Transport and Tensile Properties of Compression ... - ACS Publications

Biomacromolecules 2004, 5, 2020-2028

2020

Transport and Tensile Properties of Compression-Molded Wheat Gluten Films Mikael Ga¨llstedt,† Alessandro Mattozzi,†,‡ Eva Johansson,§ and Mikael S. Hedenqvist*,| STFI-Packforsk AB, Packaging & Logistics, Box 9, SE-164 93 Kista, Sweden, Royal Institute of Technology, Department of Fibre and Polymer Technology, SE-100 44 Stockholm, Sweden, and Department of Crop Science, The Swedish University of Agricultural Sciences, SE-230 53 Alnarp, Sweden Received April 29, 2004; Revised Manuscript Received May 25, 2004

Mechanical and transport properties were assessed on wheat gluten films with a glycerol content of 2540%, prepared by compression molding for 5-15 min at temperatures between 90 and 130 °C. Effects of storing the films up to 24 days, in 0 and 50% relative humidity (RH), were assessed by tensile measurements. The films were analyzed with respect to methanol zero-concentration diffusivity, oxygen permeability (OP), water vapor permeability (WVP), Cobb60 and sodium dodecyl sulfate (SDS) solubility coupled with sonication. The SDS solubility and methanol diffusivity were lower at the higher molding temperature. Higher glycerol content resulted in higher OP (90-95% RH), WVP, and Cobb60 values, due to the plasticizing and hygroscopic effects. Higher glycerol contents gave a lower fracture stress, lower Young’s modulus, lower fracture strain, and less strain hardening. The mold time had less effect on the mechanical properties than mold temperature and glycerol content. The fracture stress and Young’s modulus increased and the fracture strain decreased with decreasing moisture content. Introduction Wheat gluten (WG) is an interesting alternative to synthetic oxygen-barrier polymers in packaging applications.1-9 WG films have a low oxygen permeability under dry conditions due to their high contents of hydrogen bonds. The hydrogen bonds also make the films brittle and a polar plasticizer, that breaks the hydrogen bonds between the polypeptides, must therefore be used.10-12 Without plasticizer the protein is far too brittle for typical plastic applications. Since the plasticizer increases toughness and lowers the barrier properties, a compromise must therefore be made here. The most commonly used plasticizer is glycerol, but others have also been studied.13-18 Glycerol is a highly polar and effective plasticizer that is easy to disperse in the protein matrix.14 It is hygroscopic and attracts water, which in turn adds to its plasticizing efficiency.17,19-21 However, even in an originally well plasticized system, brittleness may develop with time due to plasticizer migration and the oxidation of free thiol groups.17,18,22 The size and compatibility of the plasticizer molecule and the stability of the plasticizer/protein system are therefore of major importance. Several less hygroscopic plasticizers have also been studied, but so far glycerol and triethanolamine seem to be the most efficient.13,17,18 Most of the research on WG films has been performed on solution-cast films. However, faster processing techniques such as compression molding and extrusion are, from a * To whom correspondence should be addressed. E-mail: mikaelhe@ polymer.kth.se. Tel.: +46-(0)8-790-7645. Fax: +46-(0)8-100775. † STFI-Packforsk AB, Packaging & Logistics. ‡ Present address: Royal Institute of Technology, Department of Fibre and Polymer Technology, SE-100 44 Stockholm, Sweden. § The Swedish University of Agricultural Sciences. | Royal Institute of Technology.

commercial perspective, more interesting. Only a few studies have been performed on compression-molded WG films, although it is a common process for WG in food applications.13,23-25 Therefore, this study focuses on packaging related properties of compression-molded WG films. The principles and mechanisms of compression molding are similar to those of solution casting, except that the high pressure in combination with high temperature during compression molding can affect the molecular structure.26 The structural and mechanical changes of WG during film formation (extrusion, casting and compression molding) determine the processing window.20,27-30 To develop a commercially useful material, it is important to define and, if necessary, expand the processing window.23,25,28,31-33 The outcome of solution-cast processes depends on parameters such as temperature, time, moisture content, solvent-type, plasticizer concentration, and pH.8,9,29,34,35 In extrusion processes, the specific mechanical energy, shear impact, pressure, plasticizer, time, and temperature are examples of important parameters.26-28,31,36 All of these parameters determine the extent of conformational changes, aggregation, and chemical cross-linking that occur during processing.20,27,37-39 In the absence of an external cross-linkagent,25,40 high temperature is important for obtaining good mechanical properties.26,36 Sulfhydryl, on the cystein amino acid, is responsible for the creation of disulfide cross-links during oxidation. An important part of the aggregation is a reorganization of the intramolecular disulfide bonds to intermolecular disulfide bonds via thiol-disulfide exchange reactions.41 The increase in molecular weight and the decrease in solubility of WG during heat denaturation have been investigated by several scientists.25,26,28,29,39,40,42 The extent of cross-link rearrangement and aggregation depend

10.1021/bm040044q CCC: $27.50 © 2004 American Chemical Society Published on Web 08/04/2004

Properties of Compression Molded WG Films

on, e.g., process temperature, plasticizer content and type, time, and shear rate. A dissociation of secondary bonds, during denaturation, occurs by disruption of hydrogen bonds and hydrophobic interactions. An increase in the number of chain entanglements during denaturation has also been reported.43 Extensive depolymerization, involving disulfide breakage, may also occur at high temperatures.41 This, together with too extensive an aggregation, sets the upper limit for the processing temperature window, whereas the lower limit is determined by the position of the onset of denaturation.26,29 This study presents the effects of different compressionmolding conditions on methanol diffusion, Cobb60, oxygen and water vapor permeability, and mechanical properties. For the first time, solute-concentration-dependent diffusivities (methanol) have been measured on protein films. Compression-molding time, temperature, and glycerol content have been varied. The properties of the films were studied during the first 24 days after compression molding. The increase in SDS insolubility and ultrasonic resistivity of WG has been studied in order to enable the degree of aggregation during compression molding to be determined. Experimental Section Materials. The wheat gluten (WG) powder was supplied by Ceramyl AB, Lidko¨ping, Sweden. The powder consisted of 84.8 wt % wheat gluten proteins, 8.1 wt % wheat starch, 5 wt % water, 1.34 wt % fat, and 0.76 wt % ash. Glycerol, with a concentration of g99.5 wt % and a water content of e0.5 wt %, was supplied by Karlshamns Tefac AB, Karlshamn, Sweden. Methods. Film Preparation. Dough was prepared by mixing gluten and glycerol, to a glycerol content of 25 wt %, 32.5 wt %, and 40 wt % (given as the mass of glycerol per total weight of glycerol and WG). Each blend was mortared for five minutes, with a Mortar Agate from VWR International, and compression molded to a film using a Schwabenthan Polystat 400s. 10 g of dough were placed in an aluminum frame between Mylar foils, which in turn were placed between metal plates. The frame was used to obtain square-shaped films with a side of 100 mm and a thickness of 0.5 mm. The molding temperatures were 90 or 130 °C, and the pressure was set to 100 bar, which gave an applied pressure of 1600 bar. The molding times were 5 or 15 min. The study was performed as a three level factorial analysis, with glycerol content, mold temperature, and mold time as parameters. To provide a comparison with an intermediate condition and composition, a sample with 32.5 wt % glycerol was compression molded for 10 min at 110 °C. This sample is referred to as the “central-point sample”. After molding, the plates were removed from the press and the films were allowed to cool to ambient temperature. The Mylar films were then removed and the films were separated from the frame using a scalpel. Desorption Measurements. Circular specimens with a diameter of 16 mm were punched out of the compressionmolded films. These specimens were dried in desiccators at 0% RH and room temperature for 5 days and were then

Biomacromolecules, Vol. 5, No. 5, 2004 2021

weighed. The surface area and thickness of the specimens were subsequently measured. The thicknesses were determined using a Mitutoyo thickness tester IDC-112B. Five to seven measurements were made on all specimens. In the sorption/desorption and permeability tests, these were made before exposure. The specimens were thereafter hung in a glass vessel, with the bottom covered with methanol, and a fan was used to circulate the atmosphere within the vessel. The vessel was purged continuously with dry nitrogen gas to ensure that atmospheric moisture did not enter and affect the sample. The specimens were conditioned for 3 days in the vessel in order to ensure that they were saturated with methanol. The size and mass of the saturated specimens were determined, thereafter the samples were put in another vessel and purged with dry nitrogen. The mass of the specimen was measured intermittently on an AT 261 Mettler-Toledo balance, with an accuracy of 10-5 g, until constant mass was reached. An implicit differential equation was used to fit Fick’s second law to the desorption data.44 The diffusivity was considered to be solute-concentration-dependent D ) Dc0eRC

(1)

where Dc0 is the zero-concentration diffusivity and R is the plasticization power.44,45 The deviation between the measured values and the curve was obtained as the sum of the squares of the deviations of the fitted points from the experimental points. Protein Solubility. The amount and size distribution of the soluble proteins were obtained using the size-exclusion high performance liquid chromatography (SE-HPLC), with twostep extraction. The procedure was similar to that described by Johansson et al.42 The applied method was developed by Gupta et al.46 The proteins soluble in dilute sodium dodecyl sulfate (SDS) were extracted in the first step, and additional proteins were extracted by sonication in the second step. In the first step, 11 mg of each film was suspended in 1.0 mL 0.5% SDS-phosphate buffer (pH 6.9) and vortexed for 10 s. The suspension was then stirred for 5 min at 2000 rpm and centrifuged for 30 min at 10 000 rpm to obtain the supernatant protein. In the second step, the pellet was resuspended in SDS buffer as above and sonicated in an ultrasonic disintegrator (Soniprep 150, Tamro, Mo¨lndal, Sweden) for 30 s, amplitude 5, fitted with a 3 mm exponential microtip. The samples were then centrifuged (30 min, 10 000 rpm) to obtain a supernatant of proteins. The extracts were filtered through 0.45 mm filters (Millipore, Durapore Membrane Filters) before the SE-HPLC operation. SE-HPLC analyses were performed on a Varian HPLC system using a BIOSEP SEC-4000 Phenomenex column. Separation was obtained during 30 min by loading 20 µL of sample into an eluant of 50% (v/v) acetonitrile and water containing 0.1% (v/v) trifluoroacetic acid at a flow rate of 0.2 mL min-1. Proteins were detected by UV absorption at 210 nm. However, all proteins were not dissolved using this method. Therefore, stronger extractions were tested, in which the samples were exposed for several periods of sonication, for longer times and at higher amplitudes. A last extraction step was included, with

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Biomacromolecules, Vol. 5, No. 5, 2004

30+60+60 s sonication, and the extracted proteins were separated by SE-HPLC. The SE-HPLC chromatograms were divided into four sections, representing respectively large polymeric proteins (LPP), smaller polymeric proteins (SPP), large monomeric proteins (LMP), and smaller monomeric proteins (SMP).42,47 The areas of the different sections were calculated and this enabled the percentage of total unextractable polymeric protein in the total polymeric protein and the large unextractable polymeric protein in the total large polymeric protein to be calculated.42,46 The total extractability was checked by using an extra extraction step using 0,5% SDS, 1%DTT (dithiothreitol), 1% acetic acid, 6 M urea followed by 100 °C treatment for 5 min. Oxygen Transmission Rate Measurements. The oxygen transmission rate was measured using a Mocon Ox-Tran Twin, from Modern Controls Inc., MN, at 23 °C and 9095% RH, according to ASTM D 3985-95.48 The high RH was used to evaluate if the material could endure this “extreme” condition. Specimens were mounted in an isolated diffusion cell which was subsequently purged with nitrogen (2% hydrogen) to enable the background oxygen level to be measured. The samples were conditioned until a steady state was reached. After conditioning, one side of the sample was purged with oxygen (99.95%) at atmospheric pressure. The steady-state flow rate through the specimen was measured. The oxygen permeability (OP) was calculated by normalizing the flow rate at steady state with respect to the oxygen pressure gradient over the film and the film thickness. Water Vapor Transmission Rate Measurements. The water vapor transmission rate was measured on triplicates of each sample using a Mocon Permatran-W Twin, from Modern Controls Inc., MN, at 37.8 °C and 49% RH, as described in ASTM F 1249-90.49 The temperature and relative humidity were selected based on the fact that these are very common conditions and enables the reader to compare the data with those of other materials. The specimens were tightly sandwiched between two aluminum foils, providing a 5 cm2 exposure area, and mounted in isolated diffusion cells with a saturated solution of magnesium nitrate (Sigma-Aldrich GmbH, Germany). The samples were then preconditioned in a Condition Rack for the Permatran-W Twin at a relative humifity gradient beteen 0 and 49%. The steady-state flow rate through the specimen was then measured and normalized with respect to the film thickness and the water pressure gradient over the film to yield water vapor permeability (WVP). The specimens expanded when exposed to water vapor. The WVP of these specimens is given with respect of the total swollen surface area. For the most swollen specimens, which touched and adhered to the upper wall of the desorption cell, the total exposure area was considered to be a two-based spherical segment. The first base is the edge of the aluminum foil and the second base is the part of the film that adhered to the wall. The radius of the adhesion mark was measured, and together with the height of the diffusion cell, these distances were used to calculate the effective exposed area. For specimens that did not touch the upper wall of the desorption cell, the specimen shape was considered to be a one-based spherical segment and the

Ga¨llstedt et al.

height of the sphere was measured. This enabled the specimen film area in contact with water vapor to be calculated. Water Absorbency by the Cobb60 Method. The water absorbency was determined, after 24 days in ambient condition, as described in SCAN-P 12:64.50 The apparatus used consisted of a rubber-mat baseboard and a 5 cm high metal cylinder with an inner area of 100.0 cm2. The tester is provided with a clamping device to fasten the cylinder onto the baseboard. After the initial weight of the film was determined, it was placed on top of the mat and a leak-proof seal was formed when the cylinder was clamped into position. 100 mL of distilled water was then poured into the cylinder. When determining Cobb60, the absorption time was 60 s. The water was poured out after 45 s and the test piece was removed from the instrument after an additional 15 s, the surface water was removed by pressing the film between blotting papers under a brass roller. Finally, the film was weighed and the Cobb value, X (the water absorbency in g/m2), was calculated as X ) 100(mf - mi), where mf and mi are the weights, in g/dm2, of the test piece after and before exposure to water. Two replicates of each sample were tested. Tensile Tests. An Instron 5566 tensile tester, equipped with a 100N load cell and controlled by Merlin Software on a Win98 platform, was used to determine the mechanical properties. The measurements were performed as described in ISO 37:1994.51 Dumb-bell specimens were punched to the type 2 dimensions.44 A crosshead speed of 100 mm/min was used. The specimens were conditioned for 3 days at 23 °C and 0% relative humidity (RH) in desiccators, with blue silica gel (Sigma-Aldrich GmbH, Germany), or at 23 °C and 50% RH, and subsequently measured. The different conditions were used to reveal the dependence of relative humidity. To assess the time dependence on the tensile properties, samples were also stored for 6 and 24 days prior to measurement. The thickness of each specimen was measured prior to the conditioning, by using a Mitutoyo IDC-112B. Five replicates of each sample were tested. Statistical Analysis. Statistical analysis were performed as an all-paired student-t test (p e 0.1), using JMP Software from SAS Institute Corp., NC. Results and Discussion General Aspects. The wheat gluten (WG) compression molding processing window was, as expected, narrow compared to that of many synthetic polymers. This has also been observed in other contexts using WG.23,26,29 The WG films processed at 130 °C were darker than those processed at the lower temperatures, regardless of the glycerol content. This darkening was likely a consequence of higher network density, extensive aggregation, temperature effects on pigments, and Maillard reactions.43,52,53 A deeper analysis of the cause of the color changes, and its dependence on temperature and exposure time, was not pursued here, but the cause has been investigated elsewhere.20,29,40,54 An even higher molding temperature yielded dark brown skewed films which had uneven surfaces. Therefore, 130 °C was chosen as the upper processing temperature. The low-temperature


Properties of Compression Molded WG Films

Table 1. Calculated Residual Glycerol Content, Methanol Zero-Concentration Diffusivity (Dc0), Normalized Protein Solubility, Methanol Saturation Concentration and Plastication Power for Methanol-Desorbed Compression-Molded Wheat Gluten Filmsa initial glycerol content (wt%)

25

32.5

40

10-110b

residual glycerol content (wt%) Dc0 normalized protein solubility methanol saturation concentration (g/g)c plasticization power (R)d

15-130b


6.1 (0.95) 1.83 × 10-8 (0.24 × 10-8) 0.10 0.29 (0.07) 10.2 (0.65)

3.3 (1.48) 1.29 × 10-8 (0.07 × 10-8) 0.09 0.34 (0.09) 9.23 (0.11)

15-90b


4.5 (1.20) 2.06 × 10-8 (0.53 × 10-8) 0.96 0.37 (0.08) 9.43 (0.35)

1.1 (0.72) 1.74 × 10-8 (0.32 × 10-8) 0.91 0.47 (0.13) 10.1 (2.92)

5 -130b


5.8 (0.48) 1.95 × 10-8 (0.17 × 10-8) 0.10 0.29 (0.07) 9.43 (0.50)

2.3 (1.46) 1.4 × 10-8 (0.28 × 10-8) 0.13 0.32 (0.10) 9.43 (0.72)

5-90b


5.5 (1.65) 2.21 × 10-8 (0.78 × 10-8) 1.04 0.36 (0.08) 8.89 (0.43)

1.9 (0.69) 2.27 × 10-8 (0.32 × 10-8) 0.94 0.47 (0.14) 8.35 (2.17)

5.2 (0.99) 1.75 × 10-8 0.31 0.36 (0.07) 9.38 (1.41)

a The values within parenthesis are the standard deviation. b The first number indicates the mold time (min) and the second number indicates the molding temperature (°C). c g solute/g polymer. d g polymer/g solute.

limit, ∼90 °C, was determined by the onset of gliadin polymerization.43 Below this temperature, the films were dough-like due to a low degree of denaturation. Solutioncast WG films can be denatured at lower temperatures with a proper choice of solution polarity and pH,9,29,30,34 because of the higher molecular mobility in solution. On the other hand, thermoforming of WG requires higher temperatures to give sufficient mobility to attain molecular unfolding and intermolecular disulfide cross-links.23,26,31,55 At the same time, care must be taken not to use too extensive an energy input. Too high a temperature has been shown to soften the crosslinked and aggregated gliadin.27 The molding temperatures were therefore selected, as mentioned in the Experimental Section, to be 90, 110, and 130 °C. The temperatures were higher, but the pressures, times, and moisture content were lower than those used in the study reported by Apichartsrangkoon et al.26 The moisture content of all gluten doughs in this study was about 7 wt %, and no measurable loss of moisture occurred during molding. The miscibility of the glycerol and the WG powder was essential for a homogeneous film. The 25% glycerol film, molded at 90 °C, appeared heterogeneous with color variations over the surface. The scatter in the transport and mechanical data was also highest for this blend and decreased with increasing glycerol content. The films containing 32.5 or 40 wt % glycerol were more homogeneous, and had a more uniform color. All glycerol-WG systems were mortared for the same length of time, but a lower amount of

glycerol demanded a longer mortaring time to obtain the same film homogeneity. Methanol Transport Properties. This is the first reported concentration-dependent diffusion coefficient measurement of a solute molecule in a protein matrix. Methanol was chosen in order to have a solute with appropriate polarity that would also easily evaporate during the desorption experiment. The desorption technique, in turn, was used because it is more suitable than, e.g., permeation techniques in obtaining concentration-dependent diffusivities. Further it is more flexible than permeation techniques in terms of evaluating different permeants. The zero-concentration diffusivity (Dco) was 9 × 10-9 cm2/s for the films without glycerol molded for 15 min and 8 × 10-9 cm2/s for the films molded for 5 min. These values were significantly lower than for the films with glycerol (Table 1). The film that was molded at 90 °C with 40% glycerol showed a lower Dco with increasing mold time, and the films molded at 130 °C showed the lowest Dco. No other trends were significant, as determined by the student-t analysis. Extensive glycerol migration (bleeding) occurred during methanol sorption. At the end of the sorption experiment, droplets, probably consisting mostly of glycerol and methanol, were observed on the film surface, and these were wiped off before the final film weight was determined. It was therefore not surprising that Dco increased with increasing residual glycerol content, the latter calculated from the mass of the films before and after the sorption/ desorption cycle. It should be pointed out that the increase

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Figure 1. Normalized protein solubility, i.e., the ratio of the protein solubility of the films to the protein solubility of the unprocessed wheat gluten powder exposed to the most severe treatment (the latter inserted at 25 °C), in a solution of SDS; SDS-solution and 30 s sonication, and SDS and 150 s (30 s and 60+60 s) sonication treatments. The films were molded for 5 min with 25% glycerol (b), 15 min with 25% glycerol (O), 5 min with 40% glycerol (2), and 15 min with 40% glycerol (4). The three 10 min/32.5% glycerol data points (0) refer to, from bottom to top, the SDS, the SDS/30 s, and the SDS/150 s treatments.

in diffusivity was not large enough to be significant according to the student-t test. The glycerol evaporation may also have occurred due to the increase in vapor pressure caused by the methanol. It cannot be excluded that smaller residues of gliadins were included in the exudates, although only very small quantities were expected because of the large size of the gliadin molecules. Only a small amount of glycerol was thus left in the films after the sorption/desorption cycle as seen from weight differences. In general, as observed in Table 1, the saturation concentration of methanol increased with the initial loading of glycerol and with a lower residual glycerol content. A high initial loss of glycerol eventually favored the initial uptake of methanol and as glycerol migrated, free space was replaced by methanol. The lowest methanol solubility was observed for the glycerol-free sample (0.15-0.20 g solute/g polymer). Protein Solubility and Methanol Transport Properties. To assess the degree of molecular constraints, the SDS/ sonication solubilities were analyzed. Figure 1 shows the amounts of dissolved protein monomers and polymers at the different molding temperatures. It is believed that the detergent (SDS) dissolves protein molecules based on the disruption of secondary bonds and that the shearing action of sonication primarily disrupts disulfide bridges. Sonication can also induce chemical reactions as a result of an increase in the amount of free radicals from bond scissions.43 It should be noticed that even the unmolded WG powder was difficult to dissolve using SDS and sonication, due to the aggregation in the native state. Consequently, in contrast to the gliadins in wheat flour, the gliadins in the WG powder was not easily dissolved using the “standard” SDS technique.43 Several different procedures were tested to increase protein solubility and it was found that SDS and 150 s (30 s and 2 × 60 s) sonication treatment of the unmolded powder yielded full solubility. Longer sonication led to further degradation. The protein solubility during SDS and SDS/30 s sonication

Ga¨llstedt et al.

Figure 2. Mass ratio of soluble protein polymers (LPP+SPP) and soluble protein monomers (LMP+SMP), in a solution of SDS, SDS solution and 30 s sonication, and SDS solution and 150 s (30 s and 60+60 s) sonication. The films are molded for 5 min containing 25% glycerol (b), 15 min with 25% glycerol (O), 5 min with 40% glycerol (2), and 15 min with 40% glycerol (4). The three 10 min/32.5% glycerol data points (0) refer to, from bottom to top, the SDS, the SDS/30 s, and the SDS/150 s treatments. The unprocessed wheat gluten powder is inserted at 25 °C.

decreased with increasing mold temperature. The extensive sonication treatment substantially increased the protein solubility of the samples molded at 90 °C but not that of those molded at 130 °C. Thus the degree of aggregation increased when the mold temperature was increased by 40 °C. This has also been observed by dynamic mechanical thermal analysis.27 Li and Lee claimed that the loss in solubility during extrusion was due to molecular unfolding and the exposure of hydrophobic and reactive sites on the molecules that is followed by aggregation and an increase in the molecular weight.28 Since most of the sulfur in the unmolded gluten forms inter- or intramolecular disulfide bonds, some of the increase in aggregation involves reorganization of intramolecular disulfide bonds to intermolecular disulfide bonds via thiol-disulfide exchange reactions.38 The minor effect of sonication on the samples molded at 130 °C suggests that the specimen was extensively cross-linked, also due to other cross-linking reactions, e.g., the formation of isopeptide bonds. This was also suggested by Micard et al.25 and Morel et al.38 However, due to our extraction procedure, the exact origins of the cross-links were not possible to determine here. The effects of mold time and glycerol content were insignificant compared to the effect of temperature. Figure 2 shows that it is primarily protein monomers that are dissolved at 130 °C, whereas also protein polymers are dissolved from the unmolded powder and from the samples molded at the lower temperature. It is primarily the monomers that are dissolved in the presence of SDS, whereas the polymers are dissolved first after sonication. The central point sample showed intermediate protein solubility and an intermediate polymer/monomer solubility ratio. The lower methanol zero-concentration diffusivity for films, with 40 wt % glycerol, molded at the higher temperature is thus explained by the simultaneous increase in protein aggregation (Table 1), characterized by a more dense molecular network with a larger number of intermolecular cross-links. A lower amount of glycerol resulted in a


Properties of Compression Molded WG Films Table 2. Oxygen Permeability for Compression-Molded Wheat Gluten Filmsa glycerol content (%) 25 32.5 40

5-90b

oxygen permeability (cm3 mm/(m2 day atm)) 5-130b 15-90b 15-130b 10-110b

3.06 (0.62) 1.05 (0.42) 3.50 (0.52) 2.26 (0.58) 8.83 (0.59) 12.71 (2.54) 12.03 (3.27) 13.82 (1.39) 12.67 (7.82)

a The value within parentheses are the standard deviations. b The first number indicates the mold time (minutes) and the second number the molding temperature (°C).

Table 3. Cobb60 Water Absorbency for Compression Molded Wheat Gluten Filmsa glycerol Cobb60 water absorbency (g /m2) content (%) 5-90b 5-130b 15-90b 15-130b 10-110b 25 32.5 40

47.5 (2.7) 41.0 (5.1) 48 (3.8)

41.8 (3.3) 54.2 (4.0)

73.1 (5.1) 57.7 (6.9) 78.3 (1.1) 54.8 (7.3)

a

b

The value within parentheses are the standard deviations. The first number indicates the mold time (minutes) and the second number the molding temperature (°C).

Table 4. Water Vapor Permeability for Compression Molded Wheat Gluten Filmsa glycerol content (%) 25 32.5 40

water vapor permeability (g mm/(m2 day atm)) 5-90b 5-130b 15-90b 15-130b 10-110b 1902(204)

2013 (37)

2058 (77) 2016 (349) 3097 (158)

6019 (825) 5950 (535) 5038 (1167) 4914 (715)


methanol diffusivity that was almost independent of mold time. Due to the lower degree of aggregation, the films with 40% glycerol molded at 90° in 5 min showed a lower plasticization power (R, in eq 1) than films with 25 wt % glycerol molded at 130° in 5 min. The solute saturation concentration decreases with increasing aggregation, as shown in Table 1. The product of the plasticization power and the methanol saturation concentration, the exponential term in eq 1, corresponds to the size of the methanol-induced increase in methanol diffusivity. No significant differences were observed here, except for the films with 40 wt % glycerol molded at 90° for 15 min, which was higher, and the film with 25 wt % glycerol molded at 130° for 15 min, which was lower than the other films. Oxygen and Water Vapor Permeability and Cobb60 Analysis. Films with 40 wt % glycerol had a significantly higher oxygen permeability (OP) than the films with 25 wt % glycerol, due to the plasticizing and hygroscopic effects of glycerol (Table 2). For the same reason, the Cobb60 value and water vapor permeability (WVP) were significantly higher for the 40% glycerol samples than the 25% glycerol samples, at least when the other parameters were unchanged (Tables 3 and 4). These results are well in agree with the findings of Gennadios et al,21 who claimed that hydrophilicity was the most important factor that influenced the plasticizer efficiency of glycerol. In addition, the Cobb60 value, and, although not significantly, the OP, decreased with increasing mold temperature.

Figure 3. Tensile behavior of wheat gluten films showing type I (40% glycerol, molded at 130 °C for 5 min), type II (25% glycerol, 90 °C, 5 min, stored in 3 days), and type III (25% glycerol, 90 °C, 5 min, stored in 24 days) behavior. The films were stored in 0% relative humidity.

However, Ali et al.20 reported that the WVP decreased when the treatment temperature was raised from 65 to 95 °C (for 2.5-24 h of heat treatment), for solution cast films, and suggested that this was due to an increase in the amount of covalent cross-links. In their case, the constraint imposed by a higher cross-link density seemed to dominate over other conformational changes involved in the denaturation process. It has also been reported that thermal degradation results in a decrease in the WVP.40 However, no significant effects from the molding temperature were observed (Table 4). Tensile Properties. The stress-strain behavior of the WG films, stored at 0% RH, could be divided into three types (Figure 3). Type I was observed for films with glycerol contents of 32.5 wt % and 40 wt %, independent of storage time. It was characterized by a low yield stress followed by strain hardening at higher strain. Films molded at 90 °C, with a glycerol content of 25 wt %, showed a lower fracture strain and an absence of strain hardening (type II). If the type II films were stored for 24 days, they turned brittle (type III) due to loss of moisture. All films stored at 50% RH showed type I behavior. Table 5a-d show the moisture content and tensile properties of WG films at 0 and 50% RH for different glycerol contents, mold temperatures, mold and storage times. Mold temperature and mold time had less impact on the mechanical properties than the glycerol content. As expected, due to the lower glycerol concentration and lower molecular mobility, the 25% glycerol series had the lowest fracture strain, highest fracture stress and highest Young’s modulus (Table 5a-c). For most of the films, the modulus and fracture stress were higher for films produced at a higher mold temperature. The fracture strain was lower for all films molded at a higher temperature, except for those with 25 wt % glycerol stored at 0% RH. It should be noted, as stated earlier, that the films with the lowest glycerol content (25%), especially those molded at the lowest temperature (90 °C), were more heterogeneous. The molecular mobility was probably too low to obtain sufficient protein unfolding to yield homogeneous films, and in combination with low moisture content, they

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Table 5. (a) Young’s Modulus for Compression-Molded and Stored Wheat Gluten Films; (b) Fracture Stress for Compression-Molded and Stored Wheat Gluten Films; (c) Fracture Strain for Compression-Molded and Stored Wheat Gluten Films; (d) Moisture Content for Compression-Molded Wheat Gluten Films, Stored at 0% RH or 50% RHa Part a relative humidity (%RH)

storage time (days)

glycerol content (wt %)

0 0 0 50 50 50 0 0 0 50 50 50 0 0 0 50 50 50

3 3 3 3 3 3 6 6 6 6 6 6 24 24 24 24 24 24

25 32.5 40 25 32.5 40 25 32.5 40 25 32.5 40 25 32.5 40 25 32.5 40

5-90a

Young’s modulus (MPa) 5-130a 15-90a 15-130a

197 (15)

182 (15)

176 (17)

220 (10)

1.3 (0.3) 4.6 (0.8)

10.5 (0.7) 8.1 (0.3)

1.4 (0.3) 3.9 (0.5)

13.9 (1.1) 7.9 (0.9)

0.1 (0.01) 327 (59)

3.0 (0.3) 309 (24)

0.45 (0.4) 363 (23)

4.1 (0.5) 379 (40)

4.3 (0.5) 4.6 (0.8)

17.2 (2.4) 9.0 (0.9)

5.1 (1.1) 4.8 (0.7)

24.3 (1.0) 7.8 (0.5)

0.7 (0.2) 513 (48)

3.7 (0.6) 506 (39)

0.6 (0.2) 479 (53)

3.6 (0.2) 546 (18)

10.0 (2.2) 3.6 (0.6)

20.0 (1.7) 9.0 (1.0)

10.1 (2.2) 6.5 (0.5)

26.3 (2.2) 8.1 (0.6)

0.4 (0.2)

3.6 (0.5)

0.2 (0.1)

4.1 (0.4)

10-110a 35 (2)

1.6 (0.6)

79 (3)

1.7 (0.4)

104 (11)

2.1 (0.6) Part b

relative humidity (%RH)

storage time (days)


0 0 0 50 50 50 0 0 0 50 50 50 0 0 0 50 50 50

3 3 3 3 3 3 6 6 6 6 6 6 24 24 24 24 24 24

25 32.5 40 25 32.5 40 25 32.5 40 25 32.5 40 25 32.5 40 25 32.5 40

fracture stress (MPa) 15-90a 15-130a

5-90a

5-130a

10.4 (1.3)

12.2 (0.6)

9.1 (1.0)

14.0 (0.7)

1.2 (0.05) 1.9 (0.2)

3.7 (0.1) 4.9 (0.3)

0.3 (0.04) 2.1 (0.1)

4.1 (0.2) 5.7 (0.4)

0.3 (0.03) 14.1 (1.1)

1.9 (0.2) 18.2 (1.2)

0.6 (0.1) 15.3 (1.0)

2.2 (0.2) 18.5 (4.4)

1.8 (0.1) 2.5 (0.3)

4.7 (0.2) 5.2 (0.6)

1.9 (0.1) 2.3 (0.1)

5.2 (0.2) 5.9 (0.4)

0.4 (0.1) 17.2 (2.0)

2.1 (0.1) 25.1 (1.3)

0.6 (0.1) 20.6 (1.8)

2.1 (0.1) 29.8 (1.8)

1.9 (0.1) 1.9 (0.1)

4.9 (0.3) 5.1 (0.5)

1.9 (0.1) 2.8 (0.1)

5.0 (0.3) 6.1 (0.3)

0.6 (0.1)

2.2 (0.3)

0.4 (0.1)

2.4 (0.2)

5-130a

fracture strain (%) 15-90a

15-130a

10-110a 5.1 (0.1)

1.2 (0.1)

6.7 (0.1)

1.3 (0.03)

7.1 (0.3)

1.2 (0.1) Part c

relative humidity (%RH)

storage time (days)


0 0 0 50 50 50 0 0 0 50 50 50 0 0 0 50 50 50

3 3 3 3 3 3 6 6 6 6 6 6 24 24 24 24 24 24

25 32.5 40 25 32.5 40 25 32.5 40 25 32.5 40 25 32.5 40 25 32.5 40

5-90a 10 (3)

95 (12)

31 (15)

115 (20)

660 (23) 185 (16)

322 (0.2) 106 (3.9)

680 (20) 177 (6.9)

299 (15) 94 (4.1)

274 (4.3) 4.9 (0.4)

121 (3.9) 18.8 (3.4)

228 (14) 5.7 (0.5)

102 (3.5) 17.8 (5.8)

498 (20) 159 (9.6)

310.5 (12) 103 (8.1)

507 (18) 152 (29)

279 (13) 90 (2.4)

245 (45) 3.9 (0.5)

123 (5.1) 8.1 (1.2)

235 (20) 4.9 (0.4)

103 (3.3) 8.1 (0.5)

370 (35) 176 (31)

303 (12) 103 (8.4)

380 (22) 137 (5.7)

270 (10) 96 (4.3)

238 (10)

117 (4.6)

196 (14)

103 (3.1)

10-110a 260 (10)

175 (9.7)

149 (7)

169 (3.5)

126 (10)

174 (7.4)


Properties of Compression Molded WG Films Table 5. (Continued) Part d relative humidity (%RH)

storage time (days)


0 0 0 50 50 50 0 0 0 50 50 50 0 0 0 50 50 50

3 3 3 3 3 3 6 6 6 6 6 6 24 24 24 24 24 24

25 32.5 40 25 32.5 40 25 32.5 40 25 32.5 40 25 32.5 40 25 32.5 40

moisture content (wt %) 15-90a 15-130a

5-90a

5-130a

6.1 (0.1)

6.9 (1.3)

7.0 (0.1)

7.2 (2.0)

5.9 (0.4) 10.9 (0.1)

4.7 (0.6) 11.5 (0.8)

7.1 (0.8) 11.8 (0.5)

6.1 (0.2) 10.4 (0.7)

12.1 (0.3) 5.4 (0.2)

12.0 (1.4) 6.6 (2.1)

12.2 (0.5) 6.4 (0.1)

11.8 (0.4) 6.2 (1.3)

5.4 (0.4) 11.6 (0.1)

4.1 (0.7) 12.2 (0.8)

6.7 (0.8) 12.5 (0.5)

5.7 (0.2) 11.1 (0.6)

12.9 (0.2) 5.1 (0.2)

12.9 (1.3) 6.3 (2.1)

12.6 (0.4) 6.1 (0.1)

12.7 (0.3) 5.9 (1.3)

5.3 (0.5) 12.0 (0.1)

2.7 (1.6) 12.6 (0.7)

6.5 (0.8) 12.9 (0.5)

5.6 (0.2) 11.4 (0.6)

13.2 (0.3)

13.1 (1.8)

12.8 (0.6)

12.9 (0.3)

10-110a 5.17 (0.1)

12.1 (0.5)

4.6 (0.1)

11.1 (0.3)

4.4 (0.1)

11.9 (0.3)


therefore became more brittle. A high mold temperature increased the strain hardening, which was observed as an increase in the slope of the post-yield region of the stressstrain curve. However, the effects of mold time were small compared to the effects of the glycerol content. As expected, the central point sample showed intermediate mechanical properties (Table 5a-c). The largest changes in tensile properties occurred, for the samples stored at 0% RH, within 6 days. The loss of water and migration of glycerol was observed as a decrease in Young’s modulus, decrease in fracture strain and an increase in fracture stress. The greatest increase in Young’s modulus was observed for the films containing 40% glycerol, molded at 90 °C (Table 5a). The data also show that a higher mold temperature (130 °C) in combination with the higher glycerol content (40%) yielded the smallest increase in modulus and decrease in fracture strain with storing time. Only small differences were observed for the samples stored at 50% RH. Although glycerol probably migrates also at 50% RH, the amount of water present at 50% RH is enough to resist material aging and embrittlement. Conclusions The film homogeneity, as observed by the evenness in color over the film surface, increased with increasing glycerol content. The mold time and glycerol content had only small effects on protein solubility in SDS, but an increase in the mold temperature reduced significantly the amount of soluble protein polymers, presumably due to the rearrangement of disulfide cross-links and the formation of new intermolecular secondary bonds. Sonication had only a minor effect on the samples molded at 130 °C, which indicated that the crosslinks may also have consisted of other stronger covalent bonds such as iso-peptide linkages. A higher degree of aggregation, in combination with lower glycerol content,

resulted in a lower molecular mobility. Consequently, the Young’s modulus and the fracture stress were higher, and the fracture strain lower for films molded at the higher temperatures. Higher concentration of glycerol decreased the modulus and fracture stress, and increased the fracture strain. Glycerol had a higher impact on the tensile properties than the mold temperature and mold time. The water vapor and oxygen permeability and the Cobb60 value were higher for films with higher glycerol content, due to plasticizing and hygroscopic effects. The modulus and fracture stress increased, and the fracture strain decreased during storage, most of the effects being observed within 6 days for the samples stored at 0% RH. Very few significant differences were observed during storage at 50% RH, showing that water loss was more important than glycerol migration. Extensive glycerol migration occurred during exposure to methanol vapor. In general, the methanol plasticization power increased and the methanol solubility and zero-concentration diffusivity decreased with increasing degree of aggregation (increasing mold temperature). Acknowledgment. Va¨stsvenska Lantma¨nnens Research Foundation (VL-stiftelsen) is acknowledged for financial support. Martin Svensson and Peter Baeling, Svenska Lantma¨nnen AB, Sweden, are acknowledged for valuable discussions. Bo Johansson, Reppe AB, Sweden, is thanked for supplying wheat gluten powder. Alf Svensson, Karlshamns Tefac AB, Karlshamn, Sweden, is thanked for supplying the glycerol. References and Notes (1) Lookhart, G. L.; Bietz, J. A. PBI Bull. 1997, 4-6. (2) Guilbert, S. Technology and application of edible protective films. In Food Packaging and PreserVation-Theory and Practice, Mathlouthi, M., Ed., Elsevier Applied Science Publishers: NY, 1986; pp 371-394. (3) Cuq, B.; Gontard, N.; Guilbert, S. Cereal Chem. 1998, 75 (1), 1-9. (4) Gennadios, A.; Weller, C. L. Food Technol-Chicago 1990, Oct, 6369.

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