Effects of plasticizers, pH and heating of film-forming solution on the properties of pea protein isolate films

Journal of Food Engineering 105 (2011) 295–305

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Effects of plasticizers, pH and heating of film-forming solution on the properties of pea protein isolate films Dariusz Kowalczyk ⇑, Barbara Baraniak Department of Biochemistry and Food Chemistry, University of Life Sciences in Lublin, Skromna 8, 20-704 Lublin, Poland

a r t i c l e

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Article history: Received 11 October 2010 Received in revised form 26 January 2011 Accepted 18 February 2011 Available online 23 February 2011 Keywords: Edible film Pea protein isolate WVP Solubility Light transmission Transparency Microstructure

a b s t r a c t Effects of glycerol (3–7% w/w) and sorbitol (4–8% w/w) concentration, pH (7.0, 9.0, 11.0) and heating (90 °C, 20 min) of film-forming solution (FFS) on the water vapor permeability (WVP), moisture content (MC), solubility, light transmission and transparency of pea protein isolate (PPI) films were investigated. Films plasticized with sorbitol exhibited significantly lower WVP, lower MC and higher solubility, in comparison with glycerol-plasticized films. Increasing glycerol content of the films led to increases in WVP and MC but did not affect film solubility. In contrast, increase in sorbitol content had no effect on permeability and MC but resulted in increased film solubility. Moisture sorption isotherms of PPI films suggested that the difference in WVP observed among films plasticized with glycerol and sorbitol might be due to the different hygroscopicity of these plasticizers. The pH of FFS did not have a significant effect on WVP and MC. Solubility of PPI films formed from non-heated FFS was not affected by pH, whereas solubility of films formed from heat-treated FFS generally increased when pH was increased from 7.0 to 11.0. Heating of FFS resulted in improved film transparency. All tested films were characterized by excellent ability to absorb UV radiation. Microstructural observation by scanning electron microscopy did not show differences between sorbitol- and glycerol-plasticized films. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction The constantly growing demand for disposable packaging contributes to rapid increase in the quantity of packaging waste in landfills. Individual packaging of products, which is impossible to avoid especially for perishable food-stuffs, turned packaging industry into one of the major sources of municipal waste. The global aspiration to reduce an adverse impact of synthetic packaging on the natural environment is manifested in the growth of interest in use of bio-based packaging. Materials derived from renewable resources do not burden the ecosystems and provide an alternative to the petroleum-derived polymers. Bio-based packaging can be biodegradable and/or edible, depending on formulation, formation method, and modification treatments. The application of edible films and coatings is one of the innovations of packaging technology aimed to improve quality and extend shelf life of food products by acting as barriers of gases, moisture, aroma, and oils. Edible films are defined as structures being formed separate of any eventual intended use, whereas edible coatings are thin layers of material formed directly on the surface of the food product, thereby become an integral part of food and can be eaten together with. Films can be used as covers, wraps, or separation layers; and they can be formed into casings, capsules, ⇑ Corresponding author. Tel.: +48 81 462 33 26; fax: +48 81 462 33 24. E-mail address: [email protected] (D. Kowalczyk). 0260-8774/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2011.02.037

pouches, and bags. These stand-alone films also are used as testing structures for determination of barrier, mechanical, solubility, and other properties provided by certain bio-based packaging materials. The main substances applied in the production of edible films and coatings are proteins (collagen, casein, whey protein, soy protein, gluten, zein), polysaccharides (cellulose, starch, chitosan), and lipids. For coating or packaging applications, film permeability is an important characteristic that can markedly influence the storage stability of foods by controlling mass exchange. Protein films provide excellent oxygen barrier properties but show high WVP due to their hydrophilic nature (Krochta, 2002). The poor moisture barrier properties, however, can be improved by introduction of lipids (Anker et al., 2002; Chick and Hernandez, 2002; Fabra et al., 2008; Kim and Ustunol, 2001). Protein film-formers, such as collagen and gelatin have been used for a long time in the manufacture of sausage casings and drug capsules, respectively. Plant proteins also have a great usable potential. Legume seeds are cheap sources of protein with a relatively high nutritional value, which make them a very good raw material for the production of protein preparates. At the present time, the worldwide market is dominated by soy protein, commercially available as soy flour, soy concentrate, and soy isolate. Low price, quality, and versatile applications make them difficult to compete with. Consequently, a large part of the literature is focused on soy protein-based films. Only a few studies deal with properties of films from pea protein (Choi and Han, 2001,

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2002; Gueguen et al., 1995, 1998; Viroben et al., 2000), and other legume proteins (Bamdad et al., 2006; Bourtoom, 2008; Jangchud and Chinnan, 1999a, 1999b; Liu et al., 2004). Studies by Choi and Han (2001, 2002) showed that pea proteins can be utilized to prepare edible films with water vapor permeability (WVP) and physical characteristics similar to those obtained from soy proteins, whey proteins, or zein. Price comparison made by these authors on whey protein isolate (WPI) ($13.5–27/kg), soy protein isolate (SPI) ($3–3.8/kg), corn zein ($23–35/kg) (Krochta and De MulderJohnston, 1997), and pea protein isolate (PPI) ($2.5–2.8/kg) (Choi and Han, 2001), indicates that utilization of pea protein in food production, including manufacture of the edible films and coatings could contribute to economic benefits. The lack of genetic modification in commercially available pea species (Directive, 2001/18/EC) makes pea protein a great alternative to soy protein preparates, which are mostly obtained from transgenic plants. In addition, pea, unlike soyabean, as well as cereals containing gluten, eggs, fish, peanuts, milk, lupin and products thereof, is not on the list of products that are the cause of allergies and feeding intolerance (Commission Directive, 2007/68/EC). Climatic conditions of many European countries are conducive to pea growth, therefore pea is the main protein crop cultivated in the European Union (FAOSTAT, 2010). Manufacture of biodegradable and/or edible packaging from renewable raw materials might be a great opportunity for farmers to get a new output market for their goods. However, all up to date researches show that use of protein films and coatings on an industrial-scale depends on progress in the improvement of their properties. Edible films and coatings with satisfactory properties should have sufficient structural integrity, low WVP, resistance towards water, grease, UV light, etc. In this respect various film-forming variables need to be examined to determine their effect on protein-based film properties. In previous studies, only alkaline (pH 9.0–12.5) casting process has been used to prepare the pea protein-based films (Choi and Han, 2001, 2002; Gueguen et al., 1995, 1998; Viroben et al., 2000). Such high film alkalinity is undesirable when films are intended for use in edible packaging. In the majority of literature concerning the use of edible protein-based coatings to improve quality and prolong shelf life of food products, no pH adjustment during coating formulation is applied. For this reason in our study we considered the possibility of PPI film formation at pH 7.0 (neutral). The aim of this work was to determine the influence of type and concentration of plasticizer, pH and heating of film-forming solution (FFS) on the water vapor barrier properties, moisture content, solubility, light transmission and transparency of PPI films. The effect of plasticizer type on the moisture sorption and film microstructure was also investigated. However, all factors studied in this work have been intensively investigated and published by other authors, these fundamental factors were never simultaneously combined to optimize the protein film formation. 2. Materials and methods 2.1. Materials Pea protein isolate Propulse (ProFlo)TM (82.0 ± 2.0% protein) was kindly provided by Parrheim Foods Co. currently Nutri-Pea Limited (Portage la Prairie, MB, Canada). Glycerol and sorbitol (Sigma Chemical Co., St. Louis, MO, USA) were used as plasticizers. 2.2. Film preparation Films were obtained from 10% (w/w) aqueous PPI solutions containing various amounts of plasticizers, 3–7% (w/w) glycerol or 4– 8% (w/w) sorbitol. After stirring (14,000 rpm, 2 min), the pH values

of FFS were adjusted to pH 7.0, 9.0 and 11.0 with concentrated NaOH solution. Next mixtures were degassed under vacuum to remove any dissolved air. Films were formed by casting 11 g of solution on leveled polystyrene Petri dish (Nunc, Roskilde, Denmark) with an area of 145 cm2. Following solutions were drying at room temperature (25 ± 1 °C) for about 12 h. In the case of thermally modified films the FFS (100 ml) were heated in water bath at 90 °C for 20 min, and after cooling to 25 °C were mixed again (14,000 rpm, 1 min) to obtain homogeneity. 2.3. Film thickness and conditioning Film thickness was measured to the nearest 2.54 lm with a hand-held micrometer (Mitotuyo No. 7327, Tokyo, Japan). Three to five thickness measurements were taken on each testing specimen, depending on its dimensions. All film specimens were conditioned for 48 h in a versatile environmental test chamber (MLR-350, Sanyo Electric Biomedical Co. Ltd., Japan) at 50% relative humidity (RH) and 25 °C before testing. 2.4. Water vapor permeability The WVP (g mm/m2 d kPa) was calculated as:

WVP ¼ ðWVTR LÞ=Dp

ð1Þ

2

where WVTR (g/m d) is the water vapor transmission rate of films measured at 25 °C and 50% RH gradient, L (mm) is the thickness of film specimens, and Dp (kPa) was the difference in partial water vapor pressure between the two sides of film specimens. WVTR was determined gravimetrically using a modification of the (PN-ISO 2528, 2000), also known as the cup method. The permeation cell (poly(methyl methacrylate) cups) had an internal diameter of 7.98 cm (exposed film area: 50 cm2) and an internal depth of 2 cm. Film specimens (10 cm diameter disks) were mounted onto the open circular mouths of cups filled with 10 g of anhydrous calcium chloride (0% RH). The lid was fixed by six screws, a rubber O-ring gaskets helped to assure a good seal. The cups were placed in an environmental chamber set at 25 °C and 50% RH. Weights of the cups were recorded every 2 h for a period of 10 h. The slopes of the steady state (linear) portion of weight grow versus time curves were used to calculate WVTR. 2.5. Moisture sorption isotherms Film pieces (1.5  1.5 cm) were dried at 40 °C in a vacuum chamber containing P2O5 to constant weight (for 1 week). Afterwards, film specimens were sealed in chambers with constant RH atmospheres. The equilibration chambers were set up according to ASTM E104-02 (ASTM E 104-02, 2003) and consisted of small, hermetic boxes containing saturated salt solutions and a perforated plastic plate to suspend the sample above the salt solution. The solutions of LiCl, CH3COOK, MgCl2, K2CO3, NaBr, NaCl, and KCl stored at 25 °C (± 1 °C) equilibrate with the headspace air resulting in a RH of 11.3%, 22.5%, 32.8%, 43.2%, 57.6%, 75.3% and 84.2%, respectively. The samples were removed from the chambers and weighed after incubating for 2 weeks (preliminary experiments determined the samples reached a constant mass after approximately 7–10 days). Equilibrium moisture content was calculated from the increase in mass of the dried sample after equilibration at a given RH. The water activity of each of the salt solutions was calculated as %RH/100. All tests were performed in triplicate. The experimental moisture sorption isotherm values were averaged and fitted by the Guggenheim–Anderson-de Boer (GAB) model (Eq. (2)) as follows:

M ¼ ðm0 Ckaw Þ=½ð1  kaw Þð1  kaw þ Ckaw Þ

ð2Þ

D. Kowalczyk, B. Baraniak / Journal of Food Engineering 105 (2011) 295–305

where M is the equilibrium moisture content at the water activity aw, m0 is the monolayer moisture content, C is the Guggenheim constant, and k is the corrective constant taking into account properties of multilayer molecules with respect to the bulk liquid. GAB equation parameters were calculated from the Water Analyser Series (Version 97.4, by WebbTech, Australia).

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were brittle and broke during film removal. In turn, the films casted from FFS containing 8% (w/w) glycerol were found to be very adhesive and too sticky to handle. Therefore, in the work the properties of films formed from FFS containing 3–7% (w/w) glycerol and 4–8% (w/w) sorbitol were examined. 3.1. Water vapor permeability

2.6. Moisture content (MC) and total soluble matter (TSM) Conditioned film specimens (2  2 cm) were weighed (±0.0001 g) and dried in an oven at 105 °C for 24 h. The MC value was determined percentage of initial film weight lost during drying and reported on wet basis. The TSM was expressed as the percentage of film dry matter solubilized after 24 h immersion in water. Measurement of TSM followed a modification of Rhim’s method (Rhim et al., 1998). Dried film pieces were placed in 50 ml bakers containing 30 ml of distilled water with sodium azide (0.02 w/v) to inhibit microbial growth. The beakers were covered with Parafilm ‘‘M’’ (American Can Co., Greenwich, CT, USA) and stored at 25 °C for 24 h with occasional gentle stirring. Undissolved film materials were removed from water, gently rinsed with distilled water and subsequently oven dried (105 °C for 24 h) to determine solubilized dry matter. Initial dry matter values were obtained from MC measurements for the same film. 2.7. Light transmission and film transparency The ultraviolet and visible light barrier properties of the films were measured at selected wavelengths between 200 and 800 nm, using a UV/vis spectrophotometer (Lambda 40, Perkin– Elmer, Shelton, CT, USA) according to the procedure reported by Fang et al. (2002). The transparency of the films was calculated by Eq. (3) (Han and Floros, 1997):

Transparency value ¼ A600 =x or  log T 600 =x

ð3Þ

where A600 is the absorbance at 600 nm, T600 is the transmittance at 600 nm, and x is the film thickness (mm). The greater transparency value represents the lower transparency. 2.8. Scanning electron microscopy (SEM) Studies of microstructure of films and FFS were performed by using scanning electron microscope (1430VP, LEO Electron Microscopy Ltd., Cambridge, UK). Film samples were redried under vacuum. The FFS were spread in the form of a thin layer, then submerged in liquid nitrogen and dried by sublimation. Before watching all samples were dusted with gold. 2.9. Statistical analysis Statistical analysis of four factorial (type of plasticizer, concentration of plasticizer, pH, and heating) experiment was performed for the designate of optimum conditions for PPI film making. Measurements of film property were triplicated for WVP, MC, TSM, and transparency with individually prepared films as the replicated experimental units. A statistical significance test was conducted with Tukey’s test using general linear model (GLM) procedure at 5% significance level in the STATISTICA software (Version 6.0, by StatSoft, Inc., Tulsa, USA). 3. Results and discussion The coherent PPI films were prepared when the plasticizers were added at concentrations between 3% and 8% (w/w). However, protein layers obtained from FFS containing 3% (w/w) sorbitol

The influence of type and concentration of plasticizer, pH and heating on WVP of PPI films is shown in Fig. 1. The WVP of films prepared with sorbitol was lower than glycerol-containing films (p < 0.05). At the lowest, common plasticizer level (4% w/w) values of WVP for sorbitol- and glycerol-plasticized PPI films (pH 7.0, heated) were 1.08 and 14.31 g mm/m2 d kPa, respectively. Choi and Han (2001) reported the WVP for similar PPI films prepared using glycerol (protein/glycerol ratio = 70/30 by weight; ffi4.3% plasticizer concentration) was 98.40 g mm/m2 d kPa. This value of WVP is about seven times higher that observed in our study. This difference may be caused by different test conditions (RH gradient on top/bottom sides of film = 0/100% RH) used by authors of the cited studies. The RH gradient is an important parameter in calculation of WVP, for example it has been reported that increasing the RH gradient from 0/60% to 0/80% RH resulted in about fivefold increased WVP of sodium caseinate film (McHugh et al. 1993). The WVP values of glycerol-plasticized PPI films (7.29–32.36 g mm/ m2 d kPa depending on film type) in Fig. 1 are comparable to the WVP values (8.83–39.73 g mm/m2 d kPa depending on pH and drying temperature; 0/50% RH) determined by Jangchud and Chinnan (1999a) in peanut protein films. The better water vapor barrier properties of edible films containing sorbitol as plasticizer than those containing glycerol have been reported by several authors. Wan et al. (2005) observed that soy protein films plasticized with sorbitol exhibited approximately four times lower WVP than glycerol-plasticized films. Similar results were obtained for edible films made from water-soluble fish proteins (Bourtoom, 2006). Replacement of glycerol with sorbitol also significantly reduced WVP of films prepared from whey proteins (McHugh et al., 1994), casein (Chick and Ustunol, 1998), egg albumin (Gennadios et al., 1996), and gelatin (Thomazine et al., 2005). The differences in WVP between films plasticized with glycerol and sorbitol might be due to the different hygroscopicity of these plasticizers. Glycerol exhibits greater hydrophilic character than sorbitol (Huttinger, 1978; Takahashi et al., 1984). This is supported by the differences between moisture sorption isotherms of glycerol- and sorbitol-plasticized films based on whey proteins (Kim and Ustunol, 2001), soy protein (Cho and Rhee, 2002), cassava starch (Mali et al., 2005; Müller et al., 2008), pea starch (Zhang and Han, 2008), as well as pea proteins (Fig. 2). It was observed that WVP of PPI films increased with increasing level of glycerol, while was not affected (p > 0.05) by changes in sorbitol concentration (Fig. 1). This may be explained in term of molecular size of plasticizers used. Glycerol as a low molecular weight substance can probably easier penetrate into protein network than sorbitol, thereby the increasing of glycerol addition could result in more effective disrupting of intermolecular interaction among polypeptide chains than addition of sorbitol (Fabra et al., 2008; Sothornvit and Krochta, 2000). According to Cuq et al. (1997) reorganization of the protein network due to plasticizer incorporation increases of free volume and consequently raises the water diffusion in the polymer matrix of the film. It should be noted that generally, increase of sorbitol content in PPI films from 4% to 8% doubled the WVP value (e.g. from 1.26 to 2.66 g mm/m2 d kPa, films casted at pH 7.0 from non-heated FFS); however, the difference was no significant (p > 0.05) (Fig. 1). Increasing level of hydrophilic plasticizers, favorable to adsorption and desorption of water molecules, is known to

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Fig. 1. Effect of type and concentration of plasticizer, pH and heating of FFS on water vapor permeability of PPI films. Vertical bars represent the 95% confidence interval.

Fig. 2. Effect of type of plasticizer on moisture sorption isotherms of PPI films casted at pH 7.0 from heated FFS containing 5% w/w plasticizer. The lines were derived from the GAB equation.

enhance the water permeability of protein-based films (Cuq et al., 1997; Choi and Han, 2001; Gontard et al., 1993; Sobral et al., 2001). It is generally believed that heating FFS of proteins prior to casting results in cast films with improved WVP. Heating modifies the three-dimensional structure of protein through unfolding of polypeptide chains, thus exposing internal sulfhydryl groups and hydrophobic side chains buried in the interior of the native globulin molecule (Mohammed et al., 2000). It is hence hypothesizing that intermolecular forces, involve covalent S–S bonding and hydrophobic interactions among unfolded protein strands, promote cohesion in heat-denatured films, which lead to produce less permeable protein network (Chae and Heo, 1997; Bourtoom, 2008; Liu et al., 2004; McHugh et al., 1994; Stuchell and Krochta, 1994). However, Fairley et al. (1996) reported that inhibition of sulfhydryl/disulfide interchange in WPI films, by the sulfhydryl blocking

agent, had no effect on film WVP. Also, reduction of disulfide bonds with cysteine did not affect WVP. This demonstrates that disulfide bonds might play a small role in determining the moisture barrier properties of films based on sulfur-containing proteins. Our study showed that heat denaturation of pea protein did not affect (p > 0.05) WVP of the films (Fig. 1). This result is consistent with Pérez-Gago et al. (1999) who noted no effect of heating (90 °C, 30 min) on WVP of WPI films formed at higher pH levels (6.0– 8.0). Also, Guckian et al. (2006) found that the increase in ratio of heated/unheated protein in WPI films formulation did not alter the WVP properties of the film. In contrast, studies by Hoque et al. (2010) on cuttlefish gelatin films demonstrated that heating of FFS (40–70 °C, 30 min) resulted in an increase of WVP, compared with the control film (without heating). The results reported above suggest that probably because of the differences in amino acid composition, molecular properties and film network formed between proteinaceous sources, the water barrier behavior cannot be generalized, and understanding the effect of heating treatment on WVP of films remains complex subject. Varying pH of FFS had no effect (p > 0.05) on WVP of PPI films. Any pH (6.0–9.0) effect on WVP was observed for peanut protein films as well (Jangchud and Chinnan, 1999a). Gennadios et al. (1993) reported no pH (6.0–12.0) influence in studies on SPI films. Also, WVP of myofibrillar protein films did not change with pH value (2.0–12.0) of the FFS (Shiku et al., 2003). 3.2. Moisture sorption isotherms The moisture adsorption data of PPI films casted at neutral pH (7.0) from heated FFS containing 5% (w/w) plasticizers were fitted to GAB model and isotherms are displayed in Fig. 2. At all investigated aw levels the glycerol-plasticized films had higher moisture-binding ability than sorbitol-plasticized films. The observed differences were higher with increasing levels of aw. Mathematical interpretation of isotherms showed both higher monolayer water content (m0) and stronger water association (C parameter) in the case of films containing glycerol. For both kind of films, k parameter values were the same (Table 1). As was

D. Kowalczyk, B. Baraniak / Journal of Food Engineering 105 (2011) 295–305 Table 1 GAB model constants and coefficient of determination (r2) for PPI films plasticized with different plasticizersc. Plasticizer

m0a Cb kb r2 Thickness (lm)

Glycerol

Sorbitol

0.1233 0.6604 1.0 0.734 100.3 ± 3.1

0.0465 0.5380 1.0 0.702 97.7 ± 1.5

a

monolayer moisture content (g H2O/g solids). GAB model constants. c Films casted at pH 7.0 from heated film-forming solutions containing 5% plasticizer addition. b

mentioned in Section 3.1, the differences in sorption capacity and water vapor barrier properties between glycerol- and sorbitol-containing films are probably due to different hydrophilicity of these plasticizers. Glycerol is highly hydrophilic and a strong humectant; at 25 °C and 50% RH its hygroscopicity is 25, while sorbitol is 1 mg H2O/100 mg (Takahashi et al., 1984). The GAB sorption isotherm model is the most commonly used model for edible films (Cho and Rhee, 2002; Chiou et al., 2009; Kim and Ustunol, 2001; Khwaldia et al., 2004; Mali et al., 2005; Müller et al., 2008; Zhang and Han, 2008). Therefore, this model was also selected for this study. However, coefficient of determination (r2) on level 0.7 indicates moderate fitting experimental data to GAB equation. Jangchud and Chinnan (1999b) reported that the r2 for different sorption isotherm models for peanut protein films was affected by pH levels, e.g. it was found that at pH 7.5 the GAB model was less adequate (r2 = 0.66) than BET model (r2 = 0.76) and Smith model (r2 = 0.82). 3.3. Moisture content and total soluble matter The difference in hydrophilicity of the plasticizers reflected on MC of the films. As can be expected, films plasticized with glycerol exhibited higher MC than films with sorbitol (p < 0.05) (Fig. 3).

299

Increasing the level of glycerol from 3% to 7% led to about twofold increase in MC of the films, but there was no influence of sorbitol concentration on MC (p > 0.05). These results are supported by the work of Shaw et al. (2002), where increasing plasticizer/protein ratio (from 0.5 to 1.0) led to increases (from 40% to 55%) in MC of glycerol-containing WPI films, but had no effect on MC of sorbitolcontaining films (MC ffi 20%). No significant differences (p > 0.05) were detected between MC values of PPI films produced at different pHs. Also, heating did not change MC of the films (p > 0.05) (Fig. 3). Solubility is an important property of edible films. Use of edible films and coatings as protective layers on food, especially on high aw products, requires these materials to be water resistant. In protein films the dry matter solubilized in water is likely constituted by small molecules, such as plasticizers and small polypeptides (Bamdad et al., 2006; Cuq et al., 1997; Orliac et al., 2003). The TSM of PPI films is presented in Fig. 4. The results revealed great influence of plasticizer type on this property. Plastification with glycerol allowed to obtain films with a significantly lower TSM (19.7–31.3%) than plastification with sorbitol (36.1–54.9%) (p < 0.05). A similar dependence was also reported by Kim and Ustunol (2001) who found that WPI films with sorbitol (WPI/sorbitol = 1/1) were fully soluble (100%), while films with glycerol (WPI/glicerol = 1/0.7) were only partially soluble (31.6%). Taking into account the lower solubility of sorbitol as compared with glycerol (Griffin and Lynch, 1980), the higher water resistance of glycerol–PPI films is quite surprising. Furthermore, our study showed that the increase in sorbitol content in the films led to a linear increase in TSM content (Fig. 4). It is not clear why films prepared with glycerol exhibited lower TSM than those prepared with sorbitol. One explanation might lie in the plasticizer–polymer interactions. Monterrey-Q (1998) reported that significant part of glycerol remained water-insoluble in myofibrillar protein films, suggesting occurrence of glycerol–protein interactions. Small molecular weight plasticizers probably could easier incorporate into protein chains than the large molecular weight plasticizers, which results in more efficient formation of hydrogen bonds between these hydroxyl group of plasticizer and amide groups of

Fig. 3. Effect of type and concentration of plasticizer, pH and heating of FFS on moisture content of PPI films. Vertical bars represent the 95% confidence interval.

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Fig. 4. Effect of type and concentration of plasticizer, pH and heating of FFS on total soluble matter of PPI films. Vertical bars represent the 95% confidence interval.

protein (Banker et al., 1966). In this way glycerol might be stronger linked to the macromolecules in the protein network than sorbitol. The relatively lower ability of sorbitol to associate with biopolymers could cause its lesser immobilization in film matrix during incubation in water. To explain why protein films plasticized with glycerol exhibited lower solubility than those plasticized with sorbitol, the solubility of plasticizers and proteins should be taken into account. This can be done by determining the concentration of these components in a solution remained after sample soaking. As shown in Fig. 4, glycerol content had no effect (p > 0.05) on TSM of PPI films. Also, Choi and Han (2001) found that the solubility of pea protein films was not affected by glycerol concentration. The TSM reported by these authors for glycerol-plasticized films was higher (38.7–44.6% depending on protein/plasticizer ratio) than observed in our work, probably due to difference in determining initial dry matter of the film specimens prior to solubility testing. Choi and Han, in their study have dried film specimens over anhydrous CaSO4, while in presented work film specimens were dried in an air-circulating oven. Rhim et al. (1998) proved that thermally dried (105 °C, 24 h) SPI films exhibited lower solubility than film samples not exposed to heating prior to immersion in water. The decrease in solubility is probably due to heat-induced cross-linking of proteins. Reductions in TSM of protein films following heat curing (dehydrothermal treatment) have been reported (Hernández-Muñoz et al., 2004; Kim et al., 2002; Micard et al., 2000). TSM of PPI films formed from non-heated FFS was not affected (p > 0.05) by pH, whereas films formed from heat-treated FFS at pH 11.0 generally showed higher solubility than those formed at pH 7.0 (Fig. 4). The explanation could be that, at a higher pH value, more charged ionic forms of the protein are present, which have higher ability to attract water molecules. The effect was only observed in thermally modified PPI films possibly due to conformational changes of denatured proteins; charged groups that were deeply buried in the native proteins were exposed to the solvent (water). However, Shiku et al. (2003) reported pH-depended solubility of edible films based on non-denaturated fish myofibrillar proteins. In their study TSM of the films increased when pH of

FFS was increased from 7.0 to 9.0, but no differences were found when pH was further increased to 11.0. On the other hand, Handa et al. (1999) have displayed that TSM of egg white films decreased linearly with increasing pH of film forming solution. These results indicate that the changes in protein film TSM after pH adjustment are related to psychochemical characteristics of film-formers (e.g. surface hydrophobicity, molecular weight, conformation, net charge), therefore cannot be generalized. It was observed that in few cases (depending on pH and plasticizer concentration) glycerol-plasticized PPI films formed from heat-treated FFS showed significantly lower (p < 0.05) solubility than films formed from non-heated FFS. The decrease of TSM in thermally modified films reached maximally 6.1% (for films casted at pH 7.0 from FFS containing 5% w/w glycerol) (Fig. 4). Handa et al. (1999) reported that decrease of TSM of egg white films due to heating (40 °C, 30 min) was observed at pH 11.0 and 11.5, but not at 10.5. Liu et al. (2004) observed reduction, from 50.0% to 42.7%, in soluble matters of peanut protein films after heating (70 °C, 30 min) of FFS. In turn, Pérez-Gago et al. (1999) noted that WPI films formed from non-heated FFS were totally soluble, but denaturation (90 °C, 30 min) of protein caused the films to become insoluble. The improved water resistance of heat-induced protein films is explained by creation of disulfide bonds cross-linked the film network, thus making hydrophilic groups on proteins chains less accessible by water (Handa et al., 1999; Pérez-Gago et al., 1999). 3.4. Light transmission and transparency In many applications, including food packaging, the transparency of materials is an advantage because it allows consumers to see the product before buying, and the products with an attractive appearance could be better presented by sellers. On the other hand, packaging materials should protect food from the effects of light, especially UV radiation. Our study demonstrated that PPI films perfectly meet both these requirements. All analyzed films, regardless of the differentiating factors, were characterized by excellent UV light barrier properties. The transmittance noted in the

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Fig. 5. Effect of plasticizer concentration, pH and heating of FFS on light transmission of PPI films plasticized with: (a) glycerol and (b) sorbitol.

200–280 nm light region did not exceed 1.77% and 2.13% for films plasticized with sorbitol and glycerol, respectively (Fig. 5). Similar results were found for films based on whey protein (Fang et al.,

2002; Gounga et al., 2007) and fish protein (Shiku et al., 2004). Barrier properties of proteins against UV radiation are associated with the presence of UV-absorbing chromophore, especially aromatic

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amino acids – tyrosine and tryptophan and in a less extent, phenylalanine and disulfide bonds (Aitken and Learmonth, 2000). The comparison of geometric planes created by spectral curves of PPI films showed that the light transmission was least affected by the plasticizer type and pH of FFS. The main factor influencing

the transmittance of the films was heating. Heat treatment of filmogenic solutions resulted in the increasing of light transmission of the films for visible range (350–800 nm). According to Lee et al. (2003), temperatures >50 °C can considerably increase protein solubility of some soy protein preparates. It is possible that PPI films

Fig. 6. Effect of type and concentration of plasticizer, pH and heating of FFS on transparency of PPI films. Vertical bars represent the 95% confidence interval.

Fig. 7. SEM micrographs (2000 magnification) of PPI films plasticized with: (a) glycerol, (b) sorbitol; (1) surface, (2) cross-section. Films casted at pH 7.0 from heated FFS containing 5% w/w plasticizer.

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prepared from non-heated FFS solutions could have more indissolved particles that reflect visible light, thereby the films were more opaque and exhibited the lower light transmission. It was noted that addition of plasticizers at the higher contents decreased visible light transmittance, but only in the case of PPI films from non-heated FFS. For the films formed from heated FFS this tendency was not observed (Fig. 5). Fig. 6 presents the transparency of PPI films. The higher transparency value indicates that the film is less transparent. The transparency of films obtained from heated FFS ranged from 0.86 to 1.52 A600/mm and was better than transparency of films obtained from non-heated FFS. In contrast, Choi and Han (2002) found no relationship between heating treatment and transparency of films obtained from the purified PPI (91% of protein). In their work, regardless of heating time (0–50 min, 90 °C), the average transparency was 16.71 ± 0.93 A600/mm. No differences in transparency value were also observed between gelatin films from FFS without and with heat treatment (Hoque et al., 2010). The above mentioned results suggest that the changes in film transparency after heating of FFS observed in our work could be related to thermal transformations of non-proteinic compounds, most likely carbohydrates – the second main ingredient of commercial PPI. For instance, heattreatment is known to destroy starch granules, resulting in improved transparency (Hayashi, 2004). The worse transparency of PPI films obtained by Choi and Han (2002) is probably a result of higher protein concentration in the films (higher optical density). The transparency of PPI films (from heated FFS) obtained in our study was also greater than films based on whey proteins (Gounga et al., 2007), gelatin (Rawdkuen et al., 2010) and fish proteins (Hamaguchi et al., 2007; Shiku et al., 2003, 2004). Comparison of PPI films with the commonly used synthetic films, such as low density polyethylene (3.05 A600/mm), oriented polypropylene (1.67 A600/mm), polyester (1.51 A600/mm), and polyvinylidene chloride (4.58 A600/mm) (Shiku et al., 2003) also revealed their high transparency. The transparency of PPI films prepared from heated FFS was not affected (p > 0.05) by type and concentration of plasticizer, while films made from non-heated FFS became less transparent at higher plasticizer contents (Fig. 6). In films prepared from non-denatured proteins, cohesion is mainly due to hydrogen bonding. Plasticizer molecules compete with polymer molecules for hydrogen bonds, thereby the network homogeneity of films casted from non-heated FFS could be disrupted by bulky amount of glycerol or sorbitol molecules. The reduction in degree of protein network homogeneity ultimately led to reduction in transparency (Rhim et al., 1999). In films prepared from heat-denatured proteins, besides hydrogen bonding, unfolded polypeptide chains are associated through disulfide bonds and hydrophobic interactions, thus the protein network could be more stable and the effect of plasticizer concentration on film homogeneity and then transparency is not observable. It was observed that pH had no influence on the transparency of PPI films prepared from heated FFS (p > 0.05), while it played a role as important factor in the transparency of PPI films prepared from non-heated FFS (Fig. 6). The lower transparency of films casted at pH 7.0 comparing to pH 9.0 was presumably due to incomplete solubilization of native proteins at neutral pH. On the other hand, in highly alkaline environment (pH 11.0) negative charges of nondenatured proteins could result in repulsive forces that inhibited formation of homogenous protein matrix, therefore transparency of films casted at pH 11.0 was lower than those at pH 9.0. In past studies, Okamoto (1978) concluded that no SPI film formation occurred at pH values above 10.5, which was probably caused by strong repulsive forces between highly negative charges along protein chains, preventing protein molecules from associating. Native and heat-denatured proteins differ in reactivity. Heating favorable the cross-linking of proteins, thus the pH-dependent

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Fig. 8. SEM micrographs (1000 magnification) of dehydrated FFS (5% w/w plasticizer, pH 7.0, heated) containing: (a) glycerol, (b) sorbitol.

electrostatic interactions could have negligible effect on homogeneity (transparency) of PPI films prepared from heated FFS. 3.5. Microstructure Fig. 7 presents SEM images of surface and cross-section morphology of PPI films (casted at pH 7.0 from heated FFS containing 5% w/w plasticizer) with different plasticizers. Images revealed that both films have rough and uneven surfaces, covered by round granules with characteristic depressions (Fig. 7a-1 and b-1). No marked differences were detected between the surface microstructures of glycerol- and sorbitol-plasticized films. The cross-sections of both films showed homogenous and compact microstructure (Fig. 7a-2 and b-2), which is likely caused by strong cohesion forces, appearing during slow-drying of aqueous-based materials. The porous structures created by freeze-drying of FFS allowed observation of dispersed phase particles (Fig. 8). The concave spherical objects, similar to those on the film surface, with a diameter size mainly from 5 to 30 lm were displayed. Based on studies by Soral-S´mietana et al. (1998), these structures present the particles of pea protein isolate. It implies that PPI films are agglomerates of pea protein isolate particles, linked into form a continuous matrix. 4. Conclusion The results of the present study show that it is possible to prepare pea protein-based films, not only by commonly used alkaline

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casting process, but also by casting of FFS at pH 7.0 (neutral). This is essential in production of edible coatings, which are designated to be part of the food product. No positive effect on WVP reduction was obtained either by alkaline or by heat (90 °C, 20 min) treatments. The main benefit of heat treatment of filmogenic solutions was enhanced film transparency. All tested films exhibited excellent barrier properties to UV light, which suggesting that PPI films could help to prevent the degradation of UV-sensitive food ingredients. The type of plasticizer had a great influence on WVP, MC and solubility of PPI films. The analyses performed using SEM suggest that the differences between properties of glycerol- and sorbitolplasticized PPI films are not caused by differences in microstructure. Hydrophilicity of plasticizer was found to be the most important factor in determining MC and WVP of PPI films. Our study shows that optimal conditions to form PPI films with satisfactory characteristics to utilizing in Food Industry are plastification with sorbitol at the smallest plasticizer addition, neutral pH, and the thermal treatment. Thus, combination of these filmforming conditions should be used to further modify of PPI films properties, e.g. improvement of WVP by lipid addition, can be used. Acknowledgments The authors acknowledge the financial support of this work by Project NN 312 1722 33 from the Ministerstwo Nauki i Szkolnictwa _ Wyzszego (Poland). The authors also thank Nutri-Pea Limited (Portage la Prairie, MB, Canada) previously known as Parrheim Foods for the supply of the pea protein isolate Propulse (ProFlo)TM. References Aitken, A., Learmonth, M.P., 2000. Protein determination by UV absorption. In: Walker, J.M. (Ed.), The Protein Protocols Handbook, second ed. Human Press, Totowa, NJ, pp. 3–6. Anker, M., Berntsen, J., Hermansson, A.-M., Stading, M., 2002. Improved water vapour barrier of whey protein films by addition of an acetylated monoglyceride.. Inno. Food Sci. Emer. Technol. 3, 81–92. ASTM E 104-02, 2003. Standard practice for maintaining constant relative humidity by means of aqueous solutions. Annual Book of ASTM Standards, 11.03, 1133– 1137. Bamdad, F., Goli, A.H., Kadivar, M., 2006. Preparation and characterization of proteinous film from lentil (Lens culinaris): edible film from lentil (Lens culinaris). Food Res. Int. 39, 106–111. Banker, G.S., Gore, A.Y., Swarbrick, J., 1966. Water vapor transmission properties of free polymer films. J. Pharm. Pharmacol. 18, 457–466. Bourtoom, T., 2006. Effect of plasticizer type and concentration on the properties of edible film from water-soluble fish proteins in surimi wash-water. Food Sci. Technol. Int. 12, 119–126. Bourtoom, T., 2008. Factors affecting the properties of edible film prepared from mung bean proteins. Int. Food Res. J. 15, 167–180. Chae, S.I., Heo, T.-R., 1997. Production and properties of edible film using whey protein. Biotechnol. Bioprocess Eng. 2, 122–125. Chick, J., Hernandez, R.J., 2002. Physical, thermal, and barrier characterization of casein-wax-based edible films. J. Food Sci. 67, 1073–1079. Chick, J., Ustunol, Z., 1998. Mechanical and barrier properties of lactic acid and rennet precipitated casein-based edible films. J. Food Sci. 63, 1024–1027. Chiou, B., Avena-Bustillos, R.D., Bechtel, P.J., Imam, S.H., Glenn, G.M., Orts, W.J., 2009. Effects of drying temperature on barrier and mechanical properties of cold-water fish gelatin films. J. Food Eng. 95, 327–331. Cho, S.Y., Rhee, C., 2002. Sorption characteristics of soy protein films and their relation to mechanical properties. LWT- Food Sci. Tech. 35, 151–157. Choi, W.S., Han, J.H., 2001. Physical and mechanical properties of pea–protein-based edible films. J. Food Sci. 66, 319–322. Choi, W.S., Han, J.H., 2002. Film-forming mechanism and heat denaturation effects on the physical and chemical properties of pea-protein-isolate edible films. J. Food Sci. 67, 1399–1406. Commission Directive 2007/68/EC of 27 November 2007 amending Annex IIIa to Directive 2000/13/EC of the European Parliament and of the Council as regards certain food ingredients. Official Journal of the European Union: L 310/11. Available from: . Accessed 12 Sept 2010. Cuq, B., Gontard, N., Cuq, J.L., Guilbert, S., 1997. Selected functional properties of fish myofibrillar protein-based films as affected by hydrophilic plasticizers. J. Agric. Food Chem. 45, 622–626. Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified

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