Properties of Extruded Vital Wheat Gluten Sheets ... - ACS Publications

Biomacromolecules 2009, 10, 479–488

479

Properties of Extruded Vital Wheat Gluten Sheets with Sodium Hydroxide and Salicylic Acid N. Henrik Ullsten,† Sung-Woo Cho,‡ Gwen Spencer,§ Mikael Ga¨llstedt,† Eva Johansson,§ and Mikael S. Hedenqvist*,‡ STFI-Packforsk, Box 5604, SE-11486 Stockholm, Sweden, School of Chemical Science and Engineering, Fibre and Polymer Technology, Royal Institute of Technology (KTH), SE-10044 Stockholm, Sweden, and Department of AgriculturesFarming Systems, Technology and Product Quality, The Swedish University of Agricultural Sciences, SE-230 53 Alnarp, Sweden Received June 26, 2008; Revised Manuscript Received November 28, 2008

This paper presents a novel approach to improve the barrier and mechanical properties of extruded glycerolplasticized vital wheat gluten sheets. The sheets were extruded with a single screw extruder at alkaline conditions using 3-5 wt % NaOH. Salicylic acid (SA), known to improve the extrudability of wheat gluten, was also added alone or in combination with NaOH. Oxygen transmission rate and volatile mass measurements, tensile tests, protein solubility, glycerol migration, infrared spectroscopy, and electrophoresis were used to assess the properties of the extrudate. Electrophoresis showed that the gluten/glycerol sheet and the sheet with 3 wt % NaOH and 1 wt % SA contained the same building blocks in terms of proteins and protein subunits, although the protein solubility in these samples was different. The oxygen barrier, at dry conditions, was improved significantly with the addition of NaOH. On the other hand, the addition of salicylic acid yielded poorer barrier properties. The extrudate was placed on a blotting paper and its aging properties were investigated during the first 120 days. It was observed that the extrudate with 3 wt % NaOH had the most suitable combination of properties (low oxygen permeability, large strain at break, and relatively small aging-induced changes in mechanical properties); the reason is probably due to low plasticizer migration and an optimal protein aggregation/polymerization.

Introduction Wheat gluten (WG) is a low-cost byproduct from the increasing biofuel (ethanol) industry, and an interesting possible alternative to synthetic oxygen-barrier polymers in packaging applications.1-9 WG films and sheets have a low oxygen permeability under dry conditions due to (probably to a large extent) their high contents of hydrogen bonds, but at the same time, these hydrogen bonds cause a loss in barrier properties in humid conditions. Extrusion, without solvents, is the fastest and probably the most commercially interesting processing method for the production of packaging films and sheets. However, it is among the most difficult methods as far as obtaining good quality WG films is concerned.10 Extrusion of WG based products is a wellknown technique within the food sector.11-14 Still, the extrusion methods have to be improved to yield strong and tough barrier sheets without detrimental voids, heterogeneities, or undenatured particles. The structural and rheological changes of WG during extrusion determine the processing window.15-19 Important parameters affecting the extrudability include mechanical energy, shear impact, pressure, plasticizer content, time, and temperature.13,18,20-22 These parameters determine the extent of molecular conformational changes, aggregation, and chemical cross-linking occurring during processing.15,16,23-25 It is important to have a sufficiently high extrusion temperature to achieve a high degree of denaturation and aggregation, which * To whom correspondence should be addressed. Phone: +46-8-7907645. Fax: +46-8-20-88-56. E-mail: [email protected]. † STFI-Packforsk. ‡ Royal Institute of Technology. § The Swedish University of Agricultural Sciences.

is necessary to obtain good fusion of the WG powder particles and, consequently, a homogeneous WG film.20,22 An important part of the aggregation is a reorganization of the intramolecular disulfide bonds to intermolecular disulfide bonds via thioldisulfide exchange reactions.26 Sulfhydryl, on the cystein amino acid, forms new disulfide cross-links during oxidation which, in turn, leads to aggregation. In addition, Tilley et al.27 have pointed out that tyrosine cross-link formation is an important event in the aggregation of gluten. The increase in molecular weight, number of chain entanglements, and decrease in solubility during heat denaturation have been investigated extensively.14,17,18,20,25,28-30 Extensive depolymerization, involving disulfide cleavage, may also occur at high temperatures.26 This, together with a too extensive aggregation, sets the upper limit for the processing temperature, whereas the lower limit is determined by the onset of denaturation.18,20 It has previously been shown that the upper temperature limit was increased by the use of salicylic acid (SA); probably because of its radical scavenging effect and the associated ability to reduce/delay the amount of cross-link reactions.10 Aging properties are among the most important issues that have to be understood and controlled before materials like WG can be of major use in, for example, food packaging.31 Factors that can cause time-induced brittleness include plasticizer loss or plasticizer/matrix phase separation32-34 and thiol oxidation.35,36 It is important to limit plasticizer (glycerol) migration to paper to produce WG-film/paper laminates with time-stable properties. This is why the WG samples are aged on blotting paper in this study. Vital WG powder contains starch that probably also ages. pH is well-known to affect the film forming properties of solution-cast systems. pH-values close to the isoelectric point,

10.1021/bm800691h CCC: $40.75  2009 American Chemical Society Published on Web 01/29/2009

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Biomacromolecules, Vol. 10, No. 3, 2009

Ullsten et al.

Figure 1. The protein solubility of sheets extruded with a set die temperature of 90, 105, and 120 °C, relative to that of the gluten/glycerol sheet (WGG, extruded at a set die temperature of 90 °C) and exposed to a solution of SDS (blue), SDS and 30 s sonication (yellow), or SDS and 150 s (30 s and 60 + 60s) sonication (white). All sheets were unaged.

Figure 2. Same as in Figure 1, but the sheets were aged 120 days (the reference being unaged WGG).

which for WG is about 7.5, do in general result in poor film quality.37 Gennadios et al.38 obtained homogeneous WG films at pH 2-4 and pH 9-13, whereas films were of poor quality at pH 5-6 and did not form at pH 7-8. Similar results have been reported also for extrudates made from solution, or semidry, WG components.10,39,40 The purpose of this study was to improve the oxygen barrier and mechanical properties of WG extrudates. The approach was to extrude the material at alkaline conditions using NaOH. The hypothesis was that the alkaline conditions would yield a larger extent of denatured proteins and therefore a final extrudate with a more “compact” aggregated structure. Salicylic acid, known from previous work to improve the extrudabiliy,10 was also added alone or in combination with NaOH.

Experimental Section Materials. The WG powder was kindly supplied by Reppe AB, Lidko¨ping, Sweden. According to the supplier, the powder consisted of 85.2 wt % WG proteins, 5.84 wt % wheat starch, 6.90 wt % water, 1.20 wt % fat, and 0.86 wt % ash. Glycerol with a purity of 99.5% was supplied by Karlshamns Tefac AB, Sweden. Salicylic acid (SA; 99%) was obtained from VWR International. Sodium hydroxide (>99.98%) was purchased from Merck. Methods. Material Preparation for Extrusion. The WG powder was conditioned for 1 week at room temperature and 20% relative humidity (RH) to yield a moisture content of ca 6 wt %. The NaOH pellets were ground to powder in a Retsch ZM 1 Laboratory Centrifugal Grinding Mill (Haan, Germany) and then dry-mixed with the WG powder. To obtain a homogeneous distribution of SA in the material, 10 g of SA was first ground in a mortar with an equal amount of gluten powder

Plasticized Vital Wheat Gluten Sheets


481

Figure 3. Relative protein solubility/digestability of the sheets after further extraction (extractions 4-6), 1 and 120 days after extrusion with a die temperature of 105 °C; (white square) proteins/peptides extracted with digestion ) extraction 4, (gray square) proteins extracted with SDS and DTT ) extraction 5, (black square) proteins extracted with SDS, DTT, and urea ) extraction 6. The mean standard deviation of all extractions was 1.33.

Figure 4. Infrared spectrum of the amide I region; the thin and thick curves correspond to 1 day and 120 days after extrusion at 120 °C. Curves are given as duplicates.

before it was blended with the remaining WG powder. The powder and glycerol were subsequently blended using a food processor (WATT; DUKA AB, Sweden) at the lowest speed, “speed 1: about 95 rpm”, for 20 s and thereafter at “speed 3: about 200 rpm” for 1 min. The base material was 700 g of WG and 300 g of glycerol; in addition SA and/or NaOH were added. The dough was thereafter stored at room temperature and 20% RH for 3 h to make it pelletizable. Pellets were made in a Moretto ML18/10C (Padova, Italy) granulator. Extrusion. Extrusion was performed directly after pelletization. The batch size was typically 1 kg. A single screw extruder (BX12, Axon, Sweden) was used, equipped with a flat sheet die (45 × 0.7 mm2) and a gateway screw with a 12.5 mm screw diameter, 6 mm root diameter, 12.5 mm flight, and L/D ratio of 26:1. The screw speed was 265 rpm and the set barrel temperature profile was 60-55-30 from die to hopper. The set die temperature was 90, 105, or 120 °C. The set temperatures should be regarded merely as target temperatures (chosen on the instrument panel) and the actual temperature of the material inside the barrel has not been measured. The samples were denoted as follows: WGG, base material (gluten and glycerol), 1-SA (base material with 1 wt % salicylic acid), 3-NaOH-1-SA/5-NaOH-1-SA (same as previous sample, but with an additional 3 or 5 wt % NaOH), and 3-NaOH/5-NaOH (base material with 3 or 5 wt % NaOH). The SA and NaOH wt % values refer, as for glycerol, to the mass contents relative to the mass of the glycerol/wheat gluten mixture. Aging. The extruded sheets were stored at 23 °C and 50% RH on blotting paper, Munktell 1600, Sweden, for 1, 9, 30, 60, 90, or 120 days. At the end of each aging period, the samples that were not tested mechanically were placed in plastics bags in a freezer at -20 °C to

Figure 5. Electrophoresis: (a) standard gliadins (Courto), (b) WGG extr 1, (c) WGG extr 2, (d) WGG extr 3, (e) 3-NaOH-1-SA extr 1, (f) 3-NaOH-1-SA extr 2, (g) 3-NaOH-1-SA extr 3, and (h) standard glutenin (Tamaro).

minimize further aging. The aging time in the following sections refers to aging on the blotting paper and does not include any additional storing or handling in e.g. freezing conditions. Unless stated otherwise, “unaged” refers to 1 day stored samples.

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Table 1. Oxygen Permeability of the unaged WG Sheetsa sheets

OPb

WGG 1-SA 3-NaOH-1-SA 5-NaOH-1-SA 3-NaOH 5-NaOH

213 ( 27 ORc 118 ( 73 43 ( 48 0.5 ( 0.3 0.8 ( 0.3

a Set die temperature: 105 °C. b Unit: (mm mL)/(m2 24 h atm); (-values are sample standard deviations. c Over range, i.e., >1200.

Tensile Test. Tensile tests were performed at 50% RH and 23 °C using a ZwickZ010 tensile tester, with a 500 N load-cell, and controlled by testXpert 7.1 computer software from Zwick GmbH & Co, Germany. Dumbbell-shaped specimens were punched out from the WG sheets along the extrusion direction, with a length and width of the narrow section of 16 and 4 mm, respectively. The measurements were performed as described in ASTM D 882-02 with a crosshead speed of 100 mm/min and a clamp distance of 40 mm. A total of 10 replicates of each sample were tested. The strain was calculated as the actual clamp distance divided by the initial clamp distance and the stress was calculated as the force per initial cross-sectional area of the narrow section. The modulus was calculated from the initial slope of the stress-strain curve (initial scatter excluded). Protein Solubility. The amount and size distribution of proteins in the sheets were determined using the three-step extraction procedure followed by size-exclusion high-performance liquid chromatography (SE-HPLC), developed in Ga¨llstedt et al.41 Proteins soluble in dilute sodium dodecyl sulfate (SDS) were extracted in the first step, proteins soluble in SDS after a short sonication were extracted in the second step, and additional proteins were extracted in SDS with repeated sonication. The extracts were filtered through 0.45 mm filters (Millipore, Durapore Membrane Filters) and thereafter SE-HPLC analyses were performed on a Waters HPLC system using a BIOSEP SEC-4000 Phenomenex column as described in Johansson et al.42 The amount of proteins extracted after each extraction step was normalized to the total protein solubility (after the third step) of the WG/glycerol sample (WGG). Analysis of a “similar” material, a WG sheet with 25-40 wt % glycerol, compression molded at 90 °C, have shown that more than 90% of the proteins are extracted after the third extraction step; the latter finding assuming that 100% of unprocessed WG flour is extracted after the third extraction step.41 ReVersed-Phase High-Performance Liquid Chromatography (RPHPLC). After the last step of protein extraction for SE-HPLC, the pellet was collected for further protein extraction from the following sheets: WGG, 1-SA, 3-NaOH-1-SA, and 5-NaOH-1-SA, all 1 (unaged) or 120 days aged and extruded at a die temperature of 105 °C. This extraction/digestion was carried out in three steps (extractions 4-6). The extraction/digestion buffers during the different extractions were (extraction 4) 0.7 mL buffer T (1 mM CaCl, 20 mM ammonium acetate, pH 8.3) + 0.3 mL trypsin solution (0.3 mg Trypsin in buffer T), (extraction 5) 1.0 mL 0.5% SDS and 1% 1,4-dithio-DL-threitol (DTT), and (extraction 6) 1.0 mL 6 M urea, 0.5% SDS, and 1% DTT. After each extraction, the supernatant was collected, a new buffer was added to the pellet and the sample was suspended in the buffer. For extraction 4, the samples were incubated for 24 h at 37 °C; for extractions 5 and 6, the samples were vortexed in 10 s and thereafter heated to 100 °C for 5 min. After each extraction, the samples were centrifuged for 10 min (8160 g for extraction 4 and 11420 g for extractions 5 and 6). Protein fractionation was carried out using RP-HPLC on a Waters HPLC-system with a Discovery BIO Wide Pore C8 column (Supelco) having a 5 µm particle size. The solvent flow rate was 0.8 mL/min using a column temperature of 70 °C, and the effluent was monitored at 210 nm. Elution was achieved using a gradient system formed from two solvents: (A) water containing 0.1% (v/v) trifluoroacetic acid (TFA), and (B) acetonitrile containing 0.1% (v/v) TFA. The gradient used was 28-56% solvent B from 1 to 20 min.

Ullsten et al. Protein Separation by SDS-PAGE. Two of the samples, WGG and 3-NaOH-1-SA (extruded at 105 °C), were selected for electrophoretic analyses by SDS-PAGE. Proteins were extracted in three steps (extractions 1-3), as described in the HPLC analysis section (vide supra). Five identical extractions were carried out for each of the two samples. The five identical extractions, for each sample, with each extraction buffer, were pooled after extraction. The pooled samples were thereafter dialysed for 48 h and lyophilized for 5 days. Then 16 mg of each sample was resuspended in 160 µL of a 1 M Tris HCl buffer with 0.5% SDS, 1% DTT (pH 8). Samples were vortexed and thereafter centrifuged for 5 min and the supernatants were collected. The protein content in the extracts was estimated using the Bradford method.43 A total of 32 µg protein was loaded per well on a 10% SDSPAGE gel and 21 µL of bromophenol blue was added per well. The gel was run according to Payne et al.44 and stained and destained according to Johansson et al.45 Oxygen Permeability (OP). The oxygen transmission rate was determined in accordance with ASTM D 3985-95, at 23 °C and 0% RH, using a Mocon Ox-Tran 2/20, from Modern Controls Inc., MN. The test pieces were mounted in isolated diffusion cells and subsequently purged with nitrogen gas (2% hydrogen) to measure the background oxygen leakage of the instrument. Each specimen was tightly sandwiched between two aluminum foils so that an area of 5 cm2 was exposed for the measurements. One side of the sample was exposed to flowing oxygen (99.95%) at atmospheric pressure. The oxygen transmission rate was normalized with respect to the oxygen pressure and the sheet thickness to yield the oxygen permeability. At least two replicates from each sample were measured. The tested samples were unaged (i.e., aged for 1 day) and extruded at a set die temperature of 105 °C. Equilibrium Volatile Content at 50% RelatiVe Humidity. The volatile mass was determined according to ASTM D 644-94. The test pieces were weighed and then stored for 24 h at 105 °C in a Nuˆve FN400 oven, supplied by LabRum Klimat AB, Sweden. The specimens were subsequently cooled in desiccators at 0% RH and 23 °C and then weighed to determine the mass loss (volatile mass). Infrared Spectroscopy (IR). Infrared spectra were recorded using a Perkin-Elmer Spectrum 2000 FTIR spectrometer, equipped with a normal single reflection ATR accessory (golden gate) from Specac Ltd. Kent, England. Gas Chromatography. The GC method applied in the present investigation to determine the glycerol content in the WG sheets was fully described by Olabarrieta et al.31 The glycerol content analysis after the acetylation was carried out on a Hewlett-Packard 5890 Gas Chromatograph system with a DB5-MS column (J&W, Folsom, CA; 60 m × 0.25 mm i.d.; 0.25 µm film thickness) as also described in Olabarrieta et al.31 The quantification was performed with a Turbochrom Workstation software (Perkin-Elmer Co.).

Results and Discussion Protein Solubility. From Figure 1, which presents protein solubility data (proteins soluble with the use of SDS, alone, or with sonication) on unaged sheets, the following observations were made: (1) The smallest (or second smallest) amount of SDS soluble proteins was observed for the 3-NaOH (gluten/ glycerol/NaOH) sheet. (2) After both short (30 s) and long (30 s and an extra (60 + 60 s)) sonication, the amount of soluble proteins was still smallest for this sample. (3) In contrast, the 5-NaOH sample experienced a large protein solubility; in fact, some total solubility values (i.e., the solubility after the long sonication) seemed to be unrealistically high (far above 100%). Even the 5-NaOH-1-SA sample (120 °C) showed high solubility values. (4) 1-SA showed a lower total solubility than gluten/ glycerol (WGG) at 90 and 120 °C; yet the solubility of the sheets with 3 wt % NaOH increased when 1 wt % SA was added to it (i.e., 3-NaOH-1-SA).



483

Figure 6. Stress-strain curves of the sheets, 1 day after extrusion with a set die temperature of 120 °C.

When analyzing the results of the aged samples in Figure 2, the following was observed: (1) the total protein solubility was, for all samples, reduced during aging (compare Figures 1 and 2); (2) the total solubility, independent of the choice of die temperature, was smallest for the samples containing 3 wt % NaOH and the 5-NaOH-1-SA sample; (3) in contrast to what was observed for the unaged samples, the aged 1-SA sheet showed the highest total solubility; in fact, its solubility associated with the extra sonication step was even higher after aging than before; (4) whereas the SDS solubility was higher, relative to the solubility associated with the extra sonication, for the unaged WGG and 1-SA sheets, the opposite trend was observed for several of these that were aged; (5) even after aging, the 5-NaOH sample showed somewhat suspiciously high solubility. As mentioned above, and in agreement with earlier investigations,31 the total protein solubility was reduced, although to various degrees, for all samples during aging. For example, the time-induced change in solubility was less for the 3-NaOH sheet than for the 5-NaOH sheet. In contrast to the findings in Ullsten et al.,10 SA in unaged sheets did not yield a higher total protein solubility than the WGG sheet made at high die temperatures. The reason might have been that the die temperature (120 °C) was still not high enough to yield sufficient protein aggregation to show the effect of SA on the protein extractability, as observed at 130 °C in Ullsten et al.10 Even though the denaturation temperature is far below 130 °C, it must be remembered that the sample is probably close to the target die temperature only for a very short time. The lowest protein solubility of the unaged samples was observed for the 3-NaOH sample and it also showed among the smallest change in solubility with time. The high “apparent” protein solubility of the unaged 5-NaOH samples and also of the 5-NaOH-1-SA sample, made at a set die temperature of 120 °C, were probably a combination of several reasons: As mentioned previously, 90% of the proteins are assumed to be extracted after the last extraction step of a WGG compression molded sample. However, extruded WGG experiences, relative to a compression-molded sample, a higher shear rate and its proteins are therefore probably more aggregated/cross-linked and less soluble. Hence, a possible reason for observing high “apparent” solubility values could have been, at least partly, a normalization problem, that is, that the sample solubilities were normalized to probably less-soluble, extruded WGG. In addition, the high solubility may be due to reasons

other than only pure solubility of the gluten polymers. Protein extractability was here measured through protein separation using different extraction/digestion methods in combination with HPLC analyses. In the HPLC method, the proteins were quantified with UV absorbance, which is the most accurate and most frequently used method for determining amounts and concentrations of proteins. However, different proteins might have different molar absorbance coefficients. Protein absorbance coefficients are determined largely by the composition of amino acids in the proteins, amount of disulfide bonds in the proteins and on protein folding.46 A possibility is that the new types of polymers that may form at alkaline conditions, for example, deamidated species,48,49 yield a stronger-than-expected UV-vis absorbance and, therefore, have a higher apparent solubility. Another possibility is that any or several of the other constituents reacted with NaOH and gluten to form “new” species that then appeared in the HPLC chromatogram. It is likely that these artifactic effects increase with increasing pH and becomes especially pronounced at 5 wt % NaOH. In addition, it is known that the NaOH-induced saponification of the fat in wheat, into fatty acid salts, has been observed to increase the protein solubility in water.47 Results from further protein extraction (Figure 3) showed that (1) only small quantities of proteins and peptides were released through peptide digestion of the pellet that was remaining after the first three protein extractions; a result which was not entirely unexpected because it is known that for some wheat proteins, peptide digestion is not very effective;50,51 (2) a smaller amount of protein was extracted from the aged than from the unaged NaOH containing samples; (3) using SDS, DTT, and urea, it was possible to extract, from all sheets, an additional amount. Note that the NaOH containing samples without SA were not tested here. Figure 4 shows infrared spectra of parts of the amide I region. The shoulder in the vicinity of 1616 cm-1 corresponds to the amount of amide groups in intermolecular β-sheet networks, the peak/shoulder in the vicinity of 1632 cm-1 corresponds to amide groups involved in extended β-sheets and the peak/ shoulder in the vicinity of 1652 cm-1 corresponds to the amount of amide groups that are either unordered or in an R-helix conformation.52 As also observed in an earlier investigation,31 the infrared spectrum of gluten/glycerol (WGG) change from a prominent peak in the 1652 cm-1 region and a smaller intensity in the 1616/1632 cm-1 region, toward the opposite during aging. This indicated a change from a structure of a high content of

1 3 15 ( 1 22 ( 1 20 ( 2 27 ( 3 37 ( 11 34 ( 6 40 ( 5

1 5 37 ( 7 64 ( 4 34 ( 2 46 ( 4 71 ( 10 70 ( 4 94 ( 9

die temperature, 90 °C

1 0 10 ( 1 13 ( 2 24 ( 3 31 ( 4 56 ( 10 118 ( 11 190 ( 24

0 3 31 ( 3 41 ( 2 40 ( 4 42 ( 3 42 ( 3 41 ( 3 45 ( 4

0 5 95 ( 9 104 ( 7 89 ( 8 96 ( 7 128 ( 9 131 ( 10 140 ( 11

0 0 9(2 12 ( 1 14 ( 5 23 ( 5 50 ( 5 112 ( 18 130 ( 28

1 3 17 ( 2 19 ( 1 23 ( 3 33 ( 4 48 ( 6 52 ( 5 50 ( 5

a

0 0 1.4 ( 0.1 1.4 ( 0.1 1.7 ( 0.1 2.7 ( 0.2 2.8 ( 0.3 3.3 ( 0.2 2.9 ( 0.1

1 0 1.8 ( 0.1 1.7 ( 0.1 2.3 ( 0.2 2.6 ( 0.2 3.0 ( 0.2 3.2 ( 0.3 3.5 ( 0.3

1 3 1.9 ( 0.1 2.5 ( 0.2 2.7 ( 0.3 3.0 ( 0.4 2.9 ( 0.5 3.1 ( 0.3 3.4 ( 0.4

1 5 2.0 ( 0.1 2.7 ( 0.3 2.6 ( 0.3 3.2 ( 0.4 3.4 ( 0.5 3.8 ( 0.4 3.4 ( 0.3

die temperature, 90 °C 0 3 2.6 ( 0.1 2.8 ( 0.2 3.2 ( 0.1 3.6 ( 0.2 3.7 ( 0.1 3.7 ( 0.1 3.9 ( 0.1

0 5 1.5 ( 0.1 2.4 ( 0.2 2.3 ( 0.2 2.3 ( 0.2 2.6 ( 0.2 2.6 ( 0.1 3.0 ( 0.2

0 0 1.3 ( 0.1 1.4 ( 0.1 1.7 ( 0.2 2.2 ( 0.1 2.7 ( 0.2 3.4 ( 0.3 3.0 ( 0.2

1 3 2.3 ( 0.2 2.5 ( 0.2 2.9 ( 0.2 2.9 ( 0.4 3.8 ( 0.2 4.0 ( 0.2 4.4 ( 0.2

1 5 1.9 ( 0.1 2.4 ( 0.2 2.7 ( 0.2 2.7 ( 0.3 3.0 ( 0.2 2.4 ( 0.2 3.7 ( 0.2

0 3 35 ( 4 39 ( 5 39 ( 3 37 ( 3 41 ( 4 51 ( 6 46 ( 5

0 3 2.6 ( 0.1 2.9 ( 0.2 3.2 ( 0.1 3.3 ( 0.3 3.6 ( 0.2 4.0 ( 0.2 3.9 ( 0.2

die temperature, 105 °C 1 0 1.4 ( 0.1 1.7 ( 0.2 1.9 ( 0.3 2.6 ( 0.1 2.8 ( 0.4 2.8 ( 0.2 3.4 ( 0.4

Aged, stored, and tested at 23 °C and 50% RH. (-values are sample standard deviations.

wt % SA wt % NaOH 1 day 3 days 9 days 30 days 60 days 90 days 120 days

1 5 39 ( 4 35 ( 12 30 ( 6 31 ( 5 54 ( 3 35 ( 4 76 ( 4

die temperature, 105 °C 1 0 11 ( 2 15 ( 3 19 ( 3 34 ( 5 68 ( 24 92 ( 16 179 ( 34


0 0 10 ( 1 11 ( 1 15 ( 1 43 ( 2 51 ( 10 85 ( 14 86 ( 26

Table 3. Maximum Stress (MPa) of WG Sheets Extruded at Three Different Set Die Temperaturesa

a

wt % SA wt % NaOH 1 day 3 days 9 days 30 days 60 days 90 days 120 days

Table 2. Young’s Modulus (MPa) of WG Sheets Extruded at Three Different Die Temperaturesa

0 5 1.6 ( 0.1 2.2 ( 0.3 2.4 ( 0.2 2.5 ( 0.2 2.9 ( 0.1 2.9 ( 0.2 3.1 ( 0.2

0 5 84 ( 9 85 ( 14 106 ( 6 107 ( 18 137 ( 13 134 ( 15 134 ( 14

0 0 1.3 ( 0.1 1.5 ( 0.2 1.9 ( 0.2 2.2 ( 0.2 2.6 ( 0.3 2.9 ( 0.4 2.7 ( 0.2

0 0 10 ( 2 13 ( 1 23 ( 5 35 ( 5 79 ( 5 103 ( 17 120 ( 28

1 5 26 ( 4 45 ( 12 31 ( 6 28 ( 5 29 ( 3 26 ( 4 36 ( 4

1 0 1.3 ( 0.1 1.5 ( 0.2 1.1 ( 0.2 2.5 ( 0.2 2.7 ( 0.3 2.8 ( 0.5 3.7 ( 0.3

1 3 2.0 ( 0.2 2.2 ( 0.5 2.9 ( 0.4 3.0 ( 0.5 3.3 ( 0.4 4.0 ( 0.3 3.7 ( 0.4

1 5 1.6 ( 0.1 2.3 ( 0.2 2.4 ( 0.1 2.5 ( 0.3 2.6 ( 0.1 1.6 ( 0.1 2.5 ( 0.2

0 3 34 ( 4 36 ( 5 34 ( 3 45 ( 3 46 ( 4 51 ( 6 52 ( 5

0 3 2.4 ( 0.1 3.0 ( 0.3 3.0 ( 0.2 3.3 ( 0.2 3.6 ( 0.3 3.7 ( 0.3 3.6 ( 0.3


1 3 12 ( 2 17 ( 1 25 ( 3 29 ( 4 45 ( 6 59 ( 5 54 ( 5

die temperature, 120 °C 1 0 10 ( 1 13 ( 3 18 ( 2 42 ( 6 78 ( 15 173 ( 53 228 ( 36

0 5 1.4 ( 0.1 2.6 ( 0.1 1.8 ( 0.2 2.1 ( 0.3 2.2 ( 0.2 2.3 ( 0.1 2.6 ( 0.2

0 5 64 ( 9 70 ( 14 75 ( 6 89 ( 18 129 ( 13 124 ( 15 128 ( 14

484 Biomacromolecules, Vol. 10, No. 3, 2009 Ullsten et al.


0 3 151 ( 4 156 ( 9 146 ( 9 126 ( 7 119 ( 8 119 ( 6 113 ( 10 1 5 112 ( 7 124 ( 9 119 ( 9 110 ( 12 99 ( 5 112 ( 7 94 ( 10 1 3 128 ( 14 107 ( 28 123 ( 11 112 ( 20 98 ( 12 103 ( 7 100 ( 6 1 0 81 ( 10 97 ( 10 77 ( 12 89 ( 7 64 ( 8 41 ( 20 37 ( 10 0 3 143 ( 8 146 ( 8 139 ( 7 120 ( 10 115 ( 7 120 ( 3 114 ( 5 1 5 114 ( 10 120 ( 13 119 ( 8 110 ( 7 99 ( 8 120 ( 13 94 ( 6 a


1 3 128 ( 14 126 ( 10 128 ( 7 114 ( 16 109 ( 5 108 ( 5 101 ( 3


1 0 89 ( 13 89 ( 15 92 ( 11 91 ( 8 79 ( 12 68 ( 13 61 ( 10 0 0 108 ( 11 113 ( 14 125 ( 20 109 ( 13 114 ( 18 86 ( 10 75 ( 8 0 5 36 ( 14 52 ( 15 56 ( 12 60 ( 13 45 ( 11 40 ( 8 55 ( 7 0 3 121 ( 9 124 ( 8 123 ( 5 111 ( 5 109 ( 6 106 ( 4 107 ( 4 1 5 107 ( 6 109 ( 12 112 ( 8 105 ( 13 89 ( 15 92 ( 7 70 ( 11 1 3 111 ( 13 120 ( 13 116 ( 8 107 ( 12 91 ( 14 101 ( 8 98 ( 10


1 0 101 ( 18 100 ( 12 100 ( 12 96 ( 12 82 ( 5 66 ( 9 66 ( 14 0 0 113 ( 6 121 ( 10 125 ( 12 102 ( 9 112 ( 11 93 ( 11 101 ( 8 wt % SA wt % NaOH 1 day 3 days 9 days 30 days 60 days 90 days 120 days

Table 4. Strain at Maximum Stress (%) of WG Sheets Extruded at Three Different Set Die Temperaturesa

0 5 79 ( 18 69 ( 26 61 ( 18 75 ( 13 62 ( 5 64 ( 9 75 ( 7

0 0 100 ( 12 119 ( 16 126 ( 22 106 ( 16 95 ( 13 85 ( 12 70 ( 21


0 5 98 ( 17 75 ( 17 68 ( 14 81 ( 7 50 ( 9 55 ( 7 64 ( 9


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unordered amide groups, and R-helix conformations, to a structure where β-sheet structures of both intermolecular and extended type were common. According to Pezolet et al.,52 the increase in the 1616 cm-1 peak is associated with increasing protein aggregation; thus the present data indicated that the protein structure became more aggregated with time. It is here assumed that both peaks (∼1616 cm-1 and ∼1632 cm-1) contribute to the relative increase in the 1616/1632 cm-1 shoulder. It seemed as if NaOH favored aggregation already in the unaged samples, note the less prominent ∼1652 cm-1 peak of the NaOH-containing samples (Figure 4). The shapes of the spectra of the 3-NaOH and 5-NaOH samples were somewhat different (compare both unaged and aged samples). However, these slight differences are difficult to observe, and explain, because of the many overlapping peaks and possible effects of moisture. The spectrum of the unaged 1-SA sample was similar to the unaged WGG sample, whereas the corresponding spectra of the 120-day aged samples showed, relative to the aged WGG sample, features of a more aggregated structure. This indicates a stronger time-induced aggregation in the former sample (data not shown). Strangely, the aged 1-SA sample showed higher protein solubility than for the aged WGG. This, indeed, shows that the interpretation of IR and solubility data is not straightforward in terms of explaining the structure and structure development of WG proteins. The samples containing both SA and NaOH were not discussed here. It should be noted that the spectra were obtained on undehydrated samples (only at a 120 °C set die temperature) and any effects of moisture was therefore not considered. The moisture content/volatile-mass was significantly higher, based on the sample standard deviation, for 3-NaOH and 5-NaOH relative to WGG and 1-SA. The average values, obtained from data at all, or most, times (1, 3, 9, 30, 60, 90, and 120 days) and all die temperatures, were 11 (WGG, 1-SA), 13 (3-NaOH), and 14 wt % (5-NaOH), with the latter two values being insignificantly different. Electrophoretic analyses were carried out to investigate if the protein and protein subunit composition differed between, for example, the 3-NaOH-1-SA and the WGG samples. The results showed that, after the three first extraction steps (vide supra), exactly the same proteins and protein subunits were extracted from these (Figure 5). However, the amount of extracted proteins from 3-NaOH-1-SA seemed to be substantially lower in the first extraction step than from WGG, visualized by the use of electrophoresis (Figure 5). The same result was also observed in the HPLC analyses (Figure 1). Hence, the differences in protein structure between the 3-NaOH-1-SA and the WGG sheets, indicated by electrophoresis and HPLC, did not go down to the primary protein structure of participating building blocks (proteins and protein subunits). However, note that the study was limited to only two samples extruded at 105 °C, and that the 5 wt % NaOH samples were not investigated here. Oxygen Permeability. The oxygen permeability decreased significantly in the presence of NaOH (Table 1). The 3-NaOH and 5-NaOH samples had an oxygen permeability (OP) that was more than 2 orders of magnitude lower than that of the WGG sample. It is here interesting to compare these values with those reported elsewhere. All OP data discussed below have the same unit as these in Table 1 and they were measured at 23 °C, unless stated otherwise. In addition, to make it clear, wt % values refer to weight glycerol/total-sample weight. The only report, to our knowledge, of OP data on extruded WG was presented by Hochstetter et al.53 (a value of 5 (50-75% RH) at a higher (38 wt %) glycerol content as compared to the

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Figure 7. Stress-strain curves of the sheets, 120 days after extrusion, with a set die temperature of 120 °C.

Figure 8. Residual glycerol content in the sheets, relative to the amount initially added, 1 (yellow) and 120 (blue) days after extrusion with three different die temperatures. The error bars were obtained from sample standard deviations.

present 30 wt %). The value is intermediate between these containing NaOH and SA and these with NaOH. It is, however, difficult to make a simple comparison between the present and their extrusion results; in the latter case, the material was extruded with a sizable amount of water, which was then allowed to evaporate from the sheet. Hence, the entire procedure could be considered more or less as a two-step extrusion/solution casting technique, while in the present case the OP data was measured on sheets extruded entirely without added water. OP values have also been reported for solution cast and compression molded systems.31,41,54-56 The OPs of the solution-cast films are on the same order of magnitude (0.4-1.5 (0% RH), 25-29 wt % glycerol31,54) and 1.6 to 42 within 0-100% RH (9-17 wt % glycerol55,56) as these of the NaOH films in Table 1. Interestingly compression molded films with similar glycerol content (25 wt %), exposed to moist conditions (90-95% RH) showed an OP-value of only 1.1.41 Compared to commercial gas-barrier plastics such as poly(ethylene-co-vinyl alcohol), containing only 32 mol % ethylene, the best WG films have an OP which is more than 2 orders of magnitude higher at dry conditions and 25 °C.57 On the other hand, several WG materials (vide supra) have OPs that are lower than these of dry poly(ethylene terephthalate) and polyamide 66.58 It is not obvious why the 3-NaOH and 5-NaOH samples showed similar oxygen permeabilities (Table 1). They behaved

very different in the extraction experiment. Nevertheless, it still seems that the low OP values were, in both cases, due to a significant aggregation/cross-linking. The addition of salicylic acid increased the permeability, probably since it may reduce/ delay protein aggregation/cross-linking and therefore yield a more inhomogeneous dough-like structure (vide infra).10 Tensile Tests. The stress-strain curves of all unaged sheets showed elastomeric features implying a low stiffness and strength and a large elongation at break (Figure 6). When comparing 3-NaOH and 5-NaOH, it was observed that the modulus increased with increasing content of NaOH (Table 2). The same behavior was observed for all die temperatures and all times (Table 2). When SA was added to these samples this was not always the case. The tensile strength of the unaged material increased, relative to that of the WGG sample, with the addition of 3 wt % NaOH (with or without SA). However, at 5 wt % NaOH, the unaged material became weaker again (Figure 6, Table 3). The strain at maximum stress (unaged samples) was, in general, but often within the sample standard deviation, highest for samples containing 3 wt % NaOH (Table 4); again, it can be noted that a higher (5 wt %) NaOH content resulted in adverse effects (clearly significant when comparing 5-NaOH with 3-NaOH). In most cases, the lowest extensibility was observed for the 5-NaOH and the 1-SA samples. As observed in Tables 2-4, most samples, albeit to various extents, experienced an increase in stiffness and strength and a reduction in extensibility during aging. Figure 7 and Tables 2 and 3 show that the 120 day-aged materials were significantly stiffer and had a higher maximum stress than the unaged samples. At 120 °C set die-temperature, the 3-NaOH sample was consistently, although not always significantly, the most extensible material. Considering the whole aging period, the 1-SA sheet experienced the greatest time-induced increase in Young’s modulus (at 105 °C, not significantly different WG) and it was the stiffest sample at 120 days for all die temperatures (at 105 °C, not significantly different from WG and 5-NaOH). An interesting feature was that, whereas SA had a reducing effect on the extensibility of 3-NaOH (although sometimes within the experimental error), the opposite was observed for 5-NaOH. Interestingly, although it was less extensible, the 3-NaOH-1-SA sample had a higher initial moisture content than that of 3-NaOH (data not shown). Noteworthy is that the stiffness of the unaged samples increased with increasing content of NaOH: WGG < 3-NaOH < 5-NaOH (Table 2).


To conclude, the mechanical data indicated that, in general and although often within the sample standard deviation, the most extensible material, unaged and aged, was that containing 3 wt % NaOH. It must be remembered here that the vital WG powder contains non-protein species, including bran and starch particles, and any variation in content and size of these, either existing in the original powder or due to the processing and/or the chemicals added, may affect the mechanical properties. Loss of Plasticizer. Figure 8 shows the residual amount of glycerol in, respectively, the unaged and 120-day aged WGG, 1-SA, 3-NaOH, and 3-NaOH-1-SA sheets. All these sheets lost glycerol to the blotting-paper support, and the loss was on the order of 10-20 wt % glycerol. The greatest loss, at all die temperatures, was observed for the 1-SA sheet. Indeed, this material (extruded with a 120 °C set die temperature) also experienced the greatest increase in stiffness, from 10 (day 1) to 228 MPa (day 120). The materials experiencing the smallest time-induced change in stiffness (3-NaOH, 3-NaOH-1-SA) were also the ones experiencing the smallest glycerol loss. Consequently, the change in mechanical properties during aging was, at least to a certain extent, associated with the loss of plasticizer. As observed in Figure 8, despite the same glycerolto-gluten loading in all samples, the 1-SA sample showed (although sometimes within experimental error) the smallest initial content of glycerol. Even though this could explain its low extensibility, it could not explain its relatively low modulus as compared to, for example, 3-NAOH (Tables 2 and 3). Thus, for the unaged samples, it is likely that the variation in glycerol content among the various samples played only a minor role (if any) for the mechanical behavior of 1-SA relative to these.

Conclusion A WG sheet composition with 3 wt % NaOH yielded, in relative terms, the most attractive combination of properties for further development toward packaging applications. The oxygen permeability was low (at dry conditions), the extensibility high, and the mechanical properties were relatively time-stable. This material experienced a low glycerol loss and low protein solubility. When investigating the unaged samples without SA it was observed that, when increasing from 3 to 5 wt % NaOH, the extensibility decreased, whereas the modulus increased. These are signs of a comparatively more rigid and brittle network at 5 wt % NaOH. At the same time the solubility of this protein network was high. Still the oxygen permeability was similar for both materials (3-NaOH and 5-NaOH). The reason for these observations is not easily explained. A hypothesis is that the observed variation in mechanical properties between 3-NaOH and 5-NaOH was due to differently aggregated structures because of differences in their protein polymerization behavior. The extent of aggregation was sufficiently high in both cases to yield a high oxygen barrier. 3-NaOH, however, seemed to have reached the most optimal protein aggregation/β-sheet structures, with respect to also achieving, for example, a more extensible material (vide supra). It is worth noting here that preliminary small-angle X-ray scattering results indicate that the 5-NaOH and 3-NaOH threedimensional protein structures are different. A suggestion to the reason for the high protein solubility of 5-NaOH is that the aggregated structure contained a significant amount of more polar/soluble protein species (e.g., through deamidation). However, this does not explain for that the values above 100% were observed. We can only speculate in the reason for this; it could be that, at this high NaOH content (5 wt %), a significant amount of new “chemical species” was formed that then led to


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“erroneous” HPLC solubility data. The reason for those values were extending well above 100% was probably also due that the reference data came from the extruded WGG sample, which was likely to be less protein soluble than, for example, a previously used unprocessed WG reference. Nevertheless, as stated above, these are highly speculative explanations that need to be confirmed in future work. The focus here was to investigate whether NaOH could be used to improve WG extrudate properties, and this was confirmed, at least at 3 wt % NaOH. Acknowledgment. Vinnova, the Swedish Governmental Agency for Innovation Systems, and the “Glupack” consortium are thanked for financial support.

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