75% (GT) respectively, as determined according to the Dumas method22 (Thermo Scientific, Flash 2000 NC Analyzer). Sample preparation and extrusion The Gli/GT were extruded with MPS in different ratios of 70/30, 50/50, 30/70 with 45% glycerol at 110 and 130 °C. Samples were abbreviated, Gli-MPS-45gly 30/70, in which 30%



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Gli and 70% MPS blend (1) was extruded with 45% glycerol (0.45) (wt% of the total wt. of dry blend i.e. 1:0.45). In two GT-MPS 50/50 samples a mixture of 20 parts water and 30 parts glycerol was also used, GT-MPS-20W-30gly 50/50. Samples were mixed by hand and extruded at 110 and 130 °C by Haake Minilab twin-screw extruder (Thermo Electron Corporation, Germany) at 30 rpm, in the form of strips. Protein solubility by size exclusion high performance liquid chromatography Extruded strips were chopped into particles (approx. 0.2 mm) using a scalpel for extraction of protein for SE-HPLC analysis.23 The extraction of protein was done in triplicates where 16.5 mg (±0.05 mg) of each sample was mixed with 1.4 mL buffer (0.5% SDS, 0.05M NaH2PO4, pH 6.9) in a 1.5 mL Eppendorf tube and vortexed for 10 sec (Whirli Vib 2, Labessco, Sweden). Thereafter, samples were shaken for 5 min (IKA-VIBRAX VXR, IKA, Germany) (2000 rpm, centrifuged 30 min at 12000 rpm (Legend Micro 17, Sorvall, Germany)). The supernatant was decanted and analyzed as first extraction (1Ex). The second extraction (2Ex) was performed by sonication for 30 sec (5 microns; Sanyo Soniprep 150 Ultrasonic Disintegrator, Tamro, United Kingdom) and followed by a third extraction (3Ex) (sonication 30+60+60 sec). The samples were left to cool at room temperature during each sonication interval in the 3Ex step. The 1Ex, 2Ex and 3Ex samples were analyzed using a Waters 2690 Separation Module with a Waters 996 Photodiode Array Detector (Waters, USA) according to Newson et al.23 Twenty µl of each sample was injected onto the SE-HPLC column (Biosep-SEC-S 4000, Phenomenex, USA) for analysis, with an isocratic flow of 0.2 mL/min (50% acetonitrile, 0.1% TFA; 50% H2O, 0.1% TFA). Empower Pro software (Waters, USA) was used for analysis of data (210 nm) and chromatograms were integrated and divided into two groups, polymeric proteins (PP, retention time, 7-14 min) and monomeric proteins (MP, 14-28 min).


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The area of the elution intervals represents the absorbance obtained for the protein content (available in 16.5 mg) in the composite sample. Protein polymer size by asymmetrical flow field flow fractionation The GT polymer molecular weight (Mw) in the GT-MPS composites was evaluated by measuring the molecular size of extractable protein using asymmetrical flow field flow fractionation (A4F).24 Ten mg of ground samples were dispersed and incubated at 60 °C for 15 min with 1.0 mL of 0.05 M sodium phosphate buffer (pH 6.9) containing 2% (w/v) SDS. The extracts were then sonicated for 20 sec at a power setting of 30% (full power 125W). The supernatant (centrifugation at 12,500 g at 20 °C for 15 min), was filtered through 0.45 µm filters (Gelman Sciences, France) before injection (30 µl) into the A4F/MALLS system. AF4 was accomplished with an Eclipse3 F System (Wyatt Technology, Santa Barbara, CA, USA) serially connected to a UV detector (Agilent 1200, Agilent Technologies, Germany), MALLS detector (Dawn multi-angle Heleos TM, Wyatt Technology Corporation, Europe) and an interferometric refractometer (Optilab rEX, Wyatt Technology Corporation, Europe). Absorbance was measured at 214 nm. The channel had a trapezoidal geometry and a length of 286 mm. The thickness of the spacer used in this experiment was 350 mm. The ultrafiltration membrane forming the accumulation wall was made of regenerated cellulose with a cut-off of 10 kDa (LC-10 Nadir reg. Cell, Wyatt Technology Europe, Germany). An Agilent 1200 Series Isocratic HPLC Pump (Agilent Technologies, Germany) was used with a 0.45 µm in-line filter (Gelman Sciences, France). Sodium phosphate buffer (0.05 M, pH 6.9) containing 0.1% (w/v) SDS was used as mobile phase (Gelman Sciences, France). For the fractionations using a gradient in the cross-flow, the focus time was 0.5 min at a flow rate of 2 mL.min-1, followed by a focus/injection time of 1.0 min at 0.2 mL.min-1 and a relaxation/focusing time of 0.5 min. An outflow rate (Fout) of 1.0 mL.min-1 was used and a



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cross-flow rate (Fc) decreasing exponentially from 3.0 to 0.0 mL.min-1 for 14 min. Finally, elution at a cross-flow rate of 0.0 mL/min was maintained for 9 min. All necessary constants for molecular weight distribution calculation were determined previously.24 Bovine serum albumin (BSA) standard was used to normalize the diodes of the MALLS detector. The calculated Mw (g/mol) of BSA was reasonably close to the nominal value. Nε-(carboxymethyl)lysine quantification Nε-(carboxymethyl)lysine further used as carboxymethyllysine (CML) extraction and determination of its concentration from protein-starch and protein powder samples were done according to the method developed by Niquet-Léridon et al.25 CML is a stable advance glycated end (AGE) product found as a result of glucose and lysine amino acid chemical reactions. Scanning electron microscopy The microstructure of GT-MPS and Gli-MPS composites horizontal cross-sections were studied with a scanning electron microscope (SEM) (LEO 435VP, Cambridge, UK) by secondary electron detection (acceleration voltage of 10 kV). Au/Pd (3:2) coating (JFC-1100, JEOL, Tokyo, Japan) was applied to the samples prior to SEM analysis. Light microscopy The Gli-MPS and GT-MPS films were cut into 4x4 mm approximately 0.3-0.5 mm thick pieces with a scalpel. The samples were placed on a glass slide and stained with 0.1% Light Green (Sigma, St. Louis, MO, USA) for protein and 50% Lugol solution (Fluka, GmbH, Germany) for starch. The excess stain liquid was rinsed away and samples were covered with a glass slip and immediately observed by a light microscopy (Model DMLB, Leica


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Microsystems, Bensheim, Germany) and digital pictures taken (Camera model, DC200, Leica Microsystems, Bensheim, Germany). Fourier transform infrared spectroscopy FT-IR spectroscopy was done on all the extruded samples and Gli and GT powders using a Spectrum 2000 FT-IR spectrometer (PerkinElmer inc., U.S.A) with a single reflection ATR (Golden Gate, Speac Ltd.). All the samples were dried using silica gel for at least 72 h before testing. The FT-IR absorbance spectra were first Fourier self deconvuluted (FSD) using a Spectrum software with γ = 2 and smoothing of 70%. Peak fitting was carried out with peak fitting indicated by 2nd derivative analysis (Savitzy-Golay filter, 5 points).26 Around 8-9 Gaussian peaks were fitted between 1680-1705 cm-1 using Fityk v0.9.8.27 Peak assignments were performed according to Cho et al. (2011)26 and Kuktaite et al. (2016).28 Small angle X-ray scattering Small angle X-ray scattering (SAXS) analysis was carried out using a monochromatic beam (λ = 0.91 Å; scattering vector range: q = 4π/λ sin(θ); 2θ is the scattering angle and q was 0.008 < q < 0.35Å-1) at the beamline I911-4, MAX IV Synchrotron laboratory, Lund, Sweden.29 Two dimensional images were obtained using a bi-dimensional hybrid pixel X-ray detector (Pilatus 1M, Dectris) with sample to detector distance of 1901.7 mm and an exposure time of 2 or 5 min, depending on the intensity of the beam. The obtained data were normalized and incident integrated intensity, sample absorption, parasitic scattering and detector background accounted for, using softwares such as bli9114 and Fit2D.29 Wide angle X-ray scattering Wide angle X-ray scattering analysis (WAXS) was carried out using beamline I911-2 at the MAX IV Synchrotron laboratory, Lund, Sweden.30 WAXS measurements were performed at



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wavelength of λ = 0.91 Å and a distance from the detector of 290 mm using an area-CCD detector (active area having a diameter of 165 mm). The Fit2D software was used for data analysis and parasitic scattering was subtracted from the data. Silicon powder was used as a standard for calibration.31 Tensile testing All the samples were cut to a minimum length of 70 mm (widths 3.6-4.1 mm and thickness 0.8-1.4 mm) and conditioned for 48 hours at 50% (±2) relative humidity (RH) and 23 °C temperature before testing. The width and thickness of the samples were measured using digital indicator Mitutoyo IDC 112B (Mitutoyo, Japan) at five different points and average was used for calculations. For all samples a 100 N load cell, clamp separation distance of 40 mm and crosshead speed of 10 mm/min was used in an Instron 5566 universal test machine using a Bluehill software (Instron AB, Danderyd, Sweden). All values were calculated from at least of 10 replicates of each sample. Oxygen barrier properties Samples for oxygen permeability (OP) analysis were prepared according to Muneer et al.18 Briefly, small pellets (2-4 mm) cut from extruded strips of each sample were compression molded (Polystat 400s, Servitech, Germany) for 10 min (130 °C and 200 bars) using a metal frame (100 x 100 mm). Oxygen transmission rate analysis was carried according to ASTM F1927-07, using Ox-Tran 2/21 (Mocon Inc., USA) and samples were conditioned for 16 h prior to the test at 23 °C and 50% RH and at 38 °C and 90% RH. Two replicates from each sample were analyzed and tests were carried out on a measured area of 5 cm2 using a gas flow of 10 mL/min.


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Results and discussion Protein solubility analysis The Gli-MPS and GT-MPS composites extruded at 110 and 130 °C were studied for protein polymerization behavior using SE-HPLC (Figure 1). For Gli-MPS-45gly samples, the solubility of the polymeric proteins (PP) and monomeric proteins (MP) was lower at an extrusion temperature 130 °C compared to 110 °C. The Gli solubility (for both MP and PP) increased with increasing concentration of Gli in the composite at 110 °C. Only a small increase in solubility of MP and PP with increasing Gli concentration was observed for the Gli-MPS samples extruded at 130 °C suggesting that Gli became part of an aggregated/crosslinked protein network (Figure 1). The GT-MPS composites showed rather low protein solubility for MP and PP at 110 and 130 °C. Regarding the PP protein solubility, it was found to be particularly low compared to MP at both temperatures. An increase of GT content in the composite did not affect overall GT solubility at 110°C. The decrease in solubility of MP and PP types of GT at higher processing temperatures (110 vs. 120 °C) was observed previously.32 In this study, the GT even when blended with potato starch showed a decrease in MP and PP, which is a result of higher degree of polymerization due to formation of inter-protein disulphide bonds, as well as covalent and hydrogen bonds at higher temperature.4,33 A similar behavior of WG proteins was also observed in extruded pasta samples.34 In this study, shear mixing and high extrusion temperature provided a suitable environment to form irreversible bonds as reported previously.35-38



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Molecular weight distribution of glutenins in GT-MPS composites by A4F The Mw distribution of GT in GT-MPS extrudates was analyzed by A4F (Figure 2). The molecular sizes of GT was within the range 25 x 107 to 170 x 107 g/mol. The Mw (g/mol) of the polymeric proteins was higher at 130 °C compared to 110 °C in GT-MPS-45gly (from 2.8 to 4.9). No differences in the Mw of the polymer were observed for the GT-MPS-30gly-20W 50/50 samples at the studied temperatures (Figure 2). The GT-MPS-45gly 70/30 and 50/50 samples showed an increase in the Mw of the large polymer with increasing processing temperature, probably due to higher degree of cross-links between the polymeric proteins and higher amount of proteins in the sample. Another important factor which might have affected the protein cross-linking is the presence of MPS which is present in a compact native-like conformation evidenced by its limited gelatinization in WG-MPS glycerol blends,18 as similarly observed in extruded rice pasta.34 In this study, an increase in processing temperature potentially induced the swelling of the starch in GT-MPS-30gly-20W 50/50 samples, therefore limiting the GT protein-protein interactions. The Mw of GT polymers in this study was much higher compared to other studies (5 x 106 and 2 x 107).39,40 One of the possible explanations is higher amounts of GT used in the GT-MPS composites in this study compared to the GT fraction in wheat flour.41 Results from this study suggest, that a careful selection of plasticizer (a blend of glycerol and water) can potentially regulate the cross-linking behavior of GT protein in the protein-starch composites. Previous studies have shown that excessive aggregation of proteins can be manipulated using additives such as urea and salicylic acid, which delay the aggregation reactions during extrusion.21,38 Therefore, it should also be taken into account that variation in Mw of GT depends on the sample preparation and type of sample studied.42 Determination of Nε-(carboxymethyl)lysine content


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In the present study, the content of CML was determined in order to study the AGE products resulting from non-enzymatic glycation and oxidation reactions on Gli- and GT- in the protein-starch composites (Figure 3). The CML contents of Gli- and GT-MPS samples varied greatly between the samples studied (Figure 3). The Gli-MPS-45gly 70/30 and 50/50 samples showed higher amounts of CML at 130 °C than at 110 °C (Figure 3a). The amounts of CML increased with increasing protein content in the Gli-MPS samples, and highest amount of around 80 µg/g was observed for the Gli-MPS-45gly 70/30 at 130 °C (Figure 3a). High amount of CML was also observed in the Gli (MPS free) sample at 110 °C. This result suggests that in Gli-MPS samples the protein-starch interactions resulted in glycated proteins and Amadori Dione products43 (Supporting information, Figure S1). Similarly, GT-MPS-45gly (70/30 and 50/50) and GT-MPS-30gly-20w 50/50 samples extruded at 130 °C showed higher CML content compared to 110 °C (Figure 3b). In contrast to Gli-MPS samples, a small increase in CML with increasing protein concentration was observed in GT-MPS samples at 130 °C (Figure 3b). The highest amount of CML was found in the GT-MPS 70/30 sample at 130 °C, and in GT (MPS free) samples produced at 110 and 130 °C. A slightly higher amount of CML was observed for GT-MPS-30gly-20W 50/50 at 130 °C compared to 110 °C. High processing temperature favors the formation of CML content in wheat proteins as for example, the formation of bread crust in baked bread products.44 Oxidation of the gluten proteins results in the formation of disulphide and tyrosine bonds between the protein molecules, which are probably present in our samples. The Gli and GT processed at 110 °C showed higher amounts of CML content compared to the protein-potato starch composites, possibly due to the presence of residual wheat starch in the starting material (5.8% of wheat starch in the initial WG powder). The presence of the residual wheat starch helped in the formation of glycation-derived lysine peptides (Figure S1). Similarly, in the protein-starch



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samples with highest protein content such as Gli-MPS-45gly 70/30 and in GT-MPS-45gly 70/30 samples extruded at 130 °C, the residual wheat starch most likely favored the formation of high CML content. Heating of proteins and glucose leads to the formation of Maillard products which result into glycation end-products (AGE), where CML is one of them.25 CML is commonly used to determine the protein damage in severely heated foods rich in proteins and sugars such as, glucose.25, 45-47 In this study, the measuring of CML content in the GT-MPS composites is a valuable indicator to improve understanding on chemical pathways of the glycated GT proteins and protein-starch interactions in the GT-MPS composites. Microstructure of Gli- and GT-MPS composites The microstructure of Gli- and GT-MPS samples (50/50 and 70/30, extruded at 130 °C) with either glycerol or glycerol-water was studied using SEM and LM (Figure 4). The Gli- and GT-MPS composites showed varying microstructural patterns such as, the non-homogenous morphology with few intact starch granules and voids (Figure 4a, b, e) and rather homogenous morphology with many protein-starch blended areas (circled areas; Figure 4c, d). In general, the protein-starch samples with glycerol-water showed more homogenous pattern compared to only the glycerol containing samples studied by SEM and LM. The microstructure of the Gli-MPS-45gly 70/30 sample (SEM), consisted of non-homogenous protein matrix with a few large round/oval starch granules (size ∼10 microns), a number of small starch granules (≤5 microns) and few voids (Figure 4; arrow 1, 2). In a Gli matrix (blue-green stain) (LM), only a few blended areas of protein-starch were observed (Figure 4b; arrow 3) containing mainly the intact starch granules of various sizes (violet stain) (Figure 4b; arrow 4). This observation indicates, that the conditions such as, 45% glycerol and 130 °C, were insufficient to gelatinize the MPS in Gli-MPS composites.


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The microstructure of the GT-MPS-30gly-20W 50/50 and 70/30 samples showed a homogenous protein-starch pattern observed by both SEM and LM (Figure 4c, d). Few intact starch granules were observed in the matrix and seemed better incorporated in GT-MPS30gly-20W (Figure 4c, d; dotted arrows) compared to the GT-MPS-45gly (70/30) where voids (indicated by bold arrow) and poorly incorporated starch granules (dotted arrow) were observed in the protein matrix (Figure 4e). Also, few cracks in the matrix were observed for the GT-MPS-30gly-20W samples by SEM (Figure 4c; intact arrow). For the GT-MPS-30gly20W composite, the microstructure (LM and SEM) shows the homogenous morphology of protein and starch matrix (Figure 4c, d; circled areas). This result suggests that the combination of 30 glycerol and 20 water (e.g. 130 °C) was the most satisfactory for producing a homogenous matrix of the protein-starch blends suggesting a better gelatinization of starch granules. The homogeneity of microstructure for GT-MPS-30gly20W samples observed in this study was better compared to previously observed microstructure of WG-MPS blends with glycerol-water.18 From this study we conclude that for homogenous microstructure of the protein-starch composites a combination of plasticizers (glycerol-water) is required. However, for development of tailored designs in extruded composites with highly homogenous microstructure further improvement is needed towards improving the interactions between components and better gelatinization of MPS. High pressure pre-gelatinization and higher processing temperatures (> 130 °C) for MPS starch are one of the future routes to explore. Secondary structure of proteins in protein-MPS composites Secondary structure of the proteins in Gli- and GT-MPS composites was studied using FT-IR spectroscopy in the amide I region (1600-1700 cm-1) (Figure 5). The presence of αhelices/random coils and β-sheets was observed in the amide I regions 1645-1660 cm-1 and



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1610-1635 cm-1, respectively. In all the samples, β-turns and β-sheets were also observed in the amide I region 1661-1695 (Figure S2, Table S3-S4). The samples processed at 130°C showed more developed β-sheet structure as compared to 110°C samples. Gli-MPS-45gly extruded samples (70/30, 50/50) showed the formation of greater amounts of α-helicesrandom coils and β-sheet at 130°C compared to 110°C (Figure S2, Table S3). The Gli-MPS45gly 30/70 sample processed at 110 and 130 °C showed higher content of unordered structures compared to the other samples (70/30, 50/50), most likely due to low concentration of protein in the sample and higher amount of MPS (Figure 5a; Figure S2, Table S3). The GT-MPS-45gly samples extruded at 110 °C showed the formation of both α-helices/ random coils in the 1645-1660 cm-1 region for all protein-starch ratios and a small shoulder peak in the 1620-1635 cm-1 region, for 70/30 and 50/50 samples (Figure 5b, Figure S2). The GT-MPS-45gly samples produced at 130 °C, were more aggregated as shown by the clear formation of β-sheets in the 1620-1635 cm-1 region (Figure 5b, Figure S2, Table S4). The formation of β-sheets (hydrogen bonding) in GT-MPS-45gly (70/30, 50/50) samples at 130 °C suggests that the higher temperature favors the aggregation of GT to organized secondary structures. The reorganization of the protein secondary structures i.e. the conversion of αhelices to β-sheets at higher temperatures has been reported previously in wheat proteins.26, 36 The GT-MPS-30gly20W 50/50 samples showed higher amount of β-sheets related structure when processed at 130 °C compared to 110 °C (Figure 5b, Table S4). It is important to mention, that the GT-MPS-30gly20W sample at 110°C showed a rather “balanced” β-turns to β-sheets ratio, with a small increase in β-sheets (Figure S2n, o). This phenomena suggests, that the presence of water in the composite blend favored the formation of hydrogen bonds and disulphide bonds. Higher temperature (130 °C) induced GT aggregation and resulted in the formation of even higher β-sheets vs. α-helices/random coils and unordered structures. In


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the previous study, similar aggregation effect due to temperature was observed when GT was processed with 30% glycerol at 130 °C.14

Nano-morphology of protein-starch composites Several distinct scattering peaks were observed in the Gli-MPS-45gly samples extruded at 110 and 130 °C at low and high q values by SAXS (Figure 6a, b). Three Bragg peaks (d1, d2 and d3) following a 1: √3: √4 peak positional ratio were clearly observed in all the Gli-MPS samples indicating a hexagonal structure in Gli (Figure 6a, b). This is the first time such a structure has been revealed in the protein-starch composite (Figure 6; Table S1 (Supporting information)). The formation of hexagonal structure in the WG and Gli films with either urea-glycerol11, 21 or glycerol14 has been reported in previous studies. Besides the hexagonal structure, additional peaks, d*, were observed in the Gli-MPS-45gly samples extruded at 130 °C (Figure 6b). The possible source for these peaks might be the aggregated structure of the protein with undefined structural conformation. Similar peaks have been reported at the same q-values in pristine Gli powder and Gli-glycerol compression molded films.14 At low q-values we observed the additional peak (d**) in the Gli-45gly (MPS free) samples at both 110 and 130 °C. In a study by Rasheed et al.48 this additional peak was observed in WG-glycerol material indicating the presence of a lamellar arrangement.48 The dBROAD peak observed in this study was a combined reflection of the scattering objects from the lamellar semi-crystalline structure of starch and a broad correlation distance of Gli proteins14, 18 (Figure 6a, b). The d100 peak found in this study was also observed in the WG-MPS composites.18 This peak represents the reflection of the B-type crystalline structure of starch (scattering distance between the two double helices of the amylopectin fraction in a hexagonal unit cell).49 The scattering intensity of the 100 reflection



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of the starch decreased with the decrease in starch content of the sample regardless of the extrusion temperature difference (Figure 6a, b). The GT-MPS-45gly composites did not show any specific nano-structural arrangements (Supporting information, Figure S3a, b). Two broad peaks, dBROAD and d100, were observed in all the protein-starch samples processed at 110 and 130 °C. For the GT-MPS-45gly samples processed at 130 °C, the scattering distance of the dBROAD peak increased with the decreasing protein content (Figure S3b; Table S2). For the GT-glycerol sample at 130 °C the scattering distance was found the lowest 68.8 Å, while for the GT-MPS-45gly 30/70 sample, the scattering distance was observed the highest 90.6 Å (Table S3). This observation suggests that starch effects GT aggregation behavior in the composite at the molecular scale. The addition of water-glycerol in the GT-MPS samples plasticized the protein-starch composites, and the scattering distance of dBROAD was found to be 88.5 Å at 110 °C and 69.9 Å at 130 °C, respectively (Table S2). No specific structural morphology of the GT-MPS water-glycerol samples was observed in this study (Figure S3c). Atomic structure of protein-starch composites The WAXS profiles of the Gli-MPS and GT-MPS composites and X-ray diffraction profile of MPS powder is shown in Figure 7a. MPS powder showed a large number of peaks indicating a crystalline morphology and scattering reflections of these peaks are indicated by their Miller indices as reported previously49 (Figure 7a). When comparing the morphology of the protein-MPS extrudates, the B-type crystalline (100 reflection) structure of starch was observed in all the samples studied regardless of the processing temperature and composition of the blend (Figure 7). At 130 °C, the Gli-MPS samples showed a decrease in the starch crystallinity compared to the 110 °C samples, and this was due to the improved gelatinization of starch e.g. less intense crystalline peaks observed in the WAXS profile (Figure 7b).


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For the GT-MPS samples, a morphology similar to Gli-MPS morphology was observed such as decreasing starch crystallinity with increasing protein content and processing temperature (Figure S4a, b). The use of glycerol-water in GT-MPS samples extruded at 110 and 130 °C slightly changed the morphology of the composites compared to the samples with glycerol (Figure S4a-c). The X-ray diffraction profile of the glycerol-water samples consisted of fewer crystalline starch peaks compared to the glycerol samples (Figure S4c). The use of waterglycerol in the GT-MPS composites affected the morphology of the protein-starch blend and improved the gelatinization of starch granules.18,50 The gelatinization of starch involves swelling of amorphous regions and concomitant melting of amylopectin crystals.51 Mechanical properties The mechanical properties of Gli-MPS and GT-MPS composites are shown in Figure 8. For GT-MPS-30gly-20W (50/50) samples, higher E-modulus (E-mod) and maximum stress (σmax) were observed at both 110 and 130 °C (Figure 8). For GT-MPS-45gly 30/70 samples, an increase in both the σmax and extensibility (ɛ), and no change in E-mod were observed with increasing temperature (110 to 130 °C) (Figure 8). An increase in GT content in the sample resulted in a decrease of E-mod and σmax at both temperatures, and decrease in ɛ for samples extruded at 130 °C. None of the mechanical tests were possible to perform for the GT-MPS-45gly 70/30 composite extruded at 110 °C (due to poor sample quality). The Gli-MPS-45gly composites showed different mechanical properties compared to GTMPS composites. The Gli-MPS-45gly 70/30 showed the second highest elongation of the samples produced in this study (Figure 8c). The increase in Gli content in the composites did not affect the E-mod nor the σmax. The Gli-MPS-45gly samples extruded at 110 °C were not testable after 48 h conditioning due to glycerol migration to the surface of the samples resulting in sticky surfaces and difficult to handle.



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In this study the best mechanical performance of GT-MPS-30gly-20W composites was achieved when a high amount of plasticizer (50% glycerol/water) was used at 110 °C (Figure 8). A possible explanation is that higher temperature and higher plasticizer improved starch gelatinization and its better incorporation in the protein matrix, which was obvious in the microstructure showed by SEM and LM for GT-MPS-30gly-20W vs. GT-MPS-45gly (Figure 4c, d, e).The GT protein fraction is mainly dominated by large polymers connected by disulphide bonds (resulting in low polymer solubility due to large molecular size, Figure 1 and 2). When these GT proteins and MPS are mixed with water-glycerol, the starch acts as a filler in the GT protein polymeric matrix. From our previous study18 on WG-MPS composites, we observed that a combination of two plasticizers (water and glycerol) led to higher stiffness, strength and ɛ compared to only glycerol. In this study, the added glycerolwater plasticized the GT-MPS blend, and resulted in a slightly higher E-mod, σmax and similar ɛ compared to WG-MPS composites (see Figure 4c, with improved homogeneity of the composite). In protein based systems the addition of water favors the hydrogen bonding which ultimately improves the strength and ɛ of the protein based materials.52 The use of both water and glycerol for the GT-MPS composites improved the tensile properties significantly. Oxygen permeability of protein-starch composites The oxygen permeability of selected Gli-MPS and GT-MPS composites was investigated (Table 1). The GT-MPS-45gly 30/70 sample showed the lowest OP values when measured at 23 °C and 50% RH, however at higher temperature (38 °C) and RH (90%) the values were over range (OR) (Table 1). Similar OP has been previously for MPS-45gly.18 In this study, the OP for GT-MPS composites were to a large extent determined by the amount of potato starch in the blend (Table 1).


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The Gli-MPS-45gly 70/30 and 30/70 samples showed higher OP values compared to GTMPS and MPS samples (Table 1). In this study, the OP of Gli-MPS composites at the higher temperature and RH showed reasonable properties and were still measureable; the OP increased up to 22 times compared to values measured at 23 °C and 50% RH. The attractive OP observed in Gli-MPS materials show great potential to be further explored in designing packaging materials; for example as paper coatings, multilayered films as well as in medical sector such as wound healing products, where specific oxygen permeability is required. The OP values observed in this study are comparable to other WG films, WG-starch and WG-clay based nano-composites.18, 21, 53 Conclusions In this work, the structural morphology of plasticized Gli-MPS and GT-MPS composites extruded at 110 and 130 °C was studied in relation to the protein properties, macromolecular interactions, oxygen barrier and mechanical behavior. For the first time we have shown that the hierarchically arranged hexagonal structure of Gli proteins is present in a protein-starch composite extruded at 110 and 130 °C. The analysis of secondary protein structure indicated the highest amount of β-sheets in samples with glycerol-water at 130 °C. The GT-MPS30gly-20W samples extruded at 110 °C showed a more “balanced” amount of α-helices and β-sheets compared to all other samples. The higher extrusion temperature (130 °C) favored protein cross-linking (decreased solubility and increased polymer size) compared to samples processed at 110 °C. The largest extracted GT polymers were formed in the GT-MPS-45gly 70/30 extrudates processed at 130 °C. The most oxidized proteins (the highest CML content) were found in Gli-MPS-45gly and GT-MPS-45gly 70/30 samples extruded at 130 °C. The microstructural analysis of the Gli-MPS with 45% glycerol showed non-homogenous structure with a large amount of intact starch granules and few voids. The use of a plasticizer



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blend (30% glycerol and 20% water) and an elevated temperature (e.g. 130 °C) was adequate for producing a rather homogenous matrix in GT-MPS composites. In addition, the glycerolwater blend improved the mechanical properties of the GT-MPS 50/50 composites at both 110 and 130 °C with the highest E-mod and σmax observed, compared with the samples when only glycerol was used. The GT-MPS-45ly 30/70 samples showed promising oxygen barrier properties under ambient testing conditions, which can easily compete with several synthetic or biobased polymer composites, and has a good potential for designing materials in different applications. The Gli-MPS-45gly samples showed good functional properties (extensibility and processability) promising for example, for 3D printing (Figure 9), although further development is still needed. With this study, we provide insights into the protein-starch structure and polymer biochemistry after extrusion processing, as well as characteristics of the final Gli- and GT- MPS composites for various potential end-uses. This study sufficiently improves the molecular understanding of the factors that affect the structure and physicochemical properties of the protein-starch composites. This knowledge provides a suitable base for design and tuning of the most promising protein-starch materials from this study and their future functional performance in green applications. Supporting information Table S1 and S2. Scattering distances of SAXS profiles of Gli- and GT-MPS blends processed at 110 and 130°C. Table S3 and S4. Secondary structure analysis and quantification of proteins in Gli- and GTMPS blends.


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Figure S1. Chemical pathway of carboxymethyllysine formation. Figure S2. Deconvuluted profiles of all the samples studied for secondary structure analysis. Figure S3a-c. SAXS profiles of GT-MPS-45gly and GT-MPS-30gly-20W blends. Figure S4a-c. WAXS profile of GT-MPS-45gly and GT-MPS-30gly-20W blends. Acknowledgments The Swedish Governmental Research program Trees and Crops for the Future (TC4F), The Swedish Foundation for Strategic Environmental Research MISTRA, Lyckeby Starch AB, FORMAS and Partnerskap Alnarp are acknowledged for the financial support. Acknowledgments go to M. Luisa-Prieto Linde, A.-S. Fält, P. Jacolot, L. Lequeux and K. Brismar for their assistance in the lab and William R. Newson for English editing. MAX IV laboratory (I911-4 and I711) is acknowledged for beam time. References (1) Lagrain, B.; Goderis, B.; Brijs, K.; Delcour, J. A. Molecular basis of processing wheat gluten toward biobased materials. Biomacromolecules 2010, 11, 533-541.2. (2) Yu, L.; Dean, K.; Li, L. Polymer blends and composites from renewable resources. Prog. Polym. Sci. 2006, 31, 576-602. (3) Gonzalez-Gutierrez, J.; Partal, P.; Garcia-Morales, M.; Gallegos, C. Development of highly-transparent protein/starch-based bioplastics. Bioresour. Technol. 2010, 101, 20072013. (4) Johansson, E.; Malik, A. H.; Hussain, A.; Rasheed, F.; Newson, W. R.; Plivelic, T.; Hedenqvist, M. S.; Gällstedt, M.; Kuktaite, R. Wheat Gluten Polymer Structures: The Impact of Genotype, Environment, and Processing on Their Functionality in Various Applications. Cereal Chem. J. 2013, 90, 367-376. (5) Wu, Q.; Andersson, R. L.; Holgate, T.; Johansson, E.; Gedde, U. W.; Olsson, R. T.; Hedenqvist, M. S. Highly porous flame-retardant and sustainable biofoams based on wheat gluten and in situ polymerized silica. J. Mater. Chem. A 2014, 2, 20996-21009.



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(44) Assar, S.; Moloney, C.; Lima, M.; Magee, R.; Ames, J. Determination of N ɛ(carboxymethyl)lysine in food systems by ultra performance liquid chromatography-mass spectrometry. Amino Acids 2009, 36, 317-326. (45) Charissou, A.; Ait-Ameur, L.; Birlouez-Aragon, I. Evaluation of a gas chromatography/mass spectrometry method for the quantification of carboxymethyllysine in food samples. J. Chromatogr. A 2007, 1140, 189-194. (46) Delatour, T.; Hegele, J.; Parisod, V.; Richoz, J.; Maurer, S.; Steven, M.; Buetler, T. Analysis of advanced glycation endproducts in dairy products by isotope dilution liquid chromatography–electrospray tandem mass spectrometry. The particular case of carboxymethyllysine. J. Chromatogr. A 2009, 1216, 2371-2381. (47) Ames, J. M. Determination ofNɛ-(Carboxymethyl)lysine in Foods and Related Systems. Annals of the New York Academy of Sciences 2008, 1126, 20-24. (48) Rasheed, F.; Hedenqvist, M. S.; Kuktaite, R.; Plivelic, T. S.; Gällstedt, M.; Johansson, E. Mild gluten separation–A non-destructive approach to fine tune structure and mechanical behavior of wheat gluten films. Ind. Crops Prod. 2015, 73, 90-98. (49) Nishiyama, Y.; Putaux, J.-l.; Montesanti, N.; Hazemann, J.-L.; Rochas, C. B→A Allomorphic Transition in Native Starch and Amylose Spherocrystals Monitored by In Situ Synchrotron X-ray Diffraction. Biomacromolecules 2009, 11, 76-87. (50) Lai, L. S.; Kokini, J. L. Physicochemical changes and rheological properties of starch during extrusion. (A review). Biotechnol. Prog. 1991, 7, 251-266. (51) Waigh, T. A.; Gidley, M. J.; Komanshek, B. U.; Donald, A. M. The phase transformations in starch during gelatinisation: a liquid crystalline approach. Carbohydr. Res. 2000, 328, 165-176. (52) Gontard, N.; Guilbert, S.; Cuq, J.-L. Water and Glycerol as Plasticizers Affect Mechanical and Water Vapor Barrier Properties of an Edible Wheat Gluten Film. J. Food Sci. 1993, 58, 206-211. (53) Gällstedt, M.; Mattozzi, A.; Johansson, E.; Hedenqvist, M. S. Transport and Tensile Properties of Compression-Molded Wheat Gluten Films. Biomacromolecules 2004, 5, 20202028.



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Table of content graphic Innovative gliadin/glutenin and modified potato starch green composites: Chemistry, structure and functionality induced by processing Faraz Muneer,*,† Mariette Andersson,† Kristine Koch,‡ Mikael S. Hedenqvist,§ Mikael Gällstedt,║ Tomas S. Plivelic,┴ Carolin Menzel,‡ Larbi Rhazi,┼ Ramune Kuktaite† Synopsis Tailoring the biochemistry and molecular structure of gliadin-/glutenin-modified potato starch composites can help in designing sustainable materials with specific functions.


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List of Tables Table 1. Oxygen permeability values of Gli-MPS-45gly and GT-MPS-45gly samples. Standard deviations are in brackets. Samples

Sample thickness (mm) 0.67 (0.04) 0.67 (0.02) 0.68 (0.02)

23°C, 50% RH (mm mL/m2 24h atm) Gli-MPS-45gly 70/30 10.62 (1.37) Gli-MPS-45gly 30/70 12.01 (0.24) GT-MPS-45gly 30/70 3.02 (0.19) * MPS-45gly 3.29 (1.30) ** WG-clay 6 (0.4) * 18 ** 21 Muneer et al., Kuktaite et al., OR-over range

ACS Paragon Plus Environment

38°C, 90% RH (mm mL/m2 24h atm) 226.3 (170.69) 260.4 (2.82) OR>2000 OR>2000 ----


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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List of figures Figure 1 Monomeric (MP) and polymeric protein (PP) solubility of Gli-MPS-45gly, GT-MPS45gly (30/70, 50/50, 70/30) and GT-MPS-30gly20W (50/50) samples extruded at 110 and 130 °C by SE-HPLC. Figure 2 Estimation of the protein polymer size of GT protein in GT-MPS extruded samples by A4F. Figure 3 CML content in (a) Gli-MPS and (b) GT-MPS composites extruded at 110 and 130 °C. Figure 4 SEM and LM micrographs of Gli-MPS and GT-MPS samples extruded at 130 °C with either glycerol or glycerol-water; (a) Gli-MPS-45gly (70/30), (b) Gli-MPS-45gly (50/50), (c) GT-MPS-30gly20W (70/30), (d) GT-MPS-30gly20W (50/50) and (e) GT-MPS-45gly (70/30). Bars correspond to 30 µm for SEM and 100 µm for LM. Figure 5 FT-IR spectra of Gli-MPS and GT-MPS samples extruded at 110 and 130°C; (a) GliMPS-45gly, and (b) GT-MPS-45gly samples. Spectra range 1645-1660 cm-1 corresponds to αhelices and 1620-1635 cm-1 to β-sheets. Figure 6 SAXS profiles of Gli-MPS-45gly composites extruded at 110 and 130 °C. Figure 7 WAXS profiles of Gli-MPS-45gly composites extruded at 110 and 130 °C. Figure 8 Tensile properties of Gli-MPS-45gly, GT-MPS-45gly and GT-MPS-30gly samples extruded at 110 and 130 °C; (a) E-modulus (E-mod), (b) Maximum stress (σmax) and (c) extensibility (ɛ). Figure 9 A graphical representation of the effect of processing on the nano-structure of the materials and their possible end-use.


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Figure 1

35 30

AU

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


MP PP

5 4

25 20

3

15

2

10 1

5

0

0 30/70 50/50 70/30 30/70 50/50 70/30 110°C

130°C

Gli-MPS-45gly

30/70 50/50 70/30 50/50 30/70 50/50 70/30 50/50 110°C

110°C

130°C

130°C

GT-MPS-45gly

GTMPS30gly20W

GT-MPS-45gly

GTMPS30gly20W



Figure 2

Large polymers

250

Molecular mass in weight (g/mol x107)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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200

Small polymers 150

100

50

0 30/70

50/50

70/30

50/50, 20W30gly

30/70

50/50

110°C


70/30 130°C

50/50, 20W30gly

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Figure 3

CML µg/g

a

90 80 70 60 50 40 30 20 10 0 30/70

b CML µg/g

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


50/50

70/30

30/70

50/50

70/30

110°C

130°C

Gli-MPS-45gly

Gli-MPS-45gly

100

100 110°C

Gli

Gli

90 80 70 60 50 40 30 20 10 0 30/70 50/50 70/30 50/50 110°C GT-MPS-45gly

100

30/70 50/50 70/30 50/50

110°C 110°C GTMPS30gly20W

GT

130°C

130°C

GT-MPS-45gly

GTMPS30gly20W

5


100

100 130°C

GT

GT


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 4

(a)

(b) 4 1 3 2

(d)

(c)

(e)


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Figure 5

a

70/30 50/50

AU

30/70

130°C

110°C α-helices

1 1700

1675

1650

β-sheets

1. Pristine Gliadin powder

1625

1600

v (cm-1)

b

70/30 50/50 30/70

1 2

1. GT-MPS-30gly20w (50/50) 130°C 2. GT-MPS-30gly20w (50/50) 110°C

AU

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3. Pristine Glutenin powder 130°

3

1700

1675

1650

v

1625

110°

1600

(cm-1)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6


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Int (AU)

Figure 7

Int (AU)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60




Figure 8

a

14

E-Mod (MPa)

12 10 8 6 4 2 0

2

b σmax (MPa)

1,5

1

0,5

0

c

120 100 80

ɛ (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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60 40 20 0 30/70

50/50

70/30

50/50

30/70

50/50

70/30

50/50

30/70

50/50

70/30

110°C

110°C

130°C

130°C

130°C

GT-MPS-45gly

GTMPS30gly20W

GT-MPS-45gly

GTMPS30gly20W

Gli-MPS-45gly


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Figure 9 130 °C

1 Aggregated

1. Glycerol

Protein

Starch

2. GlycerolWater

2

110 °C

Extrusion Processing

Less aggregated 50 % Glutenin 50% starch

50 % Gliadin 50% starch

Glycerol

Glycerol-Water Multilayered coating, Packaging

3D processing of a textile


Glutenin and Modified Potato Starch Green

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