Surface, mechanical and barrier properties of bio-based composite films based on chitosan and whey protein

food packaging and shelf life 1 (2014) 56–67

Available online at www.sciencedirect.com

ScienceDirect journal homepage: http://www.elsevier.com/locate/fpsl

Surface, mechanical and barrier properties of bio-based composite films based on chitosan and whey protein Mia Kurek a,b, Sabina Galus c, Fre´de´ric Debeaufort a,d,* a

PAM-PAPC, 1 Esplanade Erasme, Universite´ de Bourgogne – Agrosup Dijon, F-21000 Dijon, France Laboratory for Food Packaging, Faculty of Food Technology and Biotechnology, University of Zagreb, 6 Pierottijeva HR-10000 Zagreb, Croatia c Department of Food Engineering and Process Management, Faculty of Food Sciences, Warsaw University of Life Sciences-SGGW, 159c Nowoursynowska Street, 02-776 Warsaw, Poland d IUT-Dijon Departement Genie Biologique, 7 Boulevard Dr. Petitjean, B.P. 17867, F-21078 Dijon Cedex, France b

article info

abstract

Article history:

Mono-component and composite bilayer and blend films composed of chitosan and whey

Received 17 July 2013

protein were made. Colour, microstructure, water contact angles, swelling, water vapour

Received in revised form

sorption, barrier properties (oxygen, water vapour), water vapour diffusion coefficients and

9 December 2013

mechanical properties were determined. The influence of water vapour on barrier proper-

Accepted 2 January 2014

ties was studied in relation to the surface and structural properties. Mono-component and

Available online 21 January 2014

bilayer films were transparent with a homogeneous surface. Contrarily, blend films were

Keywords:

to mono-component and blend films. In all bilayer films, the air side (chitosan) was

Bilayer films

characterized by swelling, while the support side (whey protein) swelled after initial

translucent. Bilayer films had significantly lower water vapour permeability in comparison

Chitosan

absorption. At low relative humidities, blend films were excellent barrier to oxygen and

Whey protein

they completely lost their gas barrier performance in a humid environment. Bilayer films

Oxygen permeability

had enhanced mechanical resistance. The films with higher chitosan content showed

Surface properties

higher capacity for elongation. Lamination and blending of chitosan and whey protein is

Microstructure

a useful method to obtain new materials with desired functional properties. # 2014 Published by Elsevier Ltd.

1.

Introduction

Bio-based polymer films are principally prepared from polysaccharides, proteins and/or lipids, and they are generally biodegradable, non-toxic and edible materials. Furthermore, in certain circumstances, they can replace synthetic polymers, be used in multi-layer packaging and provide opportunities for new product development (Debeaufort, Quezada-Galo, &

Voilley, 1998). They can enhance the organoleptic properties of packaged foodstuff, supplement the nutritional value, and serve as carriers for antimicrobial and antioxidant agents. In addition, they can regulate moisture, oxygen, carbon dioxide, lipid, and aroma and flavour compounds migration between components of multi-component food products, and between food and surroundings (Sanchez-Gonzalez, Vargas, Gonza´lezMartı´nez, Chiralt, & Cha´ferm, 2009). The interest in these materials in food packaging applications has also increased

* Corresponding author at: PAM-PAPC, 1 Esplanade Erasme, Universite´ de Bourgogne – Agrosup Dijon, F-21000 Dijon, France. Tel.: +33 380 39 6547; fax: +33 380 39 6469. E-mail address: [email protected] (F. Debeaufort). Abbreviations: CS, chitosan film; WP, whey protein film; FFS, film forming solution; CS/WP, chitosan/whey protein bilayer film; CS + WP, chitosan/whey protein blend. 2214-2894/$ – see front matter # 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.fpsl.2014.01.001

food packaging and shelf life 1 (2014) 56–67

due to large surpluses of raw materials, which are produced in large amounts as by-products of agro-industrial processes. Currently, special attention is given to chitosan (CS), Nacetyl-D-glucosamine (Kurita, 2001). This polysaccharide polymer is a by-product from some crustacean industries, biodegradable; stable, it has low toxicity and it is a relatively low cost material. Whey protein isolate (WP) is also a very well-known film forming material. It is obtained from milk whey, a by-product of cheese-making (Foegeding, Davis, Doucet, & McGuffey, 2002) and has interesting mechanical properties (Kokoszka, Debeaufort, Lenart, & Voilley, 2010). Even though both protein and polysaccharide matrices generally show good film-forming abilities, their barrier and mechanical properties are naturally limited. The film properties depend on the type of material used and the process conditions employed, which in turn determine their applications (Krochta & De Mulder-Johnston, 1997; Rao, Kanatt, Chawla, & Sharma, 2010). Indeed, the barrier properties of CS and WP films, mainly with reference to moisture, are inferior to those of plastic packaging materials. Chitosan is not very permeable in a dry state, but as with other hydrophilic polymers, the permeability increases significantly with an increase in water content (Kurek, Sˇcˇetar, Voilley, Galic´, & Debeaufort, 2012). To improve physical, functional and barrier properties of both CS and WP film and with respect to the above-mentioned problems, in the present study blending and laminating these two materials has been proposed. In particular, whey proteins can interact with polysaccharide to form either soluble or insoluble complexes, depending on the colloidal properties and compatibility of protein/polysaccharide systems. These properties are related not only to the individual functionality of both components but also to the nature and strength of the interactions between them (de Souza, Bai, Gonc¸alves, & Bastos, 2009). Improvement of material formulation, its production and combination of protein and polysaccharide matrices offers opportunities for the development of new sustainable polymers with application in food industries (Dickinson, 2008). Recently, different chitosan blends were studied, e.g. quinoa protein/ chitosan (Abugoch, Tapia, Villama´n, Yazdani-Pedram, & Dı´azDosque, 2011), HDPE/chitosan (Mir, Yasin, Halley, Siddiqia, & Nicholson, 2011) and chitosan–whey protein (Ferreira, Nunes, Delgadillo, & Lopes-da-Silva, 2009). From an extensive review of the scientific literature, it was found that bilayer chitosan and whey protein films have been investigated to a much lesser extent. Thus in the present study, WP and CS laminated bilayer films with different layer thicknesses were developed. The detailed understanding of the characteristics of bilayer films is of great practical and commercial importance. Developed bilayer films were compared with blends and single composite CS and WP films. Regarding barrier properties, the critical compounds that can penetrate the packaging materials and degrade food quality are water vapour and oxygen. Thus, the water vapour and the oxygen permeability were determined. The influence of RH on the surface and on the barrier properties was tested in order to verify whether interactions between chitosan and protein enhanced properties of investigated films. In addition, the film microstructure and the mechanical properties were also determined.

2.

Materials and methods

2.1.

Materials and reagents

57

Commercial grade chitosan (France Chitine, Marseille, France, powder 652, having a molecular mass of 165 kDa, low viscosity, food grade, degree of deacetylation of >85%) was used as the carbohydrate film-forming matrix. Whey protein isolate (BiPRO, 90% protein, Davisco Foods International Inc., La Sueur, MN, USA) was used to prepare protein-based filmforming matrix. Glycerol (Fluka Chemicals, Seelze, Germany) and acetic acid (glacial 100%, Merck, Darmstadt, Germany) were used either to improve mechanical film properties or to enhance the solubilization of polymer powders. Silica gel, magnesium chloride (MgCl2), magnesium nitrate (Mg(NO3)2), sodium chloride (NaCl), potassium chloride (KCl) and deionized water were used to prepare saturated salt solutions to fix the RH for water vapour and gas permeability measurements. Deionized water was also used for surface analysis. No further purification of chemicals has been done and freshly prepared solutions were always used.

2.2.

Film preparation

2.2.1.

Chitosan films (CS)

A chitosan solution was prepared by dissolving the chitosan powder in a 1% (v/v) aqueous acetic acid, to obtain 2% (w/v) film forming solutions (FFS). To achieve complete dispersion of chitosan, the solution was stirred for 2 h at room temperature. The pH of chitosan FFS was 4.6–5. In order to improve the mechanical properties of the films, glycerol (at 30% (w/w) of chitosan) was added to the CS solution under stirring. An exact amount of the FFS (75 mL) was then poured into glass Petri dishes to obtain dry films of 80  5 mm. Films were dried in a ventilated climatic chamber (KBF 240 Binder, ODIL, France) at 25 8C and 50% RH. After drying, they were peeled off and stored in the same ventilated climatic chamber at 25 8C and 30% RH before measurements.

2.2.2.

Whey protein films (WP)

Aqueous dispersions of 2% (w/w) of whey protein isolate were heated at 80 8C for 30 min under stirring. WP solution was heated to denature the whey proteins. WP solution without heating is not able to make a continuous self-standing film after drying. Glycerol was used at 30% (w/w of protein). The pH of protein FFS was 7. An exact amount of the FFS was then poured into glass Petri dishes to obtain dry films of 80  5 mm. Films were dried and stored as described in Section 2.2.1.

2.2.3.

Chitosan/Whey protein bilayer films (CS/WP)

The bilayer films were prepared by a two-step coating technique. This method includes first the formation of one film and then, after drying, the polymer solution of the second layer is poured directly on top of the previously dried first layer. WP was used as a support layer whereas CS solution was poured on the top of it. FFS of each layer was prepared as described in Section 2.2.1. Firstly, WP films were made. Then, chitosan FFS was poured on top of the WP film. When CS was used as a supporting layer, the formation of a bilayer was

58

food packaging and shelf life 1 (2014) 56–67

impossible, due to its high swelling capacity. Thus, all bilayer films had a WP layer as the supporting one. Layering an acetic acid solution of chitosan could dissolve the pre-formed base of WP films. Thus, the thickness of each layer was checked in preliminary trials by controlling the FFS volume. The thickness of each layer and drying time were verified experimentally. We measured how much solution was needed to obtain the required layer thickness and how long a time was needed at a selected temperature and RH. For different film formulations, the thickness of each layer varied from 20 to 60 mm. Likewise, CS and WP proportions were from 25 to 75%. Finally, the thickness of all dry bilayer films was 80  5 mm. Thus, films were coded as follows: CS20/WP60 (for CS layer of 20 mm and WP of 60 mm), CS40/WP40 (for both layers of 40 mm), and CS60/ WP20 (for CS layer of 60 mm and WP of 20 mm). Finally, the system was dried at 25 8C and 50% RH in the ventilated climatic chamber (KBF 240 Binder, ODIL, France) for 24 h. After drying, the films were peeled off and stored in the same ventilated climatic chamber at 25 8C and 30% RH before measurements. All produced films were self-standing, they did not roll over or break, and they were easily peeled from the drying surface. However, to obtain the final dry material in fixed drying conditions (25 8C, 50% RH) a long time was needed (48 h). Thus for successful and not time-consuming industrial application, modification of the drying conditions is required. Glycerol was added to chitosan or whey protein film forming solution at 30% (w/v) and its amount in final dried layered films was different according to different solution needed to obtain constant film thickness (80 mm). Mono-composite chitosan and whey films, and blend films contain the same amount of plasticizer.

2.2.4.

Chitosan/Whey protein blend films (CS + WP)

Blend films were obtained by mixing chitosan and whey protein FFS in a ratio of 1:1. The pH of pure WP solution was about 7 and after mixing with CS solution the obtained blend mixture changed the pH to 5–5.5. Aggregation could occur during drying, but in this study it was not observed or measured. At this pH both chitosan (pK = 6.5) and protein (pHi = 6.8) are positively charged. This is important to avoid incompatibility between these two polymers. FFS was then poured into a glass Petri dish, dried and stored as described in previous sections.

2.3.

Film characterization

Prior to any characterization, all films were equilibrated at 30% RH and 25  1 8C. Selected analyses were made in films from different experimental series. Thus, at least 20 samples of each film were prepared.

2.3.1.

Thickness

The film thickness was measured with an electronic gauge (METRISON, Poland). The average value of five thickness measurements per film was used in all calculations.

2.3.2.

Colour measurement

The colour of the film was determined using a colorimeter (Minolta, CM Model CR-300, Japan). Hunter L*, a*, and b* values were averaged from three readings across for each sample,

and then the total colour difference (DE) was calculated according to the following equation (Ghorpade, Li, Gennadios, & Hanna, 1995): qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 (1) DE ¼ ðDL Þ2 þ ðDa Þ2 þ ðDb Þ where DL*, Da* and Db* are the differentials between a sample colour parameter and the colour parameter of a standard (L* = 92.3, a* = 0.79, b* = 4.20) used as the film background.

2.3.3.

Film microstructure

The film microstructure was examined using environmental scanning electron microscopy (ESEM, Philips XL 30 ESEM, Japan). A 5  10 mm2 film was fixed on the support using double sided adhesive tape with an angle of 908 to the surface to allow the observation of the film cross section and film surfaces. The surface in contact with the glass support during drying will be referred to as the ‘‘support side’’ and the other surface in contact with the air during drying will be referred to as the ‘‘air side’’. All the films were cut with a new razor blade to prevent as much as possible any morphological damage. The films were observed at different magnifications up to 15 000 for focusing and images were taken at magnification from 800 to 2500 with an intensity of 8 kV and absolute pressure of 230 Pa (RH 30% at 5 8C).

2.3.4.

Contact angle and wettability

The contact angle, surface hydrophobicity and wettability of films were measured by the sessile drop method, in which a droplet of the tested liquid was placed on a horizontal film surface using a DGD-DX goniometer (GBX, Romans-sur-Isere, France), equipped with the DIGIDROP image analysis software (GBX, Romans-sur-Isere, France) (Karbowiak, Debeaufort, & Voilley, 2006). Water droplets (1.5 mL approx.) were deposited on the film surface (‘air side or support side’) with a precision syringe. The experimentally acquired data were contact angle (u), droplet surface area exposed to air, droplet base area in contact with the film and droplet volume (V) as a function of time (t). The effect of evaporation was analyzed on an aluminium foil that is considered impermeable to water and aqueous solutions. The wetting kinetics lasted for 200 s. The swelling index was obtained from the drop volume kinetics and the following equation:     DV V2  V1 (2)  100 ¼  100 Swelling index ¼ V0 V0 where DV is the droplet volume variation (mL) during dt time (s) measured on the film sample. V2 is the maximal volume (mL) of the droplet, V1 is the minimal volume (mL) of the droplet and V0 is the initial volume (mL) of the droplet. Absorption flux was obtained from the drop volume kinetics taking into account the evaporation flux and the following equation:   dV  Feva Fabs ¼ AB  dt film     dV dV ¼  (3) AB  dt film AS  dt Aliuminium foil where dV is the droplet volume variation (mL) during dt time (s) measured on the film sample or the reference aluminium foil;

food packaging and shelf life 1 (2014) 56–67

AB, the base-surface area of the droplet in contact with the solid surface; AS, the surface area of the droplet in contact with the air phase, and Feva the evaporation flux from the droplet measured on the reference aluminium foil. All the films were stored in a climatic chamber (KBF 240 Binder, ODIL, France) at 50% RH and 25 8C prior to measurements. At least six measurements per film were carried out.

2.3.5. Water content, water vapour sorption kinetics and diffusion coefficient The water content was measured by determination of the weight loss of the film after drying at 105 8C for 24 h. All the measurements were made in triplicate. Saturated sodium chloride solution was used to obtain the constant humidity of the environment (75%). Films were cut in squares of 2 mm  2 mm and they were weighed periodically. The measurement was carried out at the temperature of 25  1 8C for 7 days. At least three repetitions for each type of film were made. The water vapour sorption kinetics were described by the classical Fickian diffusion equation through a membrane of L thickness (Crank, 1975): " # 1 X Mt 8 Dð2p þ 1Þ2 p2 t (4) ¼1 exp 2 2 M1 4L2 n¼0 ð2 þ 1Þ p where t is the time (s) and Mt/M1, the total amount of water vapour adsorbed by the sheet at time t. The water vapour diffusion coefficient within the films was estimated by fitting Eq. (4) to the experimental sorption kinetic data using a preestimation of D using Microsoft Excel.

2.3.6.

Water vapour permeability

The water vapour permeability (WVP) of films was determined gravimetrically using a modified ASTM E96-80 (1980) standard method, adapted to edible materials (Debeaufort, Martin-Polo, & Voilley, 1993), using the RH differentials of 33–0%, 75–30% and 100–30% and the temperature of 25  1 8C. Prior to the WVP measurements, all the film samples were equilibrated at 25  1 8C and 30% RH for 72 h. WVP (g m1 s1 Pa1) was calculated using the following equation, from the change in the cell weight versus time at the steady state: WVP ¼

Dm  e A  Dt  D p

(5)

where Dm/Dt is the weight of moisture loss per unit of time (g s1), A is the film area exposed to the moisture transfer (9.08  104 m2), e is the film thickness (m), and Dp is the water vapour pressure differential between the two sides of the film (Pa). Three replicates for each film type and RH gradient were made.

2.3.7.

Oxygen permeability

Oxygen permeability was measured using a manometric method, on a permeability testing appliance, Brugger, Type GDP-C (Brugger Feinmechanik GmbH, Germany). The increase in pressure during the test period was assessed and displayed by an external computer. Data were recorded and permeance was calculated by GDP-C software (with temperature compensation connection). The sample temperature (25 8C) was adjusted using an external thermostat (HAAKE F3 with Waterbath K). The

59

desired RH was regulated in an external saturation system, so humidified gas circulated in the permeation cell.

2.3.8.

Mechanical properties

A texture analyzer TA-XT2i (Stable Microsystems, United Kingdom) was used to measure the tensile properties according to the ASTM D882-95 method (ASTM, 1995). Prior to the test, films were cut in rectangular strips 100 mm long and 25 mm wide. Self-tightening roller grips were used as the probe to perform tensile assays. The initial grip separation and velocity were adjusted to 50 mm and 1 mm s1, respectively, considering conditioned samples with the cell load of 5 kg. The force and the distance were recorded during the extension of the strips. At least 10 samples of each film were analyzed. Young’s modulus (YM) was evaluated as the slope of the initial linear portion of stress–strain curves. The tensile strength (TS) and elongation at break (E) were calculated according to the following equations (Valenzuela, Abugoch, & Tapia, 2013): TS ¼

N mm2

(6)

where N is the maximum force at rupture of the film, mm2 is the initial cross-sectional area of the films. E¼

D f  Di  100% Di

(7)

where Df is the distance elongation at break (mm) and Di is the initial distance between the grip.

2.4.

Statistical analysis

The statistical analysis of data was performed through variance analysis (ANOVA) using the Statgraphics Plus 5.0 version program (Manugistics Corp., Rockville, MD, USA) and XLstat-Pro (win) 7.5.3. (Addinsoft, New York). The data were ranked and statistical differences were evaluated on the ranks with a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison tests. In all cases, a value of p < 0.05 was considered to be significant.

3.

Results and discussion

First of all, during the production of composite films (both bilayer and blend films) some limitations related to the film formulation had to be taken into account. It was due to the pH value for chitosan solution (pH = 4.6) and for whey protein solution (pH = 7.0). Previously it was reported that the preparation of chitosan/whey protein blend films at higher pH values (5.0–7.0) was not successful due to the insolubilization of one of the polymers or to the formation of insoluble complexes between them (Ferreira et al., 2009). Similar behaviour was also observed for chitosan/caseinate films (Pereda, Amica, & Marcovich, 2012). Based on those observations, formation of CS and WP bilayer films was justified.

3.1.

Film appearance

The transparency of edible/bio-based polymer films is a key to good film acceptance by users (Rhim, Gennadios, Weller, &

60

Values with the same superscript letters within a column are not significantly different at p < 0.05, CS – chitosan, WP – whey protein. a–h

5.90  1.05 1.89  0.49 b 1.97  0.22 b 4.01  0.55 c 0.97  0.32 a 3.97  0.71 c 1.55  1.04 2.37  0.48 b 2.30  0.23 b 0.23  0.52 c 3.45  0.26 a 0.41  0.68 c 0.34  0.25 0.99  0.10 c 0.97  0.05 c 0.58  0.45 b 1.17  0.05 a 0.48  0.05 ab 91.09  0.49 92.60  0.32 c 92.61  0.30 c 92.04  0.11 b 92.60  0.43 c 91.12  0.24 a CS CS20/WP60 CS40/WP40 CS60/WP20 WP CS + WP

Values were given as mean  standard deviation.

m2 s1)

8.20  0.30 b 8.10  0.40 ab 9.60  0.40 c 9.20  0.80 c 7.30  0.60 a 8.00  0.10 ab 3.45  0.68 0.90  0.22 abc 1.25  0.35 bcd 1.69  0.29 def 3.47  0.20 h 3.72  0.22 h 2.37  0.38 0.79  0.34 ab 1.40  0.62 bcd 1.74  0.76 defg 2.09  0.32 efg 2.31  0.50 fg 0.24  0.34 0.91  0.08 abc 0.55  0.02 a 0.91  0.33 abc 1.49  0.31 ade 1.34  0.21 bcd 38.50  5.15 1.44  0.47 a 15.10  5.02 b 35.70  7.07 cd 5.58  0.84 a 33.90  6.37 c 21.80  4.06 5.21  1.01 a 32.70  5.69 c 43.70  6.48 e 24.30  5.08 b 38.00  6.06 d 8.90  1.62 5.80  2.30 a 17.50  4.51 c 15.30  4.18 c 0.50  0.99 b 0.60  1.67 b

b a

a*

a

b*

d

DE

d

TS (MPa)

b

YM (MPa)

E (%)

d

33–0

bcd

75–30

g

100–30

h

(10

15

D WVP (1010 g m1 s1 Pa1) (DRH, %)

L*

Transparent appearance and colour changes give just partial information about the film structure. Therefore, ESEM was done in order to contribute to a better insight in the homogeneity and in the microscopic structure of different CS and WP based films. The ESEM micrographs are shown in Fig. 1. In the cross section of the bilayer film, at a microscopic level, the layers corresponding to the individual components were observed. Good adhesion and integrity of the CS and the WP layer were also noted (Fig. 1a, b). Black discontinuities through the whole film are attributed to glycerol and some impurities originally present in powder material, or to the incomplete solubility of powder grain. A similar result for chitosan/gelatin film was previously reported (Rivero et al., 2009). After drying, the whey protein part was homogeneous and compact. In contrast, the chitosan part had a characteristic layered structure (Fig. 1a, b). According to some authors this can be attributed to the fluctuations in the concentration and in the temperature during drying at the chitosan/air drying interface (solvent front migration) (Kurek, Brachais, et al., 2012; Torres, Aimoli, Beppu, & Frejlich, 2005). Cross section micrographs allow observation of film internal structures, but they also contribute to a better knowledge of the film-forming behaviour of hydrocolloid substances. Mixing of polymers in blend films yields to a more different film structure (Fig. 1). It depends on the pH conditions during film preparation. The pH control is important to avoid complex coacervation and precipitation in case of an opposite charge of two polymers. In this study, in the FFS no incompatibility occurred between CS and WP. However, blend film had a complex/heterogeneous structure that was formed during the drying step (Fig. 1). During drying, the acetic acid is probably evaporated more rapidly than water. This can lead to changes in the pH of the solvent that could induce incompatibility, explaining the final heterogeneous microstructure. According to Ferreira and co-workers, when films were prepared at pH > 6.5 a heterogeneous film formed and the phase separation phenomenon was described as a result of

Mechanical properties

Film microstructure

Colour parameters

3.2.

Film

Hanna, 2002). All the colour attributes obtained for each film sample are summarized in Table 1. Visually, all the bilayer films were transparent, similarly as pure chitosan or pure whey protein films, blend films had a translucent surface. The lightness value of all films, L*, remained fairly constant, from 91.09 to 92.61. However, the presence of the chitosan layer in bilayer films significantly increased the yellow and the green tint. Then, b* was significantly increased and a* was significantly decreased. These results were consistent with visual observations. DE, which is an indicator of global colour changes, was calculated from the other colour parameters. It ranged from 0.97 for WP to 5.90 for CS films. The colour of the blend films was comparable to the other composite materials, e.g. chitosan/guar gum films (Rao et al., 2010) and chitosan/ gelatin films (Rivero, Garcı´a, & Pinotti, 2009). To conclude, all the films were visually barely noticeable, and thus they could be used as edible films or coatings, e.g. to separate two different layers in some food products such as cookies, cakes and similar products.

Table 1 – Colour parameters, mechanical properties, water vapour permeability and water vapour diffusion coefficient of composite films from chitosan and whey protein (equilibrated at 25 8C and 30% RH).

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Fig. 1 – ESEM micrograph of cross section of chitosan/whey protein bilayer film ((CS20/WP60) (a) and CS40/WP40 (b)), cross section of chitosan/whey protein blend film (c), surface of chitosan/whey protein blend (d) and surface of chitosan/whey protein bilayer film (e).

the thermodynamic incompatibility between whey protein and chitosan (Ferreira et al., 2009). Generally, at acetic pH values whey proteins aggregate and film properties could decrease (Pe´rez-Gago & Krochta, 1999) as was observed especially in values of tensile strength and oxygen permeability at low RH of blend films. Changes in the pH of the whey protein films plasticized by glycerol may affect solution and film stability, and thus the characteristics of the whey protein layer in multisystem materials. Casting films at pH lower than the isoelectric point proved to be inefficient

since the reactivity of SH groups decreased significantly (Ferreira et al., 2009). Previous studies concerning whey protein films (Anker, Stading, & Hermansson, 2000; Kokoszka et al., 2010; McHugh, Aujard, & Krochta, 1994) showed good film-forming capacity at pH 7 and their properties have been extensively characterized under these conditions. Likewise, chitosan films showed adequate properties at acetic pH values (Kurek, Brachais, et al., 2012; Kurek, Sˇcˇetar, et al., 2012). In bilayer films, the two layers have different pH values (CS pH = 4.6–5.0, WP pH 7.0) and their stability depends on the

62

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adhesive capacity and integrity. However, to better understand stability of such bilayer films further research is necessary, principally to obtain and to characterize barrier and mechanical properties of composite films at the same pH values of each layer. The micrographs of film surfaces indicated that the air side (WP part) and the support side (CS part) surfaces of bilayer films were smooth, flat and without pores and cracks (Fig. 1d, e). On the surface of blend films heterogeneities were observed (Fig. 1d). The presence of whey protein caused discontinuities and irregularities on the film surface when it was blended with chitosan. This is consistent with the film opacity as it was already described in visual observations.

3.3.

Wettability

The contact angle of water indicates hydrophilic/hydrophobic properties of the material. First of all, film wettability was tested in order to better understand how water and relative humidity influence the surface properties and the resistance of CS and WP films. The contact angle at the time of the water droplet deposit (0 s) and at the metastable equilibrium (30 s), the water absorption rate (wettability), the swelling and the delay of swelling are given in Table 2. Furthermore, changes in the surface behaviour and kinetics observed after deposit of water droplets on different films during 90 s are shown in photos presented in Fig. 2. Practically, a large (u > 658) and a small (u < 658) contact angle represent the quantitative definition of hydrophobic and hydrophilic surfaces, respectively (Vogler, 1998). The air side and the support side of pure CS films had larger contact angles (778 and 888, respectively) compared to pure WP films (398 and 458, respectively). For liquid water contact, a dramatic change in the physical state of the film surface could also affect the contact angle measurement. Indeed, the chitosan film started to swell immediately after the deposition of the water droplet (Fig. 2). The local swelling occurred probably both because of the presence of glycerol that was oriented towards the CS film

surface and because of its hygroscopicity (Kurek, Guinault, Voilley, Galic´, & Debeaufort, 2013). In addition, for water insoluble CS partial solubilization of film constituents occurred and the film network was swollen. This phenomenon of swelling has already been observed in films made of cassava starch (Phan The, Debeaufort, Voilley & Luu, 2009) and carrageenan (Karbowiak, Debeaufort, Champion, & Voilley, 2006) but to a lesser extent. Accordingly, the water adsorption rate on the CS was not determined (Table 2). In contrast, in pure WP films the absorption of water droplets was observed on both film sides (Table 2 and Fig. 2). For soluble WP films, absorption of water leads to a loss of network that explains rapid water droplet spreading. Fabs ranged from 1.66 to 17.05  103 mL mm2 s1. This result is similar to carrageenan edible films (3.62  103 mL mm2 s1) (Karbowiak, Debeaufort, Champion, et al., 2006), and higher than that of WP films with higher protein contents (0.78  103 mL mm2 s1) (Ferreira et al., 2009). In all bilayer films, different behaviours were observed from one surface of the film (WP-casting–support side) to the other one (CS–air side). Each surface behaved in the same manner as a homologous simple film as previously described. In all bilayer films, the air side was characterized by swelling, with an increase of the drop volume (Table 2 and Fig. 2). On the support sides, films swelled after initial absorption (Table 2 and Fig. 2). Moreover, larger contact angles on the air side indicate a more hydrophobic character of CS surfaces compared to the support surface (WP). As expected, no significant changes could be attributed to the different thicknesses of layers in the bilayer films. The initial values of contact angles for blend films are found to be lower than those of CS films and higher than those of WP films (Table 2). Significant differences in the contact angles and in the Fabs were also observed between the air sides and the support sides (Table 2). It may be due to the changes in the film structure and to the orientation of macromolecular chains during film drying. Blend films followed anomalous behaviour characterized by initial absorption and delayed swelling

Table 2 – Contact angle at time 0 s (ut0) and at 30 s (ut30), absorption flux (Fabs), time delay before swelling and swelling percentage data for the analyzed films, at 25 8C and 30% RH. Film

Side

ut0 (8)

Fabs (103 mL mm2 s1)

ut30 (8) bc

a

Delay (s) c

Swelling (%)

CS

Air Support

71.29  16.18 88.46  9.41 ab

88.47  9.41 89.95  1.28 a

IS IS

0 0c

83.89  37.57 cde 237.59  42.95 b

CS20/WP60

Air Support

90.2  3.26 ab 58.95  3.17 cd

69.79  7.27 b 37.16  2.11 e

IS 9.53  5.52 ab

0c 0c

17.48  1.15 f ND

CS40/WP40

Air Support

94.26  7.33 a 49.73  1.31 d

90.7  4.62 a 37.29  1.89 e

IS 4.60  2.20 bc

0c 18.40  3.20 a

126.84  37.69 c ND

CS60/WP20

Air Support

94.38  1.72 a 54.62  0.16 cd

92.19  1.89 a 48.88  1.46 de

IS 8.05  3.94 bc

0c 5.86  1.17 bc

314.77  7.43 a ND

WP

Air Support

38.9  10.3 de 35.4  2.1 de

32.52  2.22 f 28.19  0.18 f

1.66  0.22 bc 1.52  0.37 bc

ND ND

ND ND

CS + WP

Air Support

81.57  3.69 ab 61.05  6.86 cd

63.08  1.77 bc 56.51  3.59 cd

17.05  3.07 a 2.90  0.15 bc

9.40  0.85 b 8.60  1.40 b

100.37  5.37 cd 122.35  7.86 c

Mean of at least five measurements  standard deviation. a–f Different superscripts within the column indicate significant differences between samples at p < 0.05. IS – immediate swelling; ND not detected.

food packaging and shelf life 1 (2014) 56–67

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Fig. 2 – Shape and behaviour of water droplets deposited on chitosan and whey protein based films as a function of time. CS – chitosan, WP – whey protein, CS/WP – chitosan and whey protein bilayer film, CS + WP – chitosan and whey protein blend.

(Fig. 2 and Table 2). This might indicate the orientation of WP towards surface parts since in other formulations CS parts tend to swell immediately. When one material is added to another one, it can modify the physical properties of the first one. Other authors observed that carbohydrate/protein films are characterized by a compositional gradient, with the downward film surface containing a lower amount of protein than the upward surface (Ferreira et al., 2009). However, from microstructure and surface analysis no direct conclusion on the orientation of macromolecules could be drawn in the present study.

3.4.

Mechanical properties

Mechanical properties are essential for adequate design of biopolymer packaging films that must have a certain degree of resistance. The measured values of the tensile strength (TS), the elongation at break (E) and the Young modulus (YM) are given in Table 1. As all the films were plasticized with glycerol, they were easy to handle. Glycerol reduces the interactions between polymer chains that make the chain displacement during stretching easier, which gives the film a greater ability to be

deformed without breaking. No significant differences in TS of single component films were observed. In general, protein films are brittle and susceptible to cracking. It is due to the strong cohesive energy density of the polymer (Arvanitoyannis & Biliaderis, 1998). Mechanical properties for WP films reported in the literature are lower (TS 1.00 MPa, YM 8.00  102 MPa) (Ramos et al., 2013) than those obtained in this study (TS 10.50 MPa, YM 24.30 MPa). The comparison of the data for CS films is very difficult due to different film preparation, powder composition, solubilization method and plasticizer content (Butler, Vergano, Testin, Bunn, & Wiles, 1996; Rivero et al., 2009). In the present study, CS films had a TS value around 8.91 MPa and an E of 38.50%. Elongation at break of CS film was 7 times higher than that of WP film. When comparing all film formulations, significant differences in mechanical properties were observed for different bilayer formulations (Table 1). Bilayer films with a thicker chitosan layer (40 mm and 60 mm) had the highest TS, with values of 17.50 and 15.30 MPa, respectively. Thus, increasing chitosan content led to stronger films. E and YM values were also higher. It can be explained by the strong hydrophilic nature of chitosan as observed in water vapour sorption analysis, as will be discussed in the next section (Fig. 3).

64

food packaging and shelf life 1 (2014) 56–67

Fig. 3 – Curves of water vapour sorption kinetics for chitosan and whey protein based films. CS – chitosan, WP – whey protein, CS/WP – chitosan and whey protein bilayer film, CS + WP – chitosan and whey protein blend.

Similarly, other authors have noted increase in TS and decrease in the E for soy protein films laminated by corn zein (Cho, Lee, & Rhee, 2010).

3.5. Sorption kinetics of water vapour and diffusion coefficient of water The sorption of water vapour in hydrophilic polymers is a complex phenomenon. The presence of specific interactions between water molecules and the hydrophilic sites on the polymer backbone delays water vapour diffusion through the film (Pereda et al., 2012). The kinetics of the water vapour sorption are displayed in Fig. 3. All films followed the same trend. The amount of adsorbed water increased with time. The slope of kinetic curves and thus the adsorption rate was the highest during the initial adsorption stage. CS film had a significantly higher water sorption capacity than WP films. After seven days, the final adsorbed water content was 0.19 and 0.38 gH2O gd.m.1 for pure WP and for pure CS films, respectively. During the experiment, films were plasticized by water vapour and they swelled. Then, the mobility of both the polymer segments and the water vapour was increased. Consequently, the adsorption was facilitated. Similar behaviour was previously reported for e.g. chitosan (Epure, Griffon, Pollet, & Averous, 2011) or sodium caseinate films (Fabra, Talens, & Chirald, 2010). From kinetic data, apparent diffusion coefficients of water were calculated. Results are given in Table 1. D ranged from 7.30  1015 m2 s1 for pure WP film to 9.60  1015 m2 s1 for CS/WP bilayer film with the same layer thickness (CS40/WP40). The diffusion of water vapour through a polymer matrix is a consequence of random motions of individual molecules of the

species and it depends on the geometry of the film matrix (Labuza & Hyman, 1998). For composite formulations, results obtained in this study were lower than previously reported for whey and whey-mesquite gum films (Oses et al., 2009) or protein-gum films (Tomas, Saavedra, Cruz, Pedroza-Islas, & San Martin, 2005). This can be attributed to both the different structure and the orientation of macromolecular chains in blend films. In addition, the different sorbing capacity of film forming materials can also lead to different rates of water vapour diffusion in final films, as reported in the present study.

3.6.

Water vapour permeability

Due to the direct influence on the deteriorative reactions in packed food products, WVP is the most important and extensive property of bio-based polymer films. The high WVP of most bio-based polymers makes them inappropriate for several applications unless a combination of two or more of them are employed (Arvanitoyannis & Biliaderis, 1998). The WVP values at three RH gradients (33–0%, 75–30%, and 100– 30%) are given in Table 1. The WVP of all analyzed films ranged from 0.55 to 3.72  1010 g m1 s1 Pa1. The WVP of both CS and WP has been extensively studied by other authors (Ferreira et al., 2009; Kokoszka et al., 2010; Kurek, Sˇcˇetar, et al., 2012; Labuza & Hyman, 1998). It can be noticed that WVP increased when the RH differential increased (from D33% to D45% and D70%). This non-ideal behaviour is generally attributed to a structural modification of the film. It is also related to the significant hydrogen bonding interaction with water, to the plasticization of the biopolymer by the water uptake, and to the film swelling.

food packaging and shelf life 1 (2014) 56–67

No significant changes between blend films and onecomponent films were observed (Table 1). This can indicate that the developed matrix was similar to those of the single component matrices, since permeability contributes to diffusivity and solubility of the permeant through the solid matrix (Pinotti, Garcıa, Martino, & Zaritzky, 2007). Bilayer films had significantly lower WVP in comparison to both pure and blend films. At higher RH differentials, the WVP was the highest for CS60/WP20 films. These results suggest that altering the thickness ratio of chitosan:protein in the film significantly changed its water vapour barrier properties. According to others, bilayer systems (Pe´rez-Gago, Serra, Alonso, Mateos, & del Rı´o, 2005), e.g. chitosan/collagen (Krkic´, Lazic´, Petrovic´, Gvozdenovic´, & Pejic´, 2012) or chitosan/gelatin laminated films (Rivero et al., 2009), are better water vapour barriers than blend films. In the mentioned studies, WVP was found to be in range of 1.33  1010 to 1.60  1010 g m1 s1 Pa1. These results are lower than for bilayer films reported in the present work (3.47  1010 g m1 s1 Pa1). The difference is probably due firstly to the film composition and secondly to the experimental conditions used for WVP determination.

3.7.

Oxygen permeability

Generally, bio-based polymers are aimed to be a good barrier to gases at low RH. In the determination of permeability, the effects of the moisture content have to be taken into account to reflect as much as possible the conditions of intended use. The humidity in the environment and in the packaging is often above 50%. Thus to better understand the influence of the hydration level on the oxygen permeability of CS and WP based films and to assess when exactly significant changes occur, in this study oxygen permeability was measured

65

at 0, 30, 50, 75 and 81% RH. The results are given in Fig. 4. Within the studied range, two main regimes were observed. First, in all the film samples, for RH <50%, oxygen permeability remained fairly low. In the dry state, blend film was the best barrier to oxygen with PO2 values of 1.31  1017 g m1 s1 Pa1. It was because of: - either strong compatibility between chitosan and whey protein: same charges of protein and chitosan induced a more inter-penetrated network of two biopolymers; - or incompatibility due to pH change during drying: opposite charge induces phase separation between WP and CS that creates a kind of ‘‘chemical tortuosity’’. The single films were also excellent oxygen barriers. In the dry state, PO2 of WP and CS was 6.24  1017 g m1 s1 Pa1 and 21.47  1017 g m1 s1 Pa1, respectively. Similar results have been previously observed by other authors for both CS (Park, Marsh, & Rhim, 2002) and WP films (Ramos et al., 2013). Interestingly, after the lamination process the PO2 of all bilayer formulations was increased (>48.23  1017 g m1 s1 Pa1) and no significant changes between bilayer formulations were observed. When RH increased above 75% PO2 in all samples increased exponentially to very high values. The increase was up to 122 times in blend film, followed by WP (19 times), CS (13 times) and bilayer films (10 times). This is in agreement with some previously reported results where it was found that PO2 of CS films increased by 12.9 times, when RH increased from 0% to 100% (Despond, Espuche, & Domard, 2001). There are several possible explanations. First, permeating humidified gas turns hydrophilic polymer chains into a swollen state. Actually, it absorbs water and greatly increases gas permeability (Fig. 3).

Fig. 4 – Changes in oxygen permeability (PO2) as a function of relative humidity of different chitosan and whey protein based films. a–fDifferent superscripts indicate significant differences between formulations at a given RH ( p < 0.05). CS – chitosan, WP – whey protein, CS/WP – chitosan and whey protein bilayer film, CS + WP – chitosan and whey protein blend.

66

food packaging and shelf life 1 (2014) 56–67

Then gas solubility was also improved. Accordingly, WP films absorbed less water vapour and thus the PO2 of this film formulation remained significantly lower compared to others (PO2 was equal to 124.13  1017 g m1 s1 Pa1). The plasticization by the water, the swelling and the clustering of water molecules might have induced rearrangements in the conformation, and in the mobility of the polymer chains (Gennadios, Weller, & Gooding, 1994). Transformations of chitosan polymorph and a drastic change in the chain arrangement of chitosan influenced by presence of water were previously described (Ogawa, 1991). Disruption of hydrogen bonds between molecules may create additional sites for the dissolution of oxygen and increase mobility of the O2 molecules within the polymer bulk phase (Despond et al., 2001; Gennadios & Weller, 1990). Even though similar behaviour was observed in all film formulations, bilayer films were the most permeable with PO2 values between 557.91  1017 g m1 s1 Pa1 and 653.48  1017 g m1 s1 Pa1. In bilayer films, the plasticization by water and the inclusion of water molecules in the permeating system might lead to the appearance of greater gaps between macromolecular chains at the chitosan/whey protein interface. Thus, it diminished its cohesivity and increased the PO2 of bilayer films.

4.

Conclusion

Chitosan and whey protein films showed good potential to be used as film forming matrices. Results obtained in this study indicate the possibility of production of transparent WP/CS bilayer films with higher water vapour barrier efficiency and enhanced mechanical resistance. Thus, potential industrial applications can be envisaged. On a microscopic level, good adhesion and integrity of the CS and the WP layer were observed. In all bilayer films, the air CS side was characterized by swelling, while on the support WP sides, the films swelled after initial absorption. Blend films followed an anomalous behaviour that was characterized by the initial absorption and delayed swelling. During the exposure to water vapours, water vapour plasticized the WP and CS films. Bilayer films had significantly lower water vapour permeability in comparison to both pure and blend films because it was affected by permeability of each layer. In the dry state, blend film proved to be the best barrier to oxygen followed in respective order by WP films, CS films and bilayer films. Lamination of WP and CS polymer is shown to be one of the most useful methods to obtain new materials with desired functional properties. The bilayer CS/WP films may have useful applications in the food systems where edible films should dissolve during the cooking or consumption process. In addition, these films would also have useful antimicrobial properties due to the presence of chitosan. However, further application studies are needed and envisaged.

Acknowledgements The authors wish to thank the colleagues from the PAM-PAPC Laboratory, Dijon, France, to colleagues from Department of Food Engineering and Process Management, Warsaw Poland

and to colleagues from the Laboratory of Food Packaging, Zagreb Croatia. The authors special thank Mme Aline Bonnotte for ESEM support. The authors wish to thank Prof. JP Gay for English improvement.

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