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Production and characterization of sodium caseinate edible films made by blown-film extrusion
Journal of Food Engineering 121 (2014) 39–47
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Production and characterization of sodium caseinate edible films made by blown-film extrusion Imane Belyamani ⇑, Frederic Prochazka, Gilles Assezat Université de Lyon, F-42023 Saint Etienne, France CNRS, UMR 5223, Ingénierie des Matériaux Polymères, 42023 Saint Etienne, France Université de Saint Etienne, Jean Monnet, F-42023 Saint Etienne, France
a r t i c l e
i n f o
Article history: Received 3 May 2013 Received in revised form 3 August 2013 Accepted 9 August 2013 Available online 22 August 2013 Keywords: Sodium caseinate Edible film Food packaging Twin-screw extrusion Blown-film extrusion
a b s t r a c t Caseinates are considered as an interesting raw material for making biodegradable and water-soluble packaging. However, the development of plasticized caseinates, especially sodium caseinate, has mainly focused on the formation of films obtained by solution casting. This process, consisting in drying aqueous caseinate–plasticizer solutions spread on a hydrophobic plate, is not adapted to an industrial scale production. In the present study, a co-rotating twin-screw extruder was used to produce glycerol plasticized caseinate pellets. These transparent, homogenous, smooth pellets were transformed into thin films using a classical film blowing machine. Mechanical properties of the thermoplastic material have been measured as a function of glycerol content and environmental relative humidity and found to be greatly affected by glycerol and moisture levels. Water vapor permeability (WVP) of blown films was also investigated as a function of glycerol concentration, and it showed that the higher the glycerol content is, the lower the resistance to water transfer is. The obtained extruded sodium caseinate films are transparent, handleable and can even be edible. Several applications of these materials in food or non-food packaging could be developed. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Proteins are attractive for biodegradable materials field as they possess a wide range of chemical functionalities (Petersen et al., 1999). They are based on 20 amino acid monomers; each one has a different side group that lends to it unique character (Krochta, 2002). This results in many possibilities of bindings and structures, and in complex and varied properties (Rouilly and Rigal, 2002). These characteristics make proteins excellent candidates for the development of bio-based and biodegradable packaging in order to improve food product shelf life and food quality. Soy protein (Brother and McKinney, 1939; Brandenburg et al., 1993; Guerrero et al., 2010; Kunte et al., 1997; Kumar et al., 2002; Kurose et al., 2007; Kumar and Zhang, 2009; Lodha and Netravali, 2005; Otaigbe and Adams, 1997; Sue et al., 1997; Schilling et al., 1995; Wang et al., 1996; Zhang et al., 2001), wheat gluten (Anker et al., 1972; Fischer, 2004; Li and Lee, 1996; Micard et al., 2000; Redl et al., 1999a,b), corn zein (Ha and Padua, 2001; Lawton, 2004; Padua and Wang, 2002), collagen protein (Chirita, ⇑ Corresponding author. Address: School of Polymers and High Performance Materials, University of Southern Mississippi, 118 College Drive #5050, Hattiesburg, MS 39406, United States. Tel.: +1 601 310 2734. E-mail address: [email protected] (I. Belyamani). 0260-8774/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jfoodeng.2013.08.019
2008; Deiber et al., 2011; Goissis et al., 1999; Ho et al., 2001; Maser et al., 1991; Sionkowska, 2000) and gelatin protein (Achet and He, 1995; Bigi et al., 1998, 2000, 2001; Cutter, 2006) have been studied for many years to make edible and/or biodegradable films and they were reported to be innovative materials for packaging. Among these proteins, casein, the major protein component of milk, constitutes an interesting and a suitable raw material for making bioplastics. It represents 75–80% of all milk proteins and it is organized on a micellar structure which consists of a, b and j-casein as shown in Fig. 1 (Cayot and Lorient, 1998; Kinsella, 1984). Due to their low secondary structures (alpha-helix and betasheets), caseins are random coil polypeptides with a high degree of molecular flexibility and thus able to form typical intermolecular interactions (hydrogen, electrostatic and hydrophobic bonds) (Kinsella, 1984; Swaisgood, 1982). With these properties, caseins can easily form films without further treatment (Lacroix and Cooksey, 2005). Caseinates are produced by adjusting acid-coagulated casein to pH 6.7 using sodium, calcium or potassium hydroxide (Cayot and Lorient, 1998; Kinsella, 1984). Research on plasticized casein, especially sodium caseinate, has mainly focused on the formation of films obtained by solution casting. This process consists in drying aqueous caseinate–plasticizer solutions spread on a hydrophobic plate (Arrieta et al., 2013; Avena-Bustillos and Krochta, 1993; Audic and Chaufer, 2005, 2010;
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Fig. 1. Three-dimensional molecular model for the predominant caseins in cow milk: (a) as1-casein (Kumosinski et al., 1994), (b) as2-casein (Farrell et al., 2009), (c) b-casein (Kumosinski et al., 1993a) and (d) j-casein (Kumosinski et al., 1993b).
Barreto et al., 2003; Broumand et al., 2011; Chen, 2002; Gialamas et al., 2010; Khwaldia et al., 2004; Siew et al., 1999; Schou et al., 2005). Caseinates based films show hydrophilic properties due to their high amount of polar groups that provide excellent barriers to nonpolar substances such as oxygen, carbon dioxide and aromas (Audic et al., 2003). However, it was reported to be an ineffective moisture barrier, as expected for hydrophilic materials (Guilbert, 1986; Krochta et al., 1990). Plasticizers are added to enhance workability, elasticity and flexibility of the film. Plasticization occurs in the higher molecular-mobility amorphous region, and the ability of plasticizers to interrupt hydrogen bonding along the protein chains depends on the amount and type of the plasticizer (Sothornvit and Krochta, 2005). Siew et al. (1999) have reported that the plasticizer content has a large impact on film mechanical properties. They have shown that increasing glycerol content in the sodium caseinate films reduces their tensile strength and increases the elongation at break. As with other proteins films, glycerol is the plasticizer commonly used for caseinates based films. Audic and Chaufer (2005) and Siew et al. (1999) have demonstrated that sodium caseinate plasticized with glycerol has good mechanical properties as compared to those plasticized with polyethylene glycol (PEG) and other polyols. The solution-casting method is only adequate for laboratory use; it cannot be applied in a large scale production of films that could be used for packaging, food wraps or pouches. Therefore, more efficient techniques are needed for commercial film production. Extrusion process would be an interesting process in increasing the commercial potential of biodegradable films, offering several advantages over solution-casting. Extrusion can result in a highly efficient manufacturing method with commercial potential for large-scale production of edible films due to the low moisture levels, high temperatures, and short times used (HernandezIzquierdo and Krochta, 2008). It is a continuous process where the raw materials are continuously introduced into a hopper, conveyed by a screw and pushed through a die of a desired shape. This process involves several operations at the same time: melting,
mixing, kneading, stretching and conveying (Hernandez-Izquierdo et al., 2008). These characteristics often result in films with improved mechanical, barrier, and microstructural properties (Hernandez-Izquierdo and Krochta, 2008). Extruders have been used successfully in the production of protein based films, and were found to have a significant effect on material properties. For instance, Ha and Padua (2001) have reported that the higher pressure and the shearing force that can be developed in twin-screw extrusion increase the tensile strength of the extruded zein resins based sheets. On the other hand, Pommet et al. (2003) have demonstrated that the properties of extruded wheat gluten can be tuned for a wide range of applications by changing the thermoplastic processing conditions (temperature and/or shear) to obtain the desired structure. To date, sodium caseinate based edible film has been manufactured using solution-casting method only. Extrusion process would be a highly efficient method for the continuous shaping of thermoplastic plasticized caseinate. Therefore, using extrusion to produce sodium caseinate films is a real novelty that could present the opportunity to scale-up the transformation of caseinate biodegradable/edible materials, and to our knowledge, has never been performed before. The objectives of the present study are: (a) to demonstrate that sodium caseinate could be transformed by the processes used for synthetic plastics industry: twin-screw and blown film extrusion, and (b) characterize the thermoplastic caseinate materials and blown films in terms of mechanical properties and water vapor permeability.
2. Materials and methods 2.1. Materials Sodium caseinates were purchased from Brenntag, France. It contained more than 88% proteins (dry basis), approximately 5%
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moisture, 5% of ashes and wax, less than 0.15% lactose and 1.25% Na+. Glycerol was obtained from Sigma Aldrich, France. 2.2. Processing and characterization methods 2.2.1. Twin-screw extrusion The extrusion experiments were performed using a co-rotating twin-screw extruder from Clextral (Clextral BC 21, France) with 9 heating zones as shown in Fig. 2. Each zone was heated independently and cooled with recycled water. The interpenetrate screws had a length of 900 mm, a diameter of 25 mm and the distance between screw’s axe was 21 mm. A slit die of 4 mm diameter was used. The sodium caseinate powder was introduced into the 1st zone using a K-Tron volumetric feeder (Ktron soder, Switzerland) while the glycerol and water (the only additives) were delivered separately into the 2nd zone using a piston pump (PCM pumps, PP9, France). The addition of water was used to control the shear viscosity of the melt, and then control the engine torque of the extruder. Obviously, because water and glycerol are hydrophilic plasticizers, their addition to caseinate powder makes the total viscosity decrease. This allows the powder feeding rate to increase, which results in higher total extrusion rates. The screws were configured with conveying and kneading elements, and the barrel temperature along the screw ranged from 40 to 80 °C (Prochazka and Assezat, 2012). The setup was determined to denature the protein in order to increase the potential intermolecular interactions between the protein chain and the glycerol, and then increase the molecular flexibility. The obtained extrudates were pelletized after cooling. The glycerol rate was evaluated with the following equation:
%glycerol ¼
weightglycerol 100 weightglycerol þ weightcaseinate
ð1Þ
2.2.2. Blown-film extrusion A blow-film extruder (Diani, Italy) with five zones was used for the production of thin films. The extruder consists of three zones
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and was equipped with a small compression rate (2.5) screw with a diameter of 20 mm and a length-to-diameter ratio of 25. The screw speed was set to 45 rpm, and the barrel and the die were heated to 80 °C which was sufficient enough to melt the thermoplastic pellets. The molten protein was forced through the vertical annular die of spiral mandrel type. The tube was pulled upwards from the die by a pair of nip rolls placed 1 m above the die, and the thickness of the film was controlled by the speed of the nip rollers. The thickness of the finished films averaged 30 lm and was measured using an electronic gauge (Mahr Federal, Millitron, Germany) with incertitude of 3 lm. 2.2.3. Polyacrylamide gel electrophoresis (PAGE) PAGE was performed according to Laemmli (1970). The stacking gel contained 5% of acrylamide while the separation gel had 15% of acrylamide. Detected bands were identified using a prestained SDS–PAGE Standards Broad range with the following standard: Myosin (198,510), b-galactosidase (116,254), bovine serum albumin (84,796), ovalbumin (53,896), carbonic anhydrase (37,418), soybean trypsin inhibitor (29,051), lysosyme (19,809), aprotinin (6845). The gel was photographed after it had been stained with Coomassie Brilliant Blue R250 and then destained. Prior to the electrophoresis, soluble proteins (powder and pellets) were diluted to 2.15 mg/cm3 with the sample buffer (Tris HCl 0.5 M pH 6.8). Two types of diluted samples were prepared; with and without 2-mercaptoethanol. 2.2.4. Size exclusion chromatography (SEC) SEC experiments were carried out at room temperature in water (0.1 M NaNO3 + 50 ppm NaN3 as a bacteriostatic agent) using a Thermo Sep pump operating at a flow rate of 1 mL/min in combination with an automatic injector Gilson 234 (injection volume = 0.3 mL, sample concentration 0.2 g/L). The detection proceeded through a dual flow refractive index detector (RI171 from Shodex) and a UV detector (UV2000 from Spectra Physics) operating at 280 nm. Separation was achieved using a column set constituted of two SW2000 and SW4000 TSK columns efficient for the elution of the proteins.
Fig. 2. Co-rotating twin-screw extruder, Clextral BC 21, France. The obtained sodium caseinate pellets. The blown sodium caseinate film.
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2.2.5. Tensile tests Two different apparatus were available to measure Young modulus, stress and elongation at break. For standard H shaped specimens obtained from compression molding of thermoplastic caseinate pellets, a Dy.22 Universal test machine (Adamel Lhomargy) was used. The dimensions of the specimens are: 20 mm long, 4.50 mm wide and 2 mm thick. Samples were equilibrated at 30 °C with different Relative Humidities (RH = 40%, 50%, 60%, 70%, 80% and 90%) in an environmental test chamber for 48 h and tested just after conditioning. Ten replicates were tested for each sample. For films with a large heat transferring surface and very low thickness (30 ± 3 lm), the equilibrium state reached in the environmental test chamber could be corrupted while analyzing them. To overcome this difficulty, tensile measurements of blown films were made using a homemade tensile test machine, which allows the change in environmental conditions (RH and temperature) during the test. This measuring device is compact, with a sensitive force sensor, and measuring jaws for thin films were machined and their reliability tested. Tensile measurements on thin film, fixed on the force sensor jaws, were carried out using the designed machine after being equilibrated during 1 h at 60%RH and 30 °C. The measurements were made on small H shape specimens cut from the films, and the obtained values represent the average of five replicates. 2.2.6. Adsorption isotherms The water vapor adsorption isotherms were determined at 30 °C for extruded plasticized caseinate containing 17% and 25% glycerol compared to native sodium caseinate. Samples were equilibrated at 30 °C and at different water activity ranging from 0.15 to 0.9 in an environmental test chamber for at least 48 h. The equilibrium was assumed to be reached when the change in weight did not exceed 0.0002 g. Wet samples (Ww) were then dried in a vacuum drying oven, heated to 80 °C, for 24 h in order to obtain the dried weight (Wd). Equilibrium moisture content (Xeq) was calculated from weight loss of the samples using the following equation:
Xeq ¼
Ww Wd Wd
ð2Þ
The GAB equation was used to model the water adsorption isotherms data for water activities from 0 to 0.9 (0 to 90%HR), according to Khwaldia et al. (2004), Gennadios and Weller (1994) and Van Den Berg (1985).
the film thickness, A (m2) is the film area exposed to moisture transfer and Dp (Pa) is the vapor pressure differential across the film. Four replications of the flux determination were done. 3. Results and discussion The main goal of the present work is to produce thermoplastic plasticized material from sodium caseinate. The first step of the protein transformation corresponds to the unfolding of the macromolecular chains. In order to achieve this mechanism, extrusion was used to combine the effects of temperature, pressure and shearing. Breaking the intermolecular interactions and disulfide bonds influences the functional properties of the protein (Onwulata et al., 2011). To study the possible structural modifications of sodium caseinate during process, polyacrylamide gel electrophoresis (PAGE) was carried out. The gel electrophoresis pattern of native and extruded sodium caseinate is shown in Fig. 3. The PAGE profile of the soluble proteins (columns 1 and 2) and pellets (columns 3 and 4) contain 2 major bands which were identified as as1-casein and b-casein, and 4 minor bands identified as as2-casein, j-casein, 200 kDa and 95 kDa-bands. The 200 kDa and 95 kDa-bands correspond to the interactions between different sub-units of caseins, and they are characterized by a high molecular weight. The electrophoresis analysis indicates that these complexes existed in native proteins before being processed in the extruder, and completely disappeared after the use of 2-mercaptoethanol. Thus, it could be concluded that these bands are the result of the disulfide interactions. It is important to note that these interactions still exist after shearing and heating brought by extrusion. It is mainly the lack of secondary and tertiary structures that make casein heat-stable, and in the literature is almost designated as a naturally denatured protein (Swaisgood, 1982). However, the aspect of the different bands before and after the processing seems different. Because, in this case, electrophoresis is not quantitative, size exclusion chromatography (SEC) was used to better characterize the native and extruded caseinates. Fig. 4 shows the chromatograms of native powder, pellets and films of sodium caseinate. Because calibration of size exclusion chromatography for proteins is very difficult, a combination of PAGE and SEC has allowed the interpretation of the elution profiles. The peak at a high elution volume (small molecular weights) corresponds to the individual
2.2.7. Water vapor permeability (WVP) The water vapor permeability of the films was determined gravimetrically using ASTM E96-95 (ASTM, 1995) standard method, which consists in measuring mass variation of the permeation cells over time under constant temperature and RH. Before WVP measurements, all film samples were equilibrated at room temperature. The samples were then placed between the top of the glass cell and butyl ring, and the film area exposed to moisture transfer was found to be 3.2 cm2. Each cell contained distillated water that created a fix internal humidity of 100%. The cells were then kept in an environmental test chamber maintained at 30 °C and 60%RH, which creates a RH gradient on both sides of the film of 100– 60%. Weights were taken at time zero and every 5–13 h until five data points were collected. The WVP was determined using the following equation:
WVP ¼
Dm e Dt A Dp
ð3Þ
where Dm/Dt (g s1) is the slope of the moisture loss weightversus-time plot. It was calculated using linear regression, and the correlation coefficients for all reported data were >0.94. e (mm) is
Fig. 3. The SDS–PAGE pattern of native sodium caseinate and pellets (1) native sodium caseinate without 2-mercaptoethanol, (2) native sodium caseinate with 2mercaptoethanol, (3) pellets without 2-mercaptoethanol and (4) pellets with 2mercaptoethanol.
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Fig. 4. SEC elution profiles of sodium caseinates: native (— powder), extruded (- - - pellets) and ( film). Fig. 5. Young modulus and stress at break (frame) of extruded sodium caseinate as a function of moisture and glycerol contents (j 17% Gly. 25% Gly. 33% Gly.).
casein subunits as1, as2, b and j. This peak is present for the three samples and it is much higher for the extruded pellets and films; meaning that most of the disulfide aggregates present in the native caseinate have been destroyed during the extrusion process to give the casein subunits. The two distinctive peaks at 12 and 18 mL correspond to the disulfide bridge aggregates with higher molecular weights (95 kDa and 200 kDa). They are smaller for the extruded materials than for the native protein, confirming that the structural rearrangements of casein complex probably occurred during extrusion process. It is worth mentioning that the peak below 10 mL corresponds to the excluded volume of the columns. Although it appears that the structure of the caseinates was modified by the extrusion process, mainly by the destruction of some disulfide bridge aggregates, it will be shown that the mechanical properties of the films are not affected by these modifications. A review of the literature reveals that films obtained by casting and drying aqueous caseinate–plasticizer solutions have great mechanical properties. Nevertheless, as reported by HernandezIzquierdo and Krochta (2008), the properties of protein-based materials depend on the raw materials as well as the processing and the testing conditions. In the present study, two kinds of samples were used for the mechanical properties characterization. In order to fully characterize the caseinate materials, tensile strength tests were carried out on standard H shaped specimens made from compression molding of plasticized pellets. Samples were equilibrated at different RH in an environmental test chamber, heated to 30 °C, for 48 h and tested just after conditioning. Young’s modulus (YM), stress at break (Sb) (Fig. 5) and elongation at break (Eb) (Fig. 6) have been measured as a function of relative humidity for three different glycerol contents. As can be seen, raising the glycerol concentration decreases the Young’s modulus and stress at break, while it has an increasing effect on percentage of elongation at break starting from certain glycerol content. For example, the elastic modulus of material with 33% of glycerol (120 MPa) is three times weaker than that with only 17% of glycerol (350 MPa) for similar surrounding conditions (50%RH, 30 °C). As a plasticizer, glycerol reduces the interaction between protein molecules and then raises intermolecular spacing which facilitates the mobility of protein chains. The plasticizer acts as a lubricant to
Fig. 6. Elongation at break of extruded sodium caseinate as a function of moisture and glycerol contents (j 17% Gly. 25% Gly. 33% Gly.).
facilitate the movement of the protein chains over other, thus avoiding brittleness and raising their lengthening. As shown in Figs. 5 and 6, the mechanical properties of caseinate material are greatly affected by relative humidity. With the increase in moisture content, the elastic modulus and stress at break quickly decrease, whereas the elongation at break greatly increases, particularly at low glycerol content; The material goes from a glassy state at 40%RH (YM = 270 MPa and Sb = 14 MPa) to a rubbery state at 90%RH (YM = 2 MPa and Sb = 1 MPa). The hydrophilic nature of the plasticized caseinate makes the material very sensitive to water, which acts as a plasticizer.
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Once again, because the process used for the production of thin films may change the films’ mechanical properties, the mechanical properties of films obtained with blow film extrusion were also tested. Films were produced from thermoplastic pellets plasticized with 20% of glycerol, using a blown-film extruder (described in Section 2). This glycerol content was chosen to obtain a compromise between elastic modulus and elongation at break. The obtained films were homogenous, transparent and had a good appearance. The effect of moisture on mechanical properties of sodium caseinate based materials justifies the importance of controlling the ambient humidity and temperature during the analyses, particularly in the case of films with a very low thickness (30 ± 3 lm). In fact, for these films, the equilibrium reached within the environmental test chamber could be corrupted during their analysis. The ideal is to be able to perform these measurements under controlled moisture and temperature. To suit these requirements, equipment was designed in the laboratory, as described in the experimental section. The tensile strength measurements were carried out on thin films, using the designed machine, after being equilibrated during 1 h at 60%RH and 30 °C. Values of elastic modulus, stress at break (represented as an empty diagram in Fig. 5) and elongation at break (YM = 103 ± 5 MPa, Sb = 5.0 ± 0.3 MPa and Eb = 110 ± 10%) were determined from the obtained stress–strain curve. The mechanical properties of thin films (30 ± 3 lm) are similar to those obtained with standard tensile specimens much thicker (2 mm), as shown in Figs. 5 and 6. This result shows that although the sodium caseinate based material underwent two transits to the thermal and shear treatments (twin-screw and blow-film extruders), its tensile properties do not change. It has to be noted that the SEC elution diagram (Fig. 4) gives a very similar distribution of the molecular weight for the films when compared to the pellets. The evolution of the tensile properties of caseinate based materials as a function of moisture content has been explained using native and plasticized sodium caseinate moisture adsorption isotherms (Fig. 7).
These curves describe the relationship between moisture content (Xeq) of hydrophilic materials at equilibrium with the surrounding water activity at a fixed temperature. They are of importance for hydrophilic biopolymers as they are water sensitive, and the water content could strongly influence their functionality (Yang and Paulson, 2000). As presented in Fig. 7, the plasticized sodium caseinate is far more sensitive to relative humidity than the native caseinate. The hygroscopic character of glycerol increased the water absorption of caseinate material. Moreover, the sensitivity to water activity appeared to be slightly more marked for high plasticizer content (25% Glycerol). At a water activity of 0.9, the plasticized material absorbed eight times more water (0.8 gwater/g db) than at a water activity of 0.4 (0.1 gwater/g db). The obtained isotherms curves were also used to plot the tensile properties as a function of plasticized sodium caseinate water content; knowing the RH, it becomes possible to determine the water content of the sample (Fig. 8). Fig. 8 confirms the role of glycerol which acts as a plasticizer, but more importantly the representation emphasises that water has a stronger effect as a plasticizer. In fact, at fixed water content, the elastic modulus decreased, whereas increasing water content has the same effect on mechanical properties regardless of the glycerol content. A good way to simultaneously see the influence of glycerol and water, is to represent Young modulus as a function of elongation at break for different glycerol contents (17%, 25% and 33%) with different RH (40–90% every 10%RH) (Fig. 9). The plasticizing effect of water is so important that it is the relative humidity of the sample environment which governs the mechanical properties. Regarding the strong influence of the environmental conditions on the water content of the films, it is very difficult to compare the obtained results with those reported by other research teams. This is due, in part, to differences in glycerol content, water content, caseinate sources or testing conditions. However, the values of Young Modulus, stress and strain at break presented in the present study are similar to those obtained by Audic and Chaufer (2005) or more recently by Arrieta et al. (2013). These results suggest that using extrusion to produce pellets or blown films do not affect
Fig. 7. Moisture adsorption isotherms of plasticized (s 17% Glycerol and 4 25% Glycerol) and (h native) sodium caseinate at 30 °C.
Fig. 8. Young modulus as a function of water and glycerol contents of extruded sodium caseinate (4 25% Glycerol, s 17% Glycerol and j blown film).
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Fig. 9. Young modulus as a function of elongation at break (D 33% Glycerol, s 25% Glycerol, h 17% Glycerol and d blown film). RH between 40% and 90% every 10%.
Fig. 10. Water vapor permeability (WVP) of blown films made from sodium caseinate as a function of glycerol content.
the final mechanical properties and could allow industrial scale production of caseinate films. For the same reason, i.e. water content of the material, glass transition temperature is also very difficult to measure; increasing the temperature ramp used in Differential Scanning calorimetry (DSC) or in solid state rheology (DMA), evaporates the water within the sample, which results in higher glass transition temperatures. Because of this, the glass transition temperature of the extruded sodium caseinate was estimated theoretically by using the Gordon-Taylor equation. The glass transition temperature of a sample with 20% glycerol in a 60% RH environment, estimated by Gordon-Taylor equation, is around 50 °C; changing the water content makes this glass transition temperature moving across the room temperature. The mechanical properties can then fit those of a glassy polymer in a dry environment, or those of a rubber in a very humid atmosphere.
which increased with plasticizer content resulting in higher water diffusion through the film. The values of WVP found in the literature for sodium caseinate films containing various glycerol contents are listed in Table 1. These values were compared to those measured in our laboratory for sodium caseinate blown film at 100–60% RH differential and 30 °C. These edible films were compared with those of synthetic polymer. Table 1 shows that whatever the glycerol content is, edible films are poor water vapor barriers than those of synthetic polymer. The hydrophilic nature of films, made from sodium caseinate proteins, induced interaction with water molecules. To that is added the increase in inter-chain spacing caused by inclusion of glycerol molecules between the polypeptides chains, which enhance the water vapor transfer through the film. The highly hygroscopic character of glycerol molecules, which is favourable to the absorption of water molecules, could also decrease the resistance to water vapor transmission. The higher value of WVP obtained in the present study, compared to those of the literature, could be explained by some differences in test conditions: formulation or RH gradient. Furthers measurements are under process to clarify these results.
3.1. Water vapor permeability (WVP) of blown film Moisture transfer is an important factor leading to changes in food quality during storage, because microbial growth in food proliferates when water activity is high. It was demonstrated that the content of plasticizer has an effect on mechanical properties and it is expected to have an influence on WVP as well. The results of WVP of sodium caseinate films at different content of glycerol and tested at 100–60% RH differential and 30 °C, are shown in Fig. 10. Fig. 10 shows a classical result; the higher the glycerol content, the higher WVP is. The water diffusion through the film increased from 1.15 g mm/m2 h kPa to 4.50 g mm/m2 h kPa when only 13% of glycerol is added. This could be explained by the plasticizing effect of water favoured by the glycerol content, or simply by the high content of plasticizer. This agrees with the investigations of Coupland et al. (2000), who noticed that the consequences of plasticizing action of glycerol are favourable to adsorption and absorption of water molecules by the films, so WVP will be substantially increased. In fact, plasticizing the protein matrix increased the polymer free volume and then the polymeric chain segments mobility,
Table 1 Comparison of WVP values of various edible films and synthetic films. Film
Tests conditions (temperature °C; RH gradient %)
WVP (g mm/ m2 h kPa)
Extruded NaCAS/Ga (4:1) NaCAS/G (4:1) Khwaldia et al. (2004) NaCAS/G (1.67:1) Siew et al. (1999) NaCAS/G (0.89:1) Siew et al. (1999) LDPE Shellhammer and Krochta (1997) LDPEa PVDC Shellhammer and Krochta (1997)
30 ± 1; 100/60 ± 2% 20; 0/90
1.91 ± 0.03 0.303 ± 0.001
20; 0/45
0.225
20; 0/45
0.472
27.6; 0/100
0.0013
30 ± 2; 100/60 ± 3% 24.9; 0/100
0.0051 0.0008
a The present study; NaCAS: sodium caseinate; G: glycerol; RH gradients: inside/ outside; LDPE: low-density polyethylene; PVDC: polyvinylidene chloride.
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4. Conclusion The present study clearly shows that by using twin screw extrusion, it is possible to produce thermoplastic pellets from sodium caseinate and plasticizer such as glycerol. These pellets can be used with the classical transforming plastic technics like injection, compression molding, extrusion and most particularly blowing extrusion to make films. These thin films are transparent, smooth and with no apparent surface defects or irregularities, and are also water-soluble and edible. The mechanical properties are similar to those obtained by the solution casting method and close to those of the proteins or polysaccharides based films. This material is also very sensitive to environmental moisture content and can behave like a solid material or an elastomeric one. The thermoplastic processing of sodium caseinate for film formation has promising potential for large-scale production of new biodegradable packaging. Such films could be used for several food or non-food applications. References Achet, D., He, X.W., 1995. Determination of the renaturation level in gelatin films. Polymer 36, 787–79. Anker, C.A., Foster, G.A., Loader, M.A., 1972. Method of preparing gluten containing films and coatings. US Patent 3, 653, 925. Arrieta, M.P., Peltzer, M.A., Garrigos, M.C., Jiménez, A., 2013. Structure and mechanical properties of sodium and calcium caseinate edible active films with carvacrol. J. Food Eng. 114, 486–494. Audic, J.L., Chaufer, B., Daufin, G., 2003. Non-food applications of milk components and dairy co-products: a review. Lait 83, 417–438. Audic, J.L., Chaufer, B., 2005. Influence of plasticizers and crosslinking on the properties of biodegradable films made from sodium caseinate. Eur. Polym. J. 41, 1934–1942. Audic, J.L., Chaufer, B., 2010. Caseinate based biodegradable films with improved water resistance. J. Appl. Polym. Sci. 117, 1828–1836. Avena-Bustillos, R.J., Krochta, J.M., 1993. Water vapor permeability of caseinatebased edible films as affected by pH, calcium crosslinking and lipid content. J. Food Sci. 58, 904–907. Barreto, P.L.M., Roeder, J., Crespo, J.S., Maciel, G.R., Terenzi, H., Pires, A.T.N., Soldi, V., 2003. Effect of concentration, temperature and plasticizer content on rheological properties of sodium caseinate and sodium caseinate/sorbitol solutions and glass transition of their films. Food Chem. 82, 425–431. Bigi, A., Bracci, B., Cojazzi, G., Panzavolta, S., Roveri, N., 1998. Drawn gelatin films with improved mechanical properties. Biomaterials 19, 2335–2340. Bigi, A., Borghi, M., Cojazzi, G., Fichera, A.M., Panzavolta, S., Roveri, N., 2000. Structural and mechanical properties of crosslinked drawn gelatin films. J. Therm. Anal. Calorim. 61, 451–459. Bigi, A., Cojazzi, G., Panzavolta, S., Rubini, K., Roveri, N., 2001. Mechanical and thermal properties of gelatin films at different degrees of glutaraldehyde crosslinking. Biomaterials 22, 763–768. Brandenburg, A.H., Weller, C.L., Testin, R.F., 1993. Edible films and coatings from soy protein. J. Food Sci. 58, 1086–1089. Broumand, A., Emam-Djomeh, Z., Hamedi, M., Razavi, S.H., 2011. Antimicrobial, water vapour permeability, mechanical and thermal properties of casein based Zataraia multiflora Boiss. Extract containing film. LWT-Food Sci. Technol. 44, 2316–2323. Brother, G.H., McKinney, L.L., 1939. Protein plastics from soybean products. Ind. Eng. Chem. 31, 84–87. Cayot, P., Lorient, D., 1998. Structures et Technofonctions des Protéines du Lait. Tec & Doc, Lavoisier, Paris. Chen, H., 2002. Formation and properties of casein films and coating. In: Gennadios, A. (Ed.), Protein-Based Films and Coatings. CRC Press, Florida, pp. 181–211. Chirita, M., 2008. Mechanical properties of collagen biomimetic films formed in the presence of calcium, silica and chitosan. J. Bionic Eng. 5, 149–158. Coupland, J.N., Shaw, N.B., Monahan, F.J., ÓRiordan, E.D., ÓSullivan, M., 2000. Modeling the effect of glycerol on the moisture sorption behavior of whey protein edible films. J. Food Eng. 43, 25–30. Cutter, C.N., 2006. Opportunities for bio-based packaging technologies to improve the quality and safety of fresh and further processed muscle foods. Meat Sci. 74, 131–142. Deiber, J.A., Peirotti, M.B., Ottone, M.L., 2011. Rheological characterization of edible films made from collagen colloidal particle suspensions. Food Hydrocolloids 25, 1–11. Farrell Jr., H.M., Malin, E.L., Brown, E.M., Mora-Gutierrez, A., 2009. Review of the chemistry of as2-casein and the generation of a homologous molecular model to explain its properties. J. Dairy Sci. 92, 1338–1353. Fischer, T., 2004. Effect of extrusion cooking on protein modification in wheat flour. Eur. Food Res. Technol. 218, 128–132. Gennadios, A., Weller, C.L., 1994. Moisture adsorption by grain protein films. Trans. ASAE 37, 535–539.
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Three-dimensional molecular modeling of bovine caseins: a refined, energy-minimized j-casein. J. Dairy Sci. 76, 2507–2520. Kumosinski, T.F., Brown, E.M., Farrell, H.M. Jr., 1994. Predicted energy-minimized as1-casein working model. In: Kumosinski, T.F., Liebman, M.N., (Eds.), Molecular Modeling: From Virtual Tools to Real Problems. Washing to: American Chemical Society Symposium. Kunte, L.A., Gennadios, A., Cuppett, S.L., Hanna, M.A., Weller, C.L., 1997. Cast films from soy protein isolates and fractions. Cereal Chem. 74, 115–118. Kurose, T., Urman, K., Otaigbe, J.U., Lochhead, R.Y., Thames, S.F., 2007. Effect of uniaxial drawing of soy protein isolate biopolymer film on structure and mechanical properties. Polym. Eng. Sci. 47, 374–380. Lacroix, M., Cooksey, K., 2005. Edible films and coatings from animal-origin proteins. In: Han, J.H. (Ed.), Innovations in Food Packaging. Elsevier Academic Press, London. Laemmli, U.K., 1970. 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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Production and characterization of sodium caseinate edible films made by blown-film extrusion Imane Belyamani ⇑, Frederic Prochazka, Gilles Assezat Université de Lyon, F-42023 Saint Etienne, France CNRS, UMR 5223, Ingénierie des Matériaux Polymères, 42023 Saint Etienne, France Université de Saint Etienne, Jean Monnet, F-42023 Saint Etienne, France
a r t i c l e
i n f o
Article history: Received 3 May 2013 Received in revised form 3 August 2013 Accepted 9 August 2013 Available online 22 August 2013 Keywords: Sodium caseinate Edible film Food packaging Twin-screw extrusion Blown-film extrusion
a b s t r a c t Caseinates are considered as an interesting raw material for making biodegradable and water-soluble packaging. However, the development of plasticized caseinates, especially sodium caseinate, has mainly focused on the formation of films obtained by solution casting. This process, consisting in drying aqueous caseinate–plasticizer solutions spread on a hydrophobic plate, is not adapted to an industrial scale production. In the present study, a co-rotating twin-screw extruder was used to produce glycerol plasticized caseinate pellets. These transparent, homogenous, smooth pellets were transformed into thin films using a classical film blowing machine. Mechanical properties of the thermoplastic material have been measured as a function of glycerol content and environmental relative humidity and found to be greatly affected by glycerol and moisture levels. Water vapor permeability (WVP) of blown films was also investigated as a function of glycerol concentration, and it showed that the higher the glycerol content is, the lower the resistance to water transfer is. The obtained extruded sodium caseinate films are transparent, handleable and can even be edible. Several applications of these materials in food or non-food packaging could be developed. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Proteins are attractive for biodegradable materials field as they possess a wide range of chemical functionalities (Petersen et al., 1999). They are based on 20 amino acid monomers; each one has a different side group that lends to it unique character (Krochta, 2002). This results in many possibilities of bindings and structures, and in complex and varied properties (Rouilly and Rigal, 2002). These characteristics make proteins excellent candidates for the development of bio-based and biodegradable packaging in order to improve food product shelf life and food quality. Soy protein (Brother and McKinney, 1939; Brandenburg et al., 1993; Guerrero et al., 2010; Kunte et al., 1997; Kumar et al., 2002; Kurose et al., 2007; Kumar and Zhang, 2009; Lodha and Netravali, 2005; Otaigbe and Adams, 1997; Sue et al., 1997; Schilling et al., 1995; Wang et al., 1996; Zhang et al., 2001), wheat gluten (Anker et al., 1972; Fischer, 2004; Li and Lee, 1996; Micard et al., 2000; Redl et al., 1999a,b), corn zein (Ha and Padua, 2001; Lawton, 2004; Padua and Wang, 2002), collagen protein (Chirita, ⇑ Corresponding author. Address: School of Polymers and High Performance Materials, University of Southern Mississippi, 118 College Drive #5050, Hattiesburg, MS 39406, United States. Tel.: +1 601 310 2734. E-mail address: [email protected] (I. Belyamani). 0260-8774/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jfoodeng.2013.08.019
2008; Deiber et al., 2011; Goissis et al., 1999; Ho et al., 2001; Maser et al., 1991; Sionkowska, 2000) and gelatin protein (Achet and He, 1995; Bigi et al., 1998, 2000, 2001; Cutter, 2006) have been studied for many years to make edible and/or biodegradable films and they were reported to be innovative materials for packaging. Among these proteins, casein, the major protein component of milk, constitutes an interesting and a suitable raw material for making bioplastics. It represents 75–80% of all milk proteins and it is organized on a micellar structure which consists of a, b and j-casein as shown in Fig. 1 (Cayot and Lorient, 1998; Kinsella, 1984). Due to their low secondary structures (alpha-helix and betasheets), caseins are random coil polypeptides with a high degree of molecular flexibility and thus able to form typical intermolecular interactions (hydrogen, electrostatic and hydrophobic bonds) (Kinsella, 1984; Swaisgood, 1982). With these properties, caseins can easily form films without further treatment (Lacroix and Cooksey, 2005). Caseinates are produced by adjusting acid-coagulated casein to pH 6.7 using sodium, calcium or potassium hydroxide (Cayot and Lorient, 1998; Kinsella, 1984). Research on plasticized casein, especially sodium caseinate, has mainly focused on the formation of films obtained by solution casting. This process consists in drying aqueous caseinate–plasticizer solutions spread on a hydrophobic plate (Arrieta et al., 2013; Avena-Bustillos and Krochta, 1993; Audic and Chaufer, 2005, 2010;
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Fig. 1. Three-dimensional molecular model for the predominant caseins in cow milk: (a) as1-casein (Kumosinski et al., 1994), (b) as2-casein (Farrell et al., 2009), (c) b-casein (Kumosinski et al., 1993a) and (d) j-casein (Kumosinski et al., 1993b).
Barreto et al., 2003; Broumand et al., 2011; Chen, 2002; Gialamas et al., 2010; Khwaldia et al., 2004; Siew et al., 1999; Schou et al., 2005). Caseinates based films show hydrophilic properties due to their high amount of polar groups that provide excellent barriers to nonpolar substances such as oxygen, carbon dioxide and aromas (Audic et al., 2003). However, it was reported to be an ineffective moisture barrier, as expected for hydrophilic materials (Guilbert, 1986; Krochta et al., 1990). Plasticizers are added to enhance workability, elasticity and flexibility of the film. Plasticization occurs in the higher molecular-mobility amorphous region, and the ability of plasticizers to interrupt hydrogen bonding along the protein chains depends on the amount and type of the plasticizer (Sothornvit and Krochta, 2005). Siew et al. (1999) have reported that the plasticizer content has a large impact on film mechanical properties. They have shown that increasing glycerol content in the sodium caseinate films reduces their tensile strength and increases the elongation at break. As with other proteins films, glycerol is the plasticizer commonly used for caseinates based films. Audic and Chaufer (2005) and Siew et al. (1999) have demonstrated that sodium caseinate plasticized with glycerol has good mechanical properties as compared to those plasticized with polyethylene glycol (PEG) and other polyols. The solution-casting method is only adequate for laboratory use; it cannot be applied in a large scale production of films that could be used for packaging, food wraps or pouches. Therefore, more efficient techniques are needed for commercial film production. Extrusion process would be an interesting process in increasing the commercial potential of biodegradable films, offering several advantages over solution-casting. Extrusion can result in a highly efficient manufacturing method with commercial potential for large-scale production of edible films due to the low moisture levels, high temperatures, and short times used (HernandezIzquierdo and Krochta, 2008). It is a continuous process where the raw materials are continuously introduced into a hopper, conveyed by a screw and pushed through a die of a desired shape. This process involves several operations at the same time: melting,
mixing, kneading, stretching and conveying (Hernandez-Izquierdo et al., 2008). These characteristics often result in films with improved mechanical, barrier, and microstructural properties (Hernandez-Izquierdo and Krochta, 2008). Extruders have been used successfully in the production of protein based films, and were found to have a significant effect on material properties. For instance, Ha and Padua (2001) have reported that the higher pressure and the shearing force that can be developed in twin-screw extrusion increase the tensile strength of the extruded zein resins based sheets. On the other hand, Pommet et al. (2003) have demonstrated that the properties of extruded wheat gluten can be tuned for a wide range of applications by changing the thermoplastic processing conditions (temperature and/or shear) to obtain the desired structure. To date, sodium caseinate based edible film has been manufactured using solution-casting method only. Extrusion process would be a highly efficient method for the continuous shaping of thermoplastic plasticized caseinate. Therefore, using extrusion to produce sodium caseinate films is a real novelty that could present the opportunity to scale-up the transformation of caseinate biodegradable/edible materials, and to our knowledge, has never been performed before. The objectives of the present study are: (a) to demonstrate that sodium caseinate could be transformed by the processes used for synthetic plastics industry: twin-screw and blown film extrusion, and (b) characterize the thermoplastic caseinate materials and blown films in terms of mechanical properties and water vapor permeability.
2. Materials and methods 2.1. Materials Sodium caseinates were purchased from Brenntag, France. It contained more than 88% proteins (dry basis), approximately 5%
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moisture, 5% of ashes and wax, less than 0.15% lactose and 1.25% Na+. Glycerol was obtained from Sigma Aldrich, France. 2.2. Processing and characterization methods 2.2.1. Twin-screw extrusion The extrusion experiments were performed using a co-rotating twin-screw extruder from Clextral (Clextral BC 21, France) with 9 heating zones as shown in Fig. 2. Each zone was heated independently and cooled with recycled water. The interpenetrate screws had a length of 900 mm, a diameter of 25 mm and the distance between screw’s axe was 21 mm. A slit die of 4 mm diameter was used. The sodium caseinate powder was introduced into the 1st zone using a K-Tron volumetric feeder (Ktron soder, Switzerland) while the glycerol and water (the only additives) were delivered separately into the 2nd zone using a piston pump (PCM pumps, PP9, France). The addition of water was used to control the shear viscosity of the melt, and then control the engine torque of the extruder. Obviously, because water and glycerol are hydrophilic plasticizers, their addition to caseinate powder makes the total viscosity decrease. This allows the powder feeding rate to increase, which results in higher total extrusion rates. The screws were configured with conveying and kneading elements, and the barrel temperature along the screw ranged from 40 to 80 °C (Prochazka and Assezat, 2012). The setup was determined to denature the protein in order to increase the potential intermolecular interactions between the protein chain and the glycerol, and then increase the molecular flexibility. The obtained extrudates were pelletized after cooling. The glycerol rate was evaluated with the following equation:
%glycerol ¼
weightglycerol 100 weightglycerol þ weightcaseinate
ð1Þ
2.2.2. Blown-film extrusion A blow-film extruder (Diani, Italy) with five zones was used for the production of thin films. The extruder consists of three zones
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and was equipped with a small compression rate (2.5) screw with a diameter of 20 mm and a length-to-diameter ratio of 25. The screw speed was set to 45 rpm, and the barrel and the die were heated to 80 °C which was sufficient enough to melt the thermoplastic pellets. The molten protein was forced through the vertical annular die of spiral mandrel type. The tube was pulled upwards from the die by a pair of nip rolls placed 1 m above the die, and the thickness of the film was controlled by the speed of the nip rollers. The thickness of the finished films averaged 30 lm and was measured using an electronic gauge (Mahr Federal, Millitron, Germany) with incertitude of 3 lm. 2.2.3. Polyacrylamide gel electrophoresis (PAGE) PAGE was performed according to Laemmli (1970). The stacking gel contained 5% of acrylamide while the separation gel had 15% of acrylamide. Detected bands were identified using a prestained SDS–PAGE Standards Broad range with the following standard: Myosin (198,510), b-galactosidase (116,254), bovine serum albumin (84,796), ovalbumin (53,896), carbonic anhydrase (37,418), soybean trypsin inhibitor (29,051), lysosyme (19,809), aprotinin (6845). The gel was photographed after it had been stained with Coomassie Brilliant Blue R250 and then destained. Prior to the electrophoresis, soluble proteins (powder and pellets) were diluted to 2.15 mg/cm3 with the sample buffer (Tris HCl 0.5 M pH 6.8). Two types of diluted samples were prepared; with and without 2-mercaptoethanol. 2.2.4. Size exclusion chromatography (SEC) SEC experiments were carried out at room temperature in water (0.1 M NaNO3 + 50 ppm NaN3 as a bacteriostatic agent) using a Thermo Sep pump operating at a flow rate of 1 mL/min in combination with an automatic injector Gilson 234 (injection volume = 0.3 mL, sample concentration 0.2 g/L). The detection proceeded through a dual flow refractive index detector (RI171 from Shodex) and a UV detector (UV2000 from Spectra Physics) operating at 280 nm. Separation was achieved using a column set constituted of two SW2000 and SW4000 TSK columns efficient for the elution of the proteins.
Fig. 2. Co-rotating twin-screw extruder, Clextral BC 21, France. The obtained sodium caseinate pellets. The blown sodium caseinate film.
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2.2.5. Tensile tests Two different apparatus were available to measure Young modulus, stress and elongation at break. For standard H shaped specimens obtained from compression molding of thermoplastic caseinate pellets, a Dy.22 Universal test machine (Adamel Lhomargy) was used. The dimensions of the specimens are: 20 mm long, 4.50 mm wide and 2 mm thick. Samples were equilibrated at 30 °C with different Relative Humidities (RH = 40%, 50%, 60%, 70%, 80% and 90%) in an environmental test chamber for 48 h and tested just after conditioning. Ten replicates were tested for each sample. For films with a large heat transferring surface and very low thickness (30 ± 3 lm), the equilibrium state reached in the environmental test chamber could be corrupted while analyzing them. To overcome this difficulty, tensile measurements of blown films were made using a homemade tensile test machine, which allows the change in environmental conditions (RH and temperature) during the test. This measuring device is compact, with a sensitive force sensor, and measuring jaws for thin films were machined and their reliability tested. Tensile measurements on thin film, fixed on the force sensor jaws, were carried out using the designed machine after being equilibrated during 1 h at 60%RH and 30 °C. The measurements were made on small H shape specimens cut from the films, and the obtained values represent the average of five replicates. 2.2.6. Adsorption isotherms The water vapor adsorption isotherms were determined at 30 °C for extruded plasticized caseinate containing 17% and 25% glycerol compared to native sodium caseinate. Samples were equilibrated at 30 °C and at different water activity ranging from 0.15 to 0.9 in an environmental test chamber for at least 48 h. The equilibrium was assumed to be reached when the change in weight did not exceed 0.0002 g. Wet samples (Ww) were then dried in a vacuum drying oven, heated to 80 °C, for 24 h in order to obtain the dried weight (Wd). Equilibrium moisture content (Xeq) was calculated from weight loss of the samples using the following equation:
Xeq ¼
Ww Wd Wd
ð2Þ
The GAB equation was used to model the water adsorption isotherms data for water activities from 0 to 0.9 (0 to 90%HR), according to Khwaldia et al. (2004), Gennadios and Weller (1994) and Van Den Berg (1985).
the film thickness, A (m2) is the film area exposed to moisture transfer and Dp (Pa) is the vapor pressure differential across the film. Four replications of the flux determination were done. 3. Results and discussion The main goal of the present work is to produce thermoplastic plasticized material from sodium caseinate. The first step of the protein transformation corresponds to the unfolding of the macromolecular chains. In order to achieve this mechanism, extrusion was used to combine the effects of temperature, pressure and shearing. Breaking the intermolecular interactions and disulfide bonds influences the functional properties of the protein (Onwulata et al., 2011). To study the possible structural modifications of sodium caseinate during process, polyacrylamide gel electrophoresis (PAGE) was carried out. The gel electrophoresis pattern of native and extruded sodium caseinate is shown in Fig. 3. The PAGE profile of the soluble proteins (columns 1 and 2) and pellets (columns 3 and 4) contain 2 major bands which were identified as as1-casein and b-casein, and 4 minor bands identified as as2-casein, j-casein, 200 kDa and 95 kDa-bands. The 200 kDa and 95 kDa-bands correspond to the interactions between different sub-units of caseins, and they are characterized by a high molecular weight. The electrophoresis analysis indicates that these complexes existed in native proteins before being processed in the extruder, and completely disappeared after the use of 2-mercaptoethanol. Thus, it could be concluded that these bands are the result of the disulfide interactions. It is important to note that these interactions still exist after shearing and heating brought by extrusion. It is mainly the lack of secondary and tertiary structures that make casein heat-stable, and in the literature is almost designated as a naturally denatured protein (Swaisgood, 1982). However, the aspect of the different bands before and after the processing seems different. Because, in this case, electrophoresis is not quantitative, size exclusion chromatography (SEC) was used to better characterize the native and extruded caseinates. Fig. 4 shows the chromatograms of native powder, pellets and films of sodium caseinate. Because calibration of size exclusion chromatography for proteins is very difficult, a combination of PAGE and SEC has allowed the interpretation of the elution profiles. The peak at a high elution volume (small molecular weights) corresponds to the individual
2.2.7. Water vapor permeability (WVP) The water vapor permeability of the films was determined gravimetrically using ASTM E96-95 (ASTM, 1995) standard method, which consists in measuring mass variation of the permeation cells over time under constant temperature and RH. Before WVP measurements, all film samples were equilibrated at room temperature. The samples were then placed between the top of the glass cell and butyl ring, and the film area exposed to moisture transfer was found to be 3.2 cm2. Each cell contained distillated water that created a fix internal humidity of 100%. The cells were then kept in an environmental test chamber maintained at 30 °C and 60%RH, which creates a RH gradient on both sides of the film of 100– 60%. Weights were taken at time zero and every 5–13 h until five data points were collected. The WVP was determined using the following equation:
WVP ¼
Dm e Dt A Dp
ð3Þ
where Dm/Dt (g s1) is the slope of the moisture loss weightversus-time plot. It was calculated using linear regression, and the correlation coefficients for all reported data were >0.94. e (mm) is
Fig. 3. The SDS–PAGE pattern of native sodium caseinate and pellets (1) native sodium caseinate without 2-mercaptoethanol, (2) native sodium caseinate with 2mercaptoethanol, (3) pellets without 2-mercaptoethanol and (4) pellets with 2mercaptoethanol.
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Fig. 4. SEC elution profiles of sodium caseinates: native (— powder), extruded (- - - pellets) and ( film). Fig. 5. Young modulus and stress at break (frame) of extruded sodium caseinate as a function of moisture and glycerol contents (j 17% Gly. 25% Gly. 33% Gly.).
casein subunits as1, as2, b and j. This peak is present for the three samples and it is much higher for the extruded pellets and films; meaning that most of the disulfide aggregates present in the native caseinate have been destroyed during the extrusion process to give the casein subunits. The two distinctive peaks at 12 and 18 mL correspond to the disulfide bridge aggregates with higher molecular weights (95 kDa and 200 kDa). They are smaller for the extruded materials than for the native protein, confirming that the structural rearrangements of casein complex probably occurred during extrusion process. It is worth mentioning that the peak below 10 mL corresponds to the excluded volume of the columns. Although it appears that the structure of the caseinates was modified by the extrusion process, mainly by the destruction of some disulfide bridge aggregates, it will be shown that the mechanical properties of the films are not affected by these modifications. A review of the literature reveals that films obtained by casting and drying aqueous caseinate–plasticizer solutions have great mechanical properties. Nevertheless, as reported by HernandezIzquierdo and Krochta (2008), the properties of protein-based materials depend on the raw materials as well as the processing and the testing conditions. In the present study, two kinds of samples were used for the mechanical properties characterization. In order to fully characterize the caseinate materials, tensile strength tests were carried out on standard H shaped specimens made from compression molding of plasticized pellets. Samples were equilibrated at different RH in an environmental test chamber, heated to 30 °C, for 48 h and tested just after conditioning. Young’s modulus (YM), stress at break (Sb) (Fig. 5) and elongation at break (Eb) (Fig. 6) have been measured as a function of relative humidity for three different glycerol contents. As can be seen, raising the glycerol concentration decreases the Young’s modulus and stress at break, while it has an increasing effect on percentage of elongation at break starting from certain glycerol content. For example, the elastic modulus of material with 33% of glycerol (120 MPa) is three times weaker than that with only 17% of glycerol (350 MPa) for similar surrounding conditions (50%RH, 30 °C). As a plasticizer, glycerol reduces the interaction between protein molecules and then raises intermolecular spacing which facilitates the mobility of protein chains. The plasticizer acts as a lubricant to
Fig. 6. Elongation at break of extruded sodium caseinate as a function of moisture and glycerol contents (j 17% Gly. 25% Gly. 33% Gly.).
facilitate the movement of the protein chains over other, thus avoiding brittleness and raising their lengthening. As shown in Figs. 5 and 6, the mechanical properties of caseinate material are greatly affected by relative humidity. With the increase in moisture content, the elastic modulus and stress at break quickly decrease, whereas the elongation at break greatly increases, particularly at low glycerol content; The material goes from a glassy state at 40%RH (YM = 270 MPa and Sb = 14 MPa) to a rubbery state at 90%RH (YM = 2 MPa and Sb = 1 MPa). The hydrophilic nature of the plasticized caseinate makes the material very sensitive to water, which acts as a plasticizer.
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Once again, because the process used for the production of thin films may change the films’ mechanical properties, the mechanical properties of films obtained with blow film extrusion were also tested. Films were produced from thermoplastic pellets plasticized with 20% of glycerol, using a blown-film extruder (described in Section 2). This glycerol content was chosen to obtain a compromise between elastic modulus and elongation at break. The obtained films were homogenous, transparent and had a good appearance. The effect of moisture on mechanical properties of sodium caseinate based materials justifies the importance of controlling the ambient humidity and temperature during the analyses, particularly in the case of films with a very low thickness (30 ± 3 lm). In fact, for these films, the equilibrium reached within the environmental test chamber could be corrupted during their analysis. The ideal is to be able to perform these measurements under controlled moisture and temperature. To suit these requirements, equipment was designed in the laboratory, as described in the experimental section. The tensile strength measurements were carried out on thin films, using the designed machine, after being equilibrated during 1 h at 60%RH and 30 °C. Values of elastic modulus, stress at break (represented as an empty diagram in Fig. 5) and elongation at break (YM = 103 ± 5 MPa, Sb = 5.0 ± 0.3 MPa and Eb = 110 ± 10%) were determined from the obtained stress–strain curve. The mechanical properties of thin films (30 ± 3 lm) are similar to those obtained with standard tensile specimens much thicker (2 mm), as shown in Figs. 5 and 6. This result shows that although the sodium caseinate based material underwent two transits to the thermal and shear treatments (twin-screw and blow-film extruders), its tensile properties do not change. It has to be noted that the SEC elution diagram (Fig. 4) gives a very similar distribution of the molecular weight for the films when compared to the pellets. The evolution of the tensile properties of caseinate based materials as a function of moisture content has been explained using native and plasticized sodium caseinate moisture adsorption isotherms (Fig. 7).
These curves describe the relationship between moisture content (Xeq) of hydrophilic materials at equilibrium with the surrounding water activity at a fixed temperature. They are of importance for hydrophilic biopolymers as they are water sensitive, and the water content could strongly influence their functionality (Yang and Paulson, 2000). As presented in Fig. 7, the plasticized sodium caseinate is far more sensitive to relative humidity than the native caseinate. The hygroscopic character of glycerol increased the water absorption of caseinate material. Moreover, the sensitivity to water activity appeared to be slightly more marked for high plasticizer content (25% Glycerol). At a water activity of 0.9, the plasticized material absorbed eight times more water (0.8 gwater/g db) than at a water activity of 0.4 (0.1 gwater/g db). The obtained isotherms curves were also used to plot the tensile properties as a function of plasticized sodium caseinate water content; knowing the RH, it becomes possible to determine the water content of the sample (Fig. 8). Fig. 8 confirms the role of glycerol which acts as a plasticizer, but more importantly the representation emphasises that water has a stronger effect as a plasticizer. In fact, at fixed water content, the elastic modulus decreased, whereas increasing water content has the same effect on mechanical properties regardless of the glycerol content. A good way to simultaneously see the influence of glycerol and water, is to represent Young modulus as a function of elongation at break for different glycerol contents (17%, 25% and 33%) with different RH (40–90% every 10%RH) (Fig. 9). The plasticizing effect of water is so important that it is the relative humidity of the sample environment which governs the mechanical properties. Regarding the strong influence of the environmental conditions on the water content of the films, it is very difficult to compare the obtained results with those reported by other research teams. This is due, in part, to differences in glycerol content, water content, caseinate sources or testing conditions. However, the values of Young Modulus, stress and strain at break presented in the present study are similar to those obtained by Audic and Chaufer (2005) or more recently by Arrieta et al. (2013). These results suggest that using extrusion to produce pellets or blown films do not affect
Fig. 7. Moisture adsorption isotherms of plasticized (s 17% Glycerol and 4 25% Glycerol) and (h native) sodium caseinate at 30 °C.
Fig. 8. Young modulus as a function of water and glycerol contents of extruded sodium caseinate (4 25% Glycerol, s 17% Glycerol and j blown film).
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Fig. 9. Young modulus as a function of elongation at break (D 33% Glycerol, s 25% Glycerol, h 17% Glycerol and d blown film). RH between 40% and 90% every 10%.
Fig. 10. Water vapor permeability (WVP) of blown films made from sodium caseinate as a function of glycerol content.
the final mechanical properties and could allow industrial scale production of caseinate films. For the same reason, i.e. water content of the material, glass transition temperature is also very difficult to measure; increasing the temperature ramp used in Differential Scanning calorimetry (DSC) or in solid state rheology (DMA), evaporates the water within the sample, which results in higher glass transition temperatures. Because of this, the glass transition temperature of the extruded sodium caseinate was estimated theoretically by using the Gordon-Taylor equation. The glass transition temperature of a sample with 20% glycerol in a 60% RH environment, estimated by Gordon-Taylor equation, is around 50 °C; changing the water content makes this glass transition temperature moving across the room temperature. The mechanical properties can then fit those of a glassy polymer in a dry environment, or those of a rubber in a very humid atmosphere.
which increased with plasticizer content resulting in higher water diffusion through the film. The values of WVP found in the literature for sodium caseinate films containing various glycerol contents are listed in Table 1. These values were compared to those measured in our laboratory for sodium caseinate blown film at 100–60% RH differential and 30 °C. These edible films were compared with those of synthetic polymer. Table 1 shows that whatever the glycerol content is, edible films are poor water vapor barriers than those of synthetic polymer. The hydrophilic nature of films, made from sodium caseinate proteins, induced interaction with water molecules. To that is added the increase in inter-chain spacing caused by inclusion of glycerol molecules between the polypeptides chains, which enhance the water vapor transfer through the film. The highly hygroscopic character of glycerol molecules, which is favourable to the absorption of water molecules, could also decrease the resistance to water vapor transmission. The higher value of WVP obtained in the present study, compared to those of the literature, could be explained by some differences in test conditions: formulation or RH gradient. Furthers measurements are under process to clarify these results.
3.1. Water vapor permeability (WVP) of blown film Moisture transfer is an important factor leading to changes in food quality during storage, because microbial growth in food proliferates when water activity is high. It was demonstrated that the content of plasticizer has an effect on mechanical properties and it is expected to have an influence on WVP as well. The results of WVP of sodium caseinate films at different content of glycerol and tested at 100–60% RH differential and 30 °C, are shown in Fig. 10. Fig. 10 shows a classical result; the higher the glycerol content, the higher WVP is. The water diffusion through the film increased from 1.15 g mm/m2 h kPa to 4.50 g mm/m2 h kPa when only 13% of glycerol is added. This could be explained by the plasticizing effect of water favoured by the glycerol content, or simply by the high content of plasticizer. This agrees with the investigations of Coupland et al. (2000), who noticed that the consequences of plasticizing action of glycerol are favourable to adsorption and absorption of water molecules by the films, so WVP will be substantially increased. In fact, plasticizing the protein matrix increased the polymer free volume and then the polymeric chain segments mobility,
Table 1 Comparison of WVP values of various edible films and synthetic films. Film
Tests conditions (temperature °C; RH gradient %)
WVP (g mm/ m2 h kPa)
Extruded NaCAS/Ga (4:1) NaCAS/G (4:1) Khwaldia et al. (2004) NaCAS/G (1.67:1) Siew et al. (1999) NaCAS/G (0.89:1) Siew et al. (1999) LDPE Shellhammer and Krochta (1997) LDPEa PVDC Shellhammer and Krochta (1997)
30 ± 1; 100/60 ± 2% 20; 0/90
1.91 ± 0.03 0.303 ± 0.001
20; 0/45
0.225
20; 0/45
0.472
27.6; 0/100
0.0013
30 ± 2; 100/60 ± 3% 24.9; 0/100
0.0051 0.0008
a The present study; NaCAS: sodium caseinate; G: glycerol; RH gradients: inside/ outside; LDPE: low-density polyethylene; PVDC: polyvinylidene chloride.
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