Barrier properties of sodium caseinate films as affected by lipid composition and moisture content

Journal of Food Engineering 109 (2012) 372–379

Contents lists available at SciVerse ScienceDirect

Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng

Barrier properties of sodium caseinate films as affected by lipid composition and moisture content María José Fabra a,⇑, Pau Talens a, Rafael Gavara b, Amparo Chiralt a a b

Instituto de Ingeniería de Alimentos para el Desarrollo, Departamento Tecnología de Alimentos, Universidad Politécnica de Valencia, Camino de Vera, s/n, 46022 Valencia, Spain Packaging Lab, Institute of Agrochemistry and Food Technology, CSIC, Apartado de Correos 73, Burjassot, 46100 Valencia, Spain

a r t i c l e

i n f o

Article history: Received 9 March 2011 Received in revised form 15 October 2011 Accepted 16 November 2011 Available online 25 November 2011 Keywords: Oxygen Carbon dioxide Biopolymers Permeability Oleic acid

a b s t r a c t This work analyses the effect of lipid addition (oleic acid, beeswax and oleic acid–beeswax mixtures) as well as the influence of the relative humidity (RH), or the equilibrium water content of the films, on the permeability of sodium caseinate based films to water vapor and gas (oxygen and carbon dioxide). The effect that lipid addition had on the gas and water vapor permeability was dependent both on the composition of oleic acid–beeswax mixtures and the film’s moisture content. The addition of lipid mixtures reduced water vapor transfer as compared to the control films (without lipid), whereas pure oleic acid or beeswax were less effective. Both control films and films prepared with pure beeswax showed the lowest O2 and CO2 permeability, whereas the incorporation of oleic acid exponentially increased these values. A linear increase in water vapor and gas permeability was observed when the water content of the film rose, due to its plasticizing effect, which led to an increase in the molecular mobility. Predictive equations for water vapor permeability (WVP) and gas permeability were established as a function of water content and lipid composition. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Generally, protein and polysaccharide films are quite moisture sensitive and this inherent hydrophilic nature makes them excellent barriers to non-polar substances such as oxygen or some aroma compounds (Miller and Krochta, 1997; Chen, 2002; Bertan et al., 2005; Srinivasa et al., 2007), but poor barriers to water vapor. The incorporation of lipids into protein matrices modifies the barrier properties of these films due to the presence of discontinuities in the polymer network (lipid droplets or aggregates) (Morillon et al., 2002; Fabra et al., 2008a,b) and to the development of specific interactions among lipids and protein chains. This is due to their amphiphilic nature and mainly takes place in the case of polar lipids, such as fatty acids (Fabra et al., 2010a). However, lipid addition implies an increase of the oxygen permeability due to the hydrophobic nature of lipids, which facilitates oxygen transfer (Bertan et al., 2005). The physical state of the lipid affects permeability values; the solid lipids give rise to a reduction in the gas permeability of the films (Srinivasa et al., 2007; Miller and Krochta, 1997). Fabra et al. (2010a) analyzed the interactions between oleic acid and beeswax and caseinates through the study of the glass transition of the sodium caseinate matrix and the melting properties of lipids in these composite films. They pointed out that the incorporation ⇑ Corresponding author. Tel.: +34 3877000x83613; fax: +34 963877369. E-mail address: [email protected] (M.J. Fabra). 0260-8774/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2011.11.019

of oleic acid into the protein matrix enhances the water plasticization effect from an aw value of 0.530 upwards indicating that, when the molecular mobility reaches a critical value, the bonding of the lipid to the protein chains provokes a weakening of the protein chain association reflected in a decrease of the network mechanical resistance. Nevertheless, no interactions between beeswax and caseinates were detected, both components being in separate phases. The lipid–protein interactions also affect the water sorption capacity of the films and the film barrier properties to water vapor and gases, the former, in turn, having a great influence on the barrier properties due to the water plasticizing effect and its relationship with molecular mobility. The effect of water content on water vapor permeability has been reported by different authors (Gennadios et al., 1993; Gontard et al., 1993; Alves et al., 2010; Fabra et al., 2009a). The hygroscopic character of protein based films favors the adsorption of a great number of water molecules when there are conditions of high relative humidity (RH), resulting in plasticized film matrices with great molecular mobility, which favors mass transfer phenomenon. The water plasticization effect implies the reduction of intermolecular protein interactions and the increase of intermolecular spacing. Thus, the diffusion of water molecules through the film is favored under these conditions. Water vapor transport occurs when a driving force (relative humidity gradient) is imposed in the system, which implies the establishment of a water concentration profile in the film. So, differing molecular mobility occurs through the film’s thickness and the obtained value of the transport property must be assigned

M.J. Fabra et al. / Journal of Food Engineering 109 (2012) 372–379

to the film under the experimental conditions used or to the mean concentration value of water in the film. In this sense, the use of a small driving force to obtain the transport property allows us to assign this value to a more accurate value of the moisture content (narrower concentration range) (Mauer et al., 2000; Fabra et al., 2009b, 2010b; Alves et al., 2010; Phan et al., 2009). This is a more useful method for carrying out the experiments than the use of 0/ 100%, 0/90% or 0/75% RH gradients on top/bottom of the film, which are often used to evaluate water vapor permeability values of films (Avena-Bustillos and Krochta, 1993; Chick and Ustunol, 1998; Olivas and Barbosa-Cánovas, 2008). In this paper, the permeability values of water vapor and gases (O2 and CO2) of sodium caseinate based films were obtained under different relative humidity conditions and the influence of incorporating a lipid (oleic acid, beeswax and their mixtures) to the protein matrix was analyzed as a function of the water content of the film. 2. Materials and methods 2.1. Materials Alanate 110 commercial sodium caseinate (98% purity) (NaCas) was obtained from Llorella S.A. (Barcelona, Spain). Glycerol (Gly) was purchased from Panreac Quimica, S.A. (Castellar Del Vallés, Barcelona, Spain). The lipid used was oleic acid (OA) (minimum purity 96%), which was provided by Quimica, S.A. (Castellar Del Vallés, Barcelona, Spain). Beeswax (BW) was provided by Brillocera, S.A. (Valencia, Spain). 2.2. Preparation of film-forming dispersions Film forming dispersions were based on a sodium caseinate aqueous solution with and without emulsified lipids. Control (without lipid) was prepared by dispersing sodium caseinate (8% w/w) in distillate water at a pH of 6.5. The plasticizer was added in the aqueous solution until a ratio of 1:0.3 protein:plasticizer was reached. The dispersion was homogenized for 1 min at 13,500 rpm, followed by 3 min at 20,500 rpm using an Ultra-Turrax homogenizer (Ultraturax T25, Janke & Kunkel, Germany). The lipid phase (1:0.5, protein:lipid ratio) of the film forming dispersions containing lipids was based on oleic acid and beeswax mixed in different OA:BW ratios (100:0, 70:30, 50:50, 30:70, 0:100). To prepare these emulsions, the amount of beeswax required was melted in the hot solution and was also homogenized for 1 min at 13,500 rpm, followed by 1 min at 20,500 rpm. The temperature of homogenization was 85 °C. The emulsions were cooled at room temperature and oleic acid was added in the amount required for each film composition. Each emulsion was homogenized again using a vacuum high-shear probe mixer (Ultraturax T25, Janke & Kunkel, Germany) for 2 min at 20,500 rpm. Dispersion of OA was carried out without heating, at room temperature, to avoid oxidation. All emulsions were degasified at room temperature using a vacuum pump (diaphragm vacuum pump, Wertheim, Germany). 2.3. Film formation Films were prepared by following this casting procedure: the amount of each film-forming emulsion containing 2 g of total solids was weighed and was spread evenly over a Teflon casting plate (150 mm internal diameter) resting on a level surface, and films were formed by drying for approximately 24 h at 20 °C and 45% RH. Dry films were peeled off from the casting surface and preconditioned for 2 weeks in desiccators at 25 °C and at different relative

373

humidities (33%, 53% and 75% RH) prior to testing. A digital micrometer (Palmer-Comecta, Spain, accuracy ±0.001 mm) was used to measure film thickness at five different points of the same sample, at least. 2.4. Moisture sorption isotherms Three accurately weighed film samples (1.5–2.0 g) were placed in hermetic chambers containing oversaturated salt solutions (LiCl (aw: 0.113), CH3COOK (aw: 0.225), MgCl2 (aw: 0.328), K2CO3 (aw: 0.432), Mg(NO3)2 (aw: 0.529), CuCl2 (aw: 0.675), NaCl (aw: 0.753) and KCl (aw: 0.843)) at 25 °C to maintain the relative humidity at a constant level. The samples were weighed periodically (0.00001 g precision) until they attained a constant weight, when equilibrium was assumed. Finally, the equilibrium moisture content was determined by drying them in a vacuum oven at 60 °C and 50 Torr for 3 days. Each sorption isotherm data was obtained in triplicate. 2.5. Barrier properties 2.5.1. Water vapor permeability (WVP) A modification of the ASTM E96-95 (McHugh et al., 1993) gravimetric method for measuring the WVP of flexible films was employed, using Payne permeability cups (Elcometer SPRL, Hermelle /s Argenteau, Bélgica). Deionised water or over-saturated Mg(NO3)2 and NaCl (Panreac Quimica, S.A., Castellar del Vallés, Barcelona) solutions were used inside the testing cup to reach 100%, 75% or 53% relative humidity, respectively, on one side of the film through a circular opening of 3.5 cm in diameter. Once the films were secured, the cups were placed in pre-equilibrated cabinets fitted with a variable-speed fan to reduce resistance to water vapor transport. The environment within the cabinets was held at a constant RH using over-saturated MgCl2 or Mg(NO3)2 solutions to reach 33% or 53% RH, respectively. So, the imposed RH gradients were: 100/53, 75/53 and 53/33; During WVP measurement, the side of the film in contact with the Teflon plate was placed in contact with the highest relative humidity. The cabinets were placed at a controlled temperature of 25 °C. The cups were weighed periodically (every 2 h for a day) after the steady state was reached using an analytical balance (±0.0001 g). Water vapor permeability was determined from the slope obtained from the regression analysis of the weight loss data as a function of time, once the steady state was reached. At least four replicates were obtained from each sample. The method proposed by McHugh et al. (1993) to correct the effect of concentration gradients established in the stagnant air gap inside the cup was used. 2.5.2. Oxygen permeability (O2P) The oxygen permeation rate of the sodium caseinate films was determined (in triplicate) at 33%, 53% and 90% RH and 25 °C using an OX-TRAN Model 2/21 ML Mocon (Lippke, Neuwied, Germany). Film samples were previously preconditioned for 2 weeks in the desiccators at the relative humidity level of the test using oversaturated MgCl2, Mg(NO3) and KCl solutions. Two samples were placed in the equipment for analysis and they were conditioned in the cells for 6 h, then the transmission values were determined every 45 min until equilibrium was reached. Oxygen permeability was calculated taking into account both the oxygen transmission rate as well as the thickness of the film. 2.5.3. Carbon dioxide permeability (CO2P) The carbon dioxide permeation rates of the films were determined (in triplicate) at 25 °C and at three different relative humidities (33%, 53% and 75% RH), using an isostatic permeation test described by Gavara et al. (1996). To this end, a stainless-steel cell

374

M.J. Fabra et al. / Journal of Food Engineering 109 (2012) 372–379

was used with two chambers separated by the film and a constant gas stream was passed through each chamber: the permeant gas (CO2) flowed through one of the chambers and the carrier gas (nitrogen) flowed through the other one, both at the required RH. At the chamber’s exit, the flow rate of carrier gas was measured by a mass flow meter (Dakota Instruments, Orangeburg, USA). The concentration of CO2 in this stream was analyzed by gas chromatography. An AH5890 gas chromatograph (Agilent Technologies, Barcelona, Spain), equipped with a manual injection valve and an injection loop of 500 lL, a Chromosorb 102, 80/100 mesh, 120  1/80 column (Teknokroma, Barcelona, Spain) and a thermal conductivity detector were used. 2.6. Statistical analysis Statgraphics plus for Windows 5.1 (Manugistics Corp., Rockville, MD) was used to carry out statistical analyses of data through the analysis of variance (ANOVA). Fisher0 s least significant difference (LSD) was used at the 95% confidence level. In order to establish the relationships between permeability (WVP, oxygen permeability and carbon dioxide permeability) and the contents of water and oleic acid in the film, Solver Microsoft Excel was used to obtain the equation parameters by optimizing the residual square differences between the experimental and predicted values. 3. Results 3.1. Characterization of moisture content-water activity relationships Table 1 shows the values of experimental equilibrium moisture content (dry basis) of the different films. The moisture content of the films containing lipids was referred per g of non-lipid solids, assuming that the lipid phase did not contribute to water sorption. The incorporation of lipids reduces the water sorption capacity of the film due to the fact that lipids correspond to a fraction of solids with a low water uptake capacity, especially beeswax, which is very hydrophobic (Fabra et al., 2010a). As can be observed in Table 1, the values for films with a high OA content tend to be greater than those obtained for the control film over a wide range of aw values, which means that, to some extent, OA contributes to the increase in the water uptake capacity of the matrix. This can be explained by the interactions between OA and protein, reported by Fabra et al. (2010a), which lead to the OA binding to the polymer chains, thus modifying the number of active points to water adsorption and so, increases the water uptake capacity of the matrix. Nevertheless, the films with a high BW content, where the values of the equilibrium moisture content are lower than expected for the lipid-free matrix, are observed to behave in the opposite way. This indicates that BW seems to inhibit the water sorption

capacity of the protein, which is probably due to the promotion of hydrophobic interactions between lipid and protein. The water sorption data could be used to obtain the moisture content of the films at the different relative humidities used in the permeability determination of water vapor and gases. For the water vapor permeability, the values of the moisture content on each side of the film (due to the RH gradient imposed) were determined and the mean value was considered, by assuming a linear profile for the film’s water content during the experiment. 3.2. Water vapor permeability Table 2 shows the water vapor permeability values of sodium caseinate films for the three relative humidity gradients (53/33%, 53/75% and 53/100% RH gradient on top/bottom of the film), at 25 °C. The water vapor permeability decreased when a lipid phase was incorporated in the film, regardless of the relative humidity gradient used. This was also previously reported by Fabra et al. (2008a,b) for sodium caseinate films with different protein: lipid ratios, at 5 °C and 100/58% RH). Beeswax may be seen to be slightly more efficient than oleic acid when comparing data corresponding to the films containing these pure lipids at low relative humidity levels, but this tendency did not occur when the relative humidity increased, when no significantly different values were obtained for films containing either pure oleic acid or beeswax. This could be explained by the different microstructure of the films with OA or BW. The amphiphilic nature of OA causes it to interact with protein, thus leading to a more homogeneous distribution in the matrix (Fabra et al., 2009a,b,c, 2010a). Beeswax is more heterogeneous and irregularly distributed in the film, providing hydrophilic proteinenriched zones, but with solid lipid particles which delay the transfer of water molecules at low RH. At high RH, the hydrophilic zones are greatly plasticized and water mass transfer could be favored through these zones, thus behaving in a similar way to the films containing OA. The efficiency of the lipid in control water mass transfer increased when beeswax was combined with oleic acid, especially in the ratios of 70:30 and 50:50 OA:BW. This could be explained by the protein-lipid interactions reported by Fabra et al. (2010a) for the same polymer matrix. The amphiphilic nature of oleic acid leads to the establishment of a more consolidated composite matrix acting at the interface between proteins and the highly hydrophobic beeswax compounds: esters of long chain fatty alcohols and acids, as well as long chain alkanes (Debeaufort et al., 2000; Morillon et al., 2002; Kristo et al., 2007; Fabra et al., 2008a,b). The solid state and the hydrophobic nature of beeswax help to limit water diffusion in the matrix more efficiently when it is better integrated in the continuous phase through the surfactant action of oleic acid. As expected, water vapor barrier properties of the films were affected by the RH gradient, showing the highest WVP values when

Table 1 Values of the equilibrium moisture content (We, d.b.) of the films, expressed as g of water per g of non-lipid solids, in the case of lipid containing films. aw

We (d.b.)A control film

0.113 0.225 0.330 0.430 0.520 0.675 0.755 0.845

0.038 0.049 0.067 0.085 0.117 0.215 0.286 0.452

We (g water/g non-lipid solidsB) of films containing lipids Ratio OA:BW 100:0

a–c

(0.016)a (0.014)ab (0.008)a (0.004)a (0.018)a (0.005)a (0.003)a (0.015)a

0.033 0.062 0.083 0.105 0.131 0.239 0.310 0.458

(0.009)a (0.019)ab (0.019)a (0.006)a (0.005)a (0.026)a (0.007)a (0.006)a

Ratio OA:BW 70:30 0.038 0.061 0.078 0.109 0.136 0.221 0.311 0.447

(0.012)a (0.005)a (0.021)a (0.005)a (0.016)a (0.013)a (0.017)a (0.011)a

Ratio OA:BW 50:50 0.025 0.048 0.069 0.102 0.125 0.218 0.289 0.458

(0.008)a (0.005)b (0.012)a (0.005)a (0.002)a (0.015)a (0.012)a (0.024)a

Ratio OA:BW 30:70 0.024 0.045 0.082 0.091 0.126 0.205 0.277 –C

(0.007)a (0.002)b (0.005)a (0.009)a (0.001)a (0.007)a (0.006)a

Ratio OA:BW 0:100 0.017 0.035 0.060 0.090 0.133 0.206 0.268 0.433

(0.007)a (0.004)b (0.008)a (0.003)a (0.016)a (0.006)a (0.009)a (0.007)a

Different superscripts within a file indicate significant differences among samples (p < 0.05). d.b. means ‘‘dry basis’’. B g non-lipid solids correspond to grams of protein plus glycerol; Mean value (standard deviation). C Samples of films with OA:BW ratio 30:70, equilibrated at aw 0.845, showed mould growth at the end of equilibration time and so, they were not considered.

A

375

M.J. Fabra et al. / Journal of Food Engineering 109 (2012) 372–379 Table 2 Water vapor permeability (g mm kPa1 h1 m2) values, at 25 °C and different relative humidity gradients of the studied films.

14

53/33% RH

Control 100:0 70:30 50:50 30:70 0:100

0.53 0.45 0.20 0.14 0.20 0.29

(0.03)ax (0.60)ay (0.04)az (0.02)az (0.06)az (0.06)az

75/53% RH

100/53% RH

10.02 (0.91)bx 3.42 (0.40)by 0.48 (0.08)az 0.58 (0.08)bz 1.05 (0.06)bz 3.62 (0.36)by

11.90 (0.31)cw 6.10 (0.19)cx 2.63 (0.21)by 1.59 (0.09)cz 3.05 (0.03)cy 5.71 (0.19)cx

Mean values (standard deviation). a–e Different superscripts within a file indicate significant differences among relative humidities (p < 0.05). x–z Different superscripts within a column indicate significant differences among formulations (p < 0.05).

WVP (g mm kPa -1 h-1 m -2)

12

OA:BW ratio

Control

y = 83.182x - 5.573 R² = 0.972

100:0 OA:BW ratio

y = 30.787x - 2.154 R² = 0.953

70:30 OA:BW ratio

y = 12.184x - 0.996 R² = 0.689

50:50 OA:BW ratio

y = 7.497x - 0.467 R² = 0.804

30:70 OA:BW ratio

y = 17.121x - 1.345 R² = 0.839

0:100 OA:BW ratio

y = 31.231x - 1.591 R² = 0.984

10

8

6

4

2

0 0.00

0.05

0.10

0.15

0.20

0.25

0.30

We (g water/ g non lipid solids) Fig. 1. Water vapor permeability as a function of the values of the equilibrium water content (We) referred per g of non-lipid solids.

every film. The greatest influence of the mean water content on WVP (greater slope of the fitted straight line) was obtained in the control film where a sharp increase of the values was observed in line with the mean water content increase. This effect became less marked when lipids were present in the film, especially oleic acid–beeswax mixtures, which indicates that it is not only the degree of matrix plasticization but also the presence of the hydrophobic discontinuities that affect the WVP values. These hydrophobic discontinuities allow us to control the WVP values better, even when the continuous matrix showed great molecular mobility at high water content. The development of the slope of the fitted lines as a function of the OA mass fraction in the dried film (g OA/g solids in the film) is shown in Fig. 2, where a parabolic tendency was observed, with the minimum being at 0.16 g OA/g of the dried film. By taking into account these variations of WVP as a function of the mean moisture content in the polymer matrix and the OA mass fraction, the equation shown in Table 3 was proposed to predict the WVP in these films, where coefficients were optimized by a non-linear procedure (Solver from Excel). Fig. 3 shows the predicted vs. experimental values, where it can be seen that the prediction was quite accurate, taking into account the standard deviation values of the experimental values. The degree of molecular mobility in the system can be related with the difference between the system temperature (T) and the slope (WVP units⋅g non lipid solids / g water)

the greatest driving force (53/100% RH) was applied, which also corresponded to the highest mean moisture content of the film. As reported in several works (Lim et al., 1999; Fabra et al., 2009a; Alves et al., 2010), an increased water content in the film structure implies both an increase in molecular mobility, due to the plasticization effect, and also in all the properties which depend on molecular diffusion, such as transport properties (Roos, 1995). This effect was also observed in fatty acid–sodium caseinate films using two different RH gradients (53/100% and 53/33% RH) associated to two different mean water contents in the films (Fabra et al., 2009a). Similarly, Mauer et al., 2000 reported that b-casein films showed higher WVP values when a RH gradient of 53/76% was applied, as compared with the lower values obtained for a RH gradient of 53/11%. However, the influence of the mean moisture content of the film was much less marked when lipids were present in the matrix, especially when these were mixtures of oleic acid and beeswax. This seems to indicate that the hydrophobic character of the dispersed phase greatly contributes to the control of the water transport, despite the increase in the molecular mobility in the matrix. As mentioned above, the oleic acid–beeswax mixtures were more efficient at reducing water vapor transport in the films than the pure lipids due to the greater hydrophobic nature of beeswax and the surfactant character of oleic acid, which promotes interfacial interactions with the protein matrix. The WVP values of sodium caseinate films were in the same order as those reported for other protein films, such as why protein measured at 25 °C/0/11% RH (0.22 g mm m2 h1 kPa1) (McHugh et al., 1994), soy protein and soy protein–beeswax films measured at 5 °C/58/100% RH (6.7 and 3.8 g mm m2 h1 kPa1, respectively) (Monedero et al., 2009) and wheat gluten and wheat gluten-lauric acid films measured at 25 °C/0/100% RH (4.01 and 1.0.6 g mm m2 h1 kPa1, respectively) (Pommet et al., 2003). Fig. 1 shows the relationship between WVP values and the mean water content of the films, expressed per g of non-lipid solids. The mean water content of the film was estimated by assuming that each side of the film was equilibrated at the respective aw of the gas phase in contact. Since an aw gradient in the film was established during the WVP determination, both an aw profile and the corresponding water content profile occur throughout the film thickness. To estimate a mean value of the water content in the film, a linear profile was assumed for aw and a mean value was determined from the two extreme values. The mean water content of the film was estimated from the mean aw value and the corresponding sorption isotherm data. In every case, the relationships observed between WVP and the mean water content of the film were practically linear and, in every case, WVP is zero at a determined mean moisture content value that is very close in all film samples. This critical mean moisture content value (We = 0.073 g water/g non-lipid solids) was established as the mean value for

35 30

y = 1509.9x 2 -470.55x + 45.29 R² = 0.985

25 20 15 10 5 0 0

0.05

0.1

0.15

0.2

0.25

0.3

X OA (g oleic acid / g dried film) Fig. 2. Slope of the fitted lines obtained from Fig. 1 as a function of the oleic acid mass fraction in the dried film.

M.J. Fabra et al. / Journal of Food Engineering 109 (2012) 372–379

7 6

films with OA

WVP (g mm kPa -1h-1m-2) predicted

glass transition temperature (Tg), at a determined moisture content of the product (Roos, 1995). The greater the temperature difference, the more molecular mobility there is. The Tg values of both the caseinate films and also the composite films of oleic acid– caseinate and beeswax–caseinate were determined as a function of the moisture content in a previous work (Fabra et al., 2010a), and so WVP values can be correlated with the TgT values at the corresponding moisture content of the films. Fig. 4 shows the relationships between these values which were obtained for the different samples. Tg could not be obtained for samples containing oleic acid–beeswax mixtures, because the melting endotherm of beeswax occurs over a wide temperature range, overlapping the glass transition of the amorphous matrix (Fabra et al., 2010a). However, the Tg values for pure beeswax were considered to be the same as for the lipid-free film, since no changes were observed in the melting behavior of beeswax in the presence of caseinate. Therefore, it can be assumed that sodium caseinate and beeswax are in two different phases (Fabra et al., 2010a). This assumption could not be made for films prepared with OA: BW mixtures, due to the oleic acid interacting with the sodium caseinate matrix modifying the glass transition temperature values. Table 4 shows that Tg values are higher than the temperature test (25 °C) in every case, which means that films are in the glassy state. Nevertheless, the lower the Tg value (and so, the smaller the temperature difference), the greater the molecular mobility in the caseinate matrix and the higher the WVP values. In fact, Fig. 4 shows that WVP values increased sharply as the TgT difference decreased coinciding with the approximation of the system to the rubbery state. A linear relationship for the WVP and the TgT difference was obtained for the three cases, although a different straight-line could be fitted for each kind of film. The steepest slope was obtained for the control film which indicates the extreme sensitivity of this film to the Tg changes induced by water. However, the gentlest slope was obtained for the oleic acid–caseinate film, which suggests that this lipid makes the matrix less sensitive to the increase in molecular mobility which contributes negatively to water transport across the film.

5 4 3 2 1 0 0

1

2

3

4

5

6

7

WVP (gmm kPa-1h-1m-2) experimental Fig. 3. Comparison between experimental and predicted values of the WVP of films with oleic acid.

14 y = -2.694x + 82.649 R² = 0.991

Control

12 100:0 OA:BW ratio

WVP (g mm kPa -1 h-1 m -2)

376

10

0:100 OA:BW ratio

8 y = -1.211x + 37.016 R² = 0.980 6

4

y = -0.293x + 8.705 R² = 0.987

2

0

3.3. Oxygen and carbon dioxide permeabilities

0

5

10

15

20

25

30

35

Tg-T (ºC)

The influence of both the oleic acid–beeswax mixtures and relative humidity on the oxygen and carbon dioxide permeability of sodium caseinate films is shown in Figs. 5 and 6. No data could be obtained for films in which the lipid phase was composed of pure oleic acid, probably because the liquid oleic acid droplets seem to be removed during the gas permeability analysis giving rise to voids in the film, which makes the permeability analysis difficult with the equipment used. For both gases, the control film (lipid-free) showed the lowest O2P and CO2P values, while lipid addition modified the behavior of these barrier properties. Oleic acid promotes oxygen and carbon dioxide permeability more efficiently than beeswax, which can be mainly attributed to its liquid state at room temperature favoring molecular mobility and diffusion phenomena through the lipid phase. Thus, O2 and CO2 can permeate easily through the films containing a higher content of oleic acid. On the other hand, previous works (Fabra et al., 2008a,b, 2010a) reported that oleic acid has a

Fig. 4. Values of water vapor permeability vs. TgT for lipid-free films and films containing pure oleic acid or beeswax (ratio OA:BW of 100–0 and 0:100, respectively).

plasticizing effect on sodium caseinate matrices, which also helps to promote diffusion phenomena through the matrix and so, to decrease gas barrier efficiency. The same trend was observed when the permeability of aroma compounds in sodium caseinate–oleic acid–beeswax films were analyzed (Fabra et al., 2008a,b). On the contrary, increasing the amount of beeswax in the lipid phase reduced the gas (oxygen and carbon dioxide) permeability of the films containing lipids due to its highly crystalline structure, which limits the diffusion of both gases in the lipid phase. So, it can be concluded that the diffusion of gases occurs preferentially through the liquid lipid phase when oleic acid is present in the film, instead of through the sodium caseinate matrix or beeswax particles.

Table 3 Predictive equations for water vapor, oxygen and carbon dioxide permeability as a function of the content of water and oleic acid in the film. Type of film

WVP (g mm kPa1 h1 m2)

O2P  1013 (cm3 m1 s1 Pa1)

CO2P  1013 (cm3 m1 s1 Pa1)

Films without oleic acid Films with oleic acid

56.15 xw

425.34 xw

0:073 þ ð957x2OA þ 290:36xOA þ 26:96ÞW e

16:01 199W e þ 5:0  1013 xOA

2412W e þ 9:0  1013 x15:04 OA

xw: water mass fraction in the film, xOA: oleic acid mass fraction in the dried film, We: g water/g non-lipid solids.

377

M.J. Fabra et al. / Journal of Food Engineering 109 (2012) 372–379 Table 4 Values of the mean moisture content of the films (lipid-free solid basis) and the corresponding Tg values for some films. OA:BW ratio

Control 100:0 70:30 50:50 30:70 0:100 a

53/33% RH

75/53% RH

We (g water/g non-lipid solids)

Tg (°C)

0.078 0.078 0.074 0.066 0.077 0.057

55.4 53.6

a

55.4

100/53% RH

We (g water/g non-lipid solids)

Tg (°C)

0.174 0.203 0.191 0.189 0.178 0.179

52.2 41.8

52.2

a

We (g water/g non-lipid solids)

Tg (°C)a

0.219 0.253 0.253 0.239 0.231 0.225

51.1 34.5

51.1

Obtained from Fabra et al. (2010a).

O2P ×1013 (cm -3 m -1 s-1 Pa-1)

300

250

33% HR 75% HR

200 90% HR 150

100

50

0 70:30

50:50

30:70

0:100

Control

OA:BW ratio Fig. 5. Oxygen permeability  1013 (cm3 m1 s1 Pa1) values of studied films at the different values of the relative humidity.

CO2 P×1013 (cm -3 m -1 s-1 Pa-1)

2500

33%

2000

53% 90%

1500

1000

500

0 70:30

50:50

30:70

0:100

Control

OA:BW ratio Fig. 6. Carbon dioxide permeability  1013 (cm3 m1 s1 Pa1) values of studied films at the different values of the relative humidity.

O2P and CO2P levels rose notably when the relative humidity increased, which is coherent with the promotion of both the water uptake in the film and molecular mobility. Water molecules interact with polar groups of the protein strands, leading to an increase in the water content in the film as the relative humidity increases, thus reducing intermolecular protein interactions and increasing intermolecular spacing. The disruption of hydrogen bonds may create additional sites for the dissolution of oxygen or carbon dioxide and increase the mobility of the O2 and CO2 molecules within

the polymer bulk phase (Gennadios and Weller, 1990; Gontard et al., 1993, 1996; Mújica-Paz and Gontard, 1997). As commented on above, the effect of relative humidity on carbon dioxide permeability was similar to that of oxygen permeability, but more intense. This can be explained by the fact that CO2 is more soluble in water (S = 1.49 g CO2/kg H2O at 25 °C and 1 atm; Dodds et al., 1956) than oxygen (8.27 103 g O2/kg H2O at 25 °C and 1 atm, Weiss, 1970). So, CO2 permeates faster than oxygen. Gontard et al. (1996) and Alves et al. (2010) observed similar behavior when working with wheat gluten films and composite films based on kappa-carrageenan/pectin blends and mica flakes, respectively. Fig. 7 was obtained by taking into account the equilibrium water content (wet basis) of each film at each relative humidity, where the linear increase of both O2P and CO2P with the water mass fraction can be observed for each kind of film. Gontard et al. (1996) also reported that O2P increased quite linearly with water content (from values greater than 20%) in gluten films. Whereas the straight line intercept increased sharply when the content of oleic acid in the lipid phase rose, a very similar slope was obtained in every case for a determined gas. Taking this behavior into account, a model was established to estimate O2P and CO2P as a function of the film moisture and oleic acid contents. Since the mass fraction of total lipids is constant in the dried films (0.28), only the mass fraction of OA in the dried film was considered. So, both the mass fraction of OA and BW are correlated. Two different groups of samples could be considered: films without oleic acid (control film and films with pure beeswax) and films containing this compound. The behavior of the former could be modeled using the linear equation as a function of the film moisture content described in Table 3, where the greater contribution of the water content in the CO2P is reflected in the higher value of the obtained coefficient. Gas permeability of films containing oleic acid also increased linearly in line with the water content of the film, but a sharp increase was also observed when the concentration of this fatty acid increased in the lipid phase. Straight lines could be fitted to each kind of film for both O2P and CO2P to describe the water content effect. Among the slope values of the fitted lines, it is remarkable that no significant differences (p > 0.05) were found for a determined gas, although a notable increase in the straight line intercept was observed in line with the oleic acid concentration. In fact, exponential relationships were observed between the intercept values and the oleic acid mass fraction in the film for both gases. On the basis of these results, an empirical model was obtained to predict O2P and CO2P whose parameters were optimized by a non-linear procedure (Solver from EXCEL). Table 3 shows the obtained equation where the first linear term includes the influence of the water mass fraction of the film and the second exponential term the effect of the oleic acid mass fraction in the film. The influence of oleic acid concentration was greater on O2P than on CO2P, probably due to the greater sensitivity of CO2P to the water content, which masked the effect of the fatty acid. Fig. 8 shows the comparison of experimental vs. predicted values, where the relatively close fit of the values can be observed.

378

M.J. Fabra et al. / Journal of Food Engineering 109 (2012) 372–379

(a) 300

(b) 2000 1800

CO2P ×1013 (cm3 m -1 s-1 Pa-1)

O2P ×1013 (cm3 m -1 s-1 Pa-1)

250

200

150

100

50

1600 1400 1200 1000 800 600 400 200

0

0

0.0

0.1

0.2

0.3

0.4

0.5

0.0

0.1

0.2

0.3

70:30

0.4

0.5

0.6

0.7

xw (w.b)

xw (w.b.)

50:50

30:70

0:100

control

Fig. 7. P O2 (a) and P CO2 (b) values vs. equilibrium water mass fraction in the film at the equilibrium for lipid-free films and films containing oleic acid–beeswax mixtures.

2000

CO2P ×1013 (cm -3 m-1 s-1 Pa-1)predicted

O2P ×1013 (cm -3 m -1 s-1 Pa-1)predicted

300

films with OA

250

films without OA (*)

200

150

100

50

0 0

50

100

150

200

250

300

1800

films with OA

1600 1400

films without OA (*)

1200 1000 800 600 400 200 0 0

200

400

600

800

1000 1200 1400 1600 1800 2000

P CO2 ×1013 (c.c. m -1 s-1 Pa-1)experimental

P O2 ×10 13 (c.c. m -1 s-1 Pa-1)experimental

Fig. 8. Comparison between experimental and predicted values of the P O2 and P CO2 :

4. Conclusion

Acknowledgements

Oleic acid–beeswax mixtures produce a greater decrease of the water vapor permeability values of sodium caseinate based films than pure lipid compounds, due to the combination of the highly hydrophobic nature of the wax and the surfactant action of the oleic acid, which allows a better integration of components in the polymer matrix. Linear relationships can be established between the WVP values and the water content of the films. Likewise, WVP values decreased linearly when the TgT difference decreased, coinciding with the trend of the matrix towards the rubbery state and the promotion of molecular mobility. The oxygen and carbon dioxide permeability of sodium caseinate films increased linearly with the water content of the films, although that of CO2 rose more markedly due to the higher water solubility of this gas. The incorporation of oleic acid in the matrix provoked an exponential increase of both the O2P and CO2P due to its liquid state and to the protein–lipid interactions which contribute to the plasticization of the polymer matrix. Gas permeability values could be estimated from the water and oleic acid contents of each film.

The authors acknowledge the financial support from the Spanish Ministerio de Educación y Ciencia throughout the project AGL200701009 and AGL2010-20694, con-financed with FEDER founds. Author M.J. Fabra thanks the Campus de Excelencia Internacional VLC/CAMPUS for their support.

References Alves, V.D., Costa, N., Colehoso, I.M., 2010. Barrier properties of biodegradable composite films base don kappa-carrageenan/pectin blends and mica flakes. Carbohydrates Polymers 79, 269–276. ASTM E96-95, 1990. Standard test methods for water vapor transmission of materials. In: Annual Book of ASTM Standards. American Society for Testing and Materials, Philadelphia, PA. Avena-Bustillos, R.J., Krochta, J.M., 1993. Water vapor permeability of caseinatebased edible films as affected by pH, calcium cross-linking and lipid content. Journal of Food Science 58, 904–907. Bertan, L.C., Tanada-Palmu, P.S., Siani, A.C., Grosso, C.R.F., 2005. Effect of fatty acids and ‘‘Brazilian elemi’’ on composite films based on gelatin. Food Hydrocolloids 19 (1), 73–82.

M.J. Fabra et al. / Journal of Food Engineering 109 (2012) 372–379 Chen, H., 2002. Formation and properties of casein films and coatings. In: Gennadios, A. (Ed.), Protein-Based Films and Coatings. CRC Press, New York, pp. 181–211. Chick, J., Ustunol, Z., 1998. Mechanical and barrier properties of lactic acid and rennet precipitated casein-based edible films. Journal of Food Science 63, 1024– 1027. Debeaufort, F., Quezada-Gallo, J.A., Delporte, B., Voilley, A., 2000. Lipid hydrophobicity and physical state effects on the properties of bilayer edible films. Journal of Membrane Science 180, 47–55. Dodds, W.S., StutzmanSollami, L.F., Sollami, J., 1956. Carbon dioxide solubility in water. Industrial and Engineering Chemistry, 92–95. Fabra, M.J., Talens, P., Chiralt, A., 2008a. Tensile properties and water vapor permeability of sodium caseinate films containing oleic acid–beeswax mixtures. Journal of Food Engineering 85 (1), 393–400. Fabra, M.J., Hambleton, A., Talens, P., Debeaufort, F., Chiralt, A., Voilley, A., 2008b. Aroma barrier properties of sodium caseinate based films. Biomacromolecules 9, 1406–1410. Fabra, M.J., Jiménez, A., Atarés, L., Talens, P., Chiralt, A., 2009a. Effect of fatty acids and beeswax addition on properties of sodium caseinate dispersions and films. Biomacromolecules 10 (6), 1500–1507. Fabra, M.J., Hambleton, A., Talens, P., Debeaufort, F., Chiralt, A., Voilley, A., 2009b. Influence of interactions on water and aroma permeabilities of iotacarrageenan-oleic acid–beeswax films used for flavor encapsulation. Carbohydrates Polymers 76 (2), 325–332. Fabra, M.J., Talens, P., Chiralt, A., 2009c. Microstructure and optical properties of sodium caseinate films containing oleic acid–beeswax mixtures. Food Hydrocolloids 23 (3), 676–683. Fabra, M.J., Talens, P., Chiralt, A., 2010a. Water sorption isotherms and phase transitions of sodium caseinate-lipid as affected by lipid interactions. Food Hydrocolloids 24, 384–391. Fabra, M.J., Talens, P., Chiralt, A., 2010b. Properties of sodium caseinate films containing lipids. In: Hollingworth, S. (Ed.), Food Hydrocolloids: Characteristics, Properties and Structures. Novapublishers. Gavara, R., Catala, R., Hernandez-Muñoz, P., Hernandez, R.J., 1996. Evaluation of permeability through permeation experiments. Isostatic and quasi-isostatic methods compared. Packaging Technology and Science 9, 215–234. Gennadios, A., Weller, C.L., Testin, R.F., 1993. Temperature effect on oxygen permeability of edible protein-based films. Journal of Food Science 58 (1), 212–214, 219. Gennadios, A., Weller, C.L., 1990. Edible films and coatings from wheat and corn proteins. Food Technology 44 (10), 63–69. Gontard, N., Guilbert, S., Cuq, J.L., 1993. Water and glycerol as plasticizers affect mechanical and water vapor barrier properties of an edible wheat gluten film. Journal of Food Science 58 (1), 206–211.

379

Gontard, N., Thibault, R., Cuq, B., Guilbert, S., 1996. Influence of relative humidity and film composition on oxygen and carbon dioxide permeabilities of edible films. Journal of Agricultural and Food Chemistry 44 (4), 1064–1069. Kristo, E., Biliaderis, C.G., Zampraka, A., 2007. Water vapor barrier and tensile properties of composite caseinate-pullulan films: biopolymer composition effects and impact of beeswax lamination. Food Chemistry 101 (2), 753–764. Lim, L.T., Mine, Y., Tung, M.A., 1999. Barrier and tensile properties of transglutaminase cross-linked gelatin films as affected by relative humidity, temperature and glycerol content. Journal of Food Science 64 (4), 616–620. Mauer, L.J., Smith, D.E., Labuza, T.P., 2000. Water vapor permeability, mechanical, and structural properties of edible b-casein films. International Dairy Journal 10 (5–6), 353–358. McHugh, T.H., Aujard, J.F., Krochta, J.M., 1994. Plasticized whey protein edible films: water vapor permeability properties. Journal of Food Science 59 (2), 416–419, 423. Mc Hugh, T.H., Avena-Bustillos, R., Krochta, J.M., 1993. Hydrophobic edible films: modified procedure for water vapor permeability and explanation of thickness effects. Journal of Food Science 58 (4), 899–903. Miller, K.S., Krochta, J.M., 1997. Oxygen and aroma barrier properties of edible films: a review. Trends in Food Science and Technology 8, 228–237. Monedero, F.M., Fabra, M.J., Talens, P., Chiralt, A., 2009. Effect of oleic acid–beeswax mixtures on mechanical, optical and barrier properties of soy protein isolate based film. Journal of Food Engineering 91, 509–515. Morillon, V., Debeaufort, F., Blond, G., Capelle, M., Voilley, A., 2002. Factors affecting the moisture permeability of lipid-based edible films: a review. Critical Review in Food Science and Nutrition 42 (1), 67–89. Mújica-Paz, H., Gontard, N., 1997. Oxygen and carbon dioxide permeability of wheat gluten film: effect of relative humidity and temperature. Journal of Agricultural and Food Chemistry 45, 4101–4105. Olivas, G.I., Barbosa-Cánovas, G.V., 2008. Alginate-calcium films: water vapor permeability and mechanical properties as affected by plasticizer and relative humidity. Lebensmittel-Wissenschaft und-Technologie 41, 359–366. Phan, T.D., Debeaufort, F., Voilley, A., Luu, D., 2009. Biopolymer interactions affect the functional properties of edible films based on agar, cassava starch and arabinolyxan blends. Journal of Food Engineering 90, 548–558. Pommet, M., Redl, A., Morel, M.H., Guilbert, S., 2003. Study of wheat gluten plastification with fatty acids. Polymer 44, 115–122. Roos, Y.H., 1995. Phase Transitions in Food. Academic Press, San Diego, CA. Srinivasa, P.C., Ramesh, M.N., Tharanathan, R.N., 2007. Effect of plasticizers and fatty acids on mechanical and permeability characteristics of chitosan films. Food Hydrocolloids 21, 1113–1122. Weiss, R.F., 1970. The solubility of nitrogen, oxygen and argon in water and seawater. Deep Sea Research and Oceanographic Abstracts 17 (4), 721–735.