Biodegradable Zein-Based Blend Films : Structural , Mechanical and Barrier Properties

Packaging is essential for food transport and distribution, enabling quality preservation and protection against external chemical, physical and microbiological contamination (1–3). Nowadays, plastic materials are the most frequently used food packaging due to their thermostability, fl exibility, lightness and low price (3–5); nonetheless, as a result of the global environmental concern, new biodegradable materials, mainly fi lms and coatings, have been developed in order to reduce plastic packaging usage (6–8). In comparison with plastics, biodegradable fi lms and coatings are quickly degraded by the action of microorganisms as a consequence of their natural origin (3,8).


Introduction
Packaging is essential for food transport and distribution, enabling quality preservation and protection against external chemical, physical and microbiological contamination (1)(2)(3).Nowadays, plastic materials are the most frequently used food packaging due to their thermostability, fl exibility, lightness and low price (3)(4)(5); nonetheless, as a result of the global environmental concern, new biodegradable materials, mainly fi lms and coatings, have been developed in order to reduce plastic packaging usage (6)(7)(8).In comparison with plastics, biodegradable fi lms and coatings are quickly degraded by the action of microorganisms as a consequence of their natural origin (3,8).
In general, biodegradable materials are prepared from biopolymers such as proteins, carbohydrates or lipids.For instance, zein, the main corn protein, is one of the materials that have been used to produce biodegradable fi lms and coatings for food and pharmaceutical applications (8)(9)(10).Zein-based materials have glossy appearance and low water solubility, they are tough, greaseproof, hydrophobic and resistant to microbial att ack (8,11), although britt le (12,13).
Several studies have demonstrated that preparing biodegradable materials from two or more biopolymers, known as composites or blends, allows for improving their functional properties compared to single biopolymer fi lms (5,6,14).Some composite and blend biodegradable materials that have recently been reported are: (i) cassava starch-based fi lms with clay nanoparticles (3), (ii) pea starch and peanut protein isolate blend fi lms (5), (iii) nanocrystalline cellulose-reinforced chitosan-based nanocomposite fi lms (6), and (iv) zein-wax composite and zein-fatt y acid blend fi lms (12).These composite and blend materials showed bett er homogeneity or improved mechanical and water vapour barrier properties than the single biopolymer ones.16) reported that zein-based fi lms blended with oleic acid had bett er water vapour barrier properties and fl exibility.Barbosa De Almeida et al. (17) reported the improvement of zein-oleic acid fi lm homogeneity when diff erent concentrations of xanthan gum were added; nevertheless, their eff ect on the fi lm's functional properties was not evaluated.Following this idea, the aim of the current study is to evaluate the eff ect of adding xanthan gum on the structural and functional properties of zein-oleic acid blend fi lms.

Film composition and preparation
Zein-based fi lms blended with oleic acid (Z-OA) and zein-based fi lms blended with oleic acid and xanthan gum (Z-OA-XG) were prepared by adding 20 % (by mass per volume) zein to 95 % aqueous ethanol during 5 min of mechanical stirring at (65±0.5) °C.Glycerol, emulsifi er and oleic acid were added to the solution at 10, 5 and 70 % (by mass), respectively; then 0.05 % (by mass per volume) xanthan gum was added to form the Z-OA-XG solution and then the Z-OA and Z-OA-XG solutions were mechanically stirred for 10 min.A volume of 50 mL of solution was poured onto a rectangular glass plate and air-dried overnight at room conditions.Films were peeled off and conditioned according to ASTM D618-13 (18) at 57.6 % relative humidity (RH) and (24±2) °C for at least 40 h prior to analyses.

Film thickness
Thickness was measured with a digital micrometer (model P54; Digimess Instruments Ltd., Derby, UK) with 0.001 mm precision.Five thickness measurements were taken, one at the centre and four around the perimeter.The average thickness value was used in further calculations.

Light permeability (opacity)
Opacity was assessed at 600 nm in a spectrophotometer (model SP-220; Bioespectro Equipar, Curitiba, PR, Brazil).Film samples were cut into 9 mm×43 mm rectangular shapes and placed in the internal side of the spectrophotometer cell (14,19).Six specimens of each fi lm were tested and opacity was calculated according to the following equation: /1/ where x is the average fi lm thickness in mm and A 600 nm is the absorbance at 600 nm.

Water solubility
Film water solubility was defi ned as the content of dried matt er solubilized aft er 24 h of immersion in water.Films were cut into 2-cm (diameter) disks and dried in oven at (105±2) °C for 24 h.Samples were weighed (initial mass, m i ) and immersed into 50 mL of distilled water at (27±2) °C for 24 h under agitation in an orbital shaker (model MA-410; Marconi, Piracicaba, SP, Brazil) at 76 rpm.Aft er 24 h of immersion, the samples were taken out and dried (fi nal mass, m f ) under the same conditions mentioned before, to determine the mass of the dried matt er that was not solubilized in the water (14,20).Three specimens of each fi lm were tested and water solubility was calculated based on the following equation:

Water vapour permeability
Water vapour permeability (WVP) of the fi lms was measured gravimetrically according to the ASTM E96/96M-14 standard, desiccant method (21).Test cells were covered and sealed by the fi lm samples and placed in a controlled chamber (desiccator) maintained at RH=51 % by a saturated solution of calcium nitrate.Silica gel activated at 200 °C was used to maintain RH=0 % inside the test cells.Desiccator was stored at (24±2) °C.Three specimens of each fi lm were tested and WVP was calculated according to the following equation: /3/ where S is the saturation of vapour pressure at test temperature (2985 Pa at 24 °C), R 1 is RH in the test desiccator expressed as a fraction, R 2 is RH inside the test cell expressed as a fraction and x is the average fi lm thickness.WVT is the water vapour transmission, calculated as follows: /4/ where m g is the mass change (gain) of the test cell, t is the time needed for the mass change and a is the test area (12.57cm 2 ).The ratio of m g /t was obtained from the slope of the linear portion of the plot of m g vs. t.

Oxygen permeability
The oxygen transmission rate (OTR) was measured according to the ASTM F1927-14 standard (22), using an oxygen permeation instrument (OX-TRAN ® model 2/20; Mocon, Minneapolis, MN, USA).OTR of the fi lm was determined under control conditions (23 °C and RH=0 %).The fi lm was placed between two sides of the test chamber, one side was exposed to carrier gas containing 98 % N 2 and 2 % H 2 while the other side was exposed to test gas (pure O 2 ).The sensor monitored the exit port of the carrier gas side measuring the amount of present oxygen.OTR was calculated according to the following equation: where V(O 2 ) is the measured volume of oxygen, t is the time required for reaching the stationary state and a is the transfer fi lm area (100 cm 2 ).Oxygen permeability (OP) was calculated as follows: /6/ where x is the fi lm average thickness and p is the partial oxygen pressure.

Mechanical test
Mechanical properties were evaluated according to the ASTM D882-12 standard (23).Elongation at break (η), tensile strength (σ) and Young's modulus (YM) were tested with a texture analyzer (TA.XT Plus; Stable Microsystems Ltd., Godalming, Surrey, UK) using the Exponent soft ware and the A/TG probe (Stable Microsystems Ltd.).Test fi lms were cut with a guillotine according to ASTM D6287-09 standard (24) into rectangular strips 100 mm long and 15 mm wide.Thickness was measured in fi ve points along the sample in order to assure thickness uniformity.Initial grip separation and crosshead speed were set at 50 mm and 500 mm/min, respectively.Stress and strain were recorded during the extension of the strips and minimum 5 specimens were analyzed.

Microstructure
Film superfi cial and cross-section microstructure were observed under high vacuum and constant temperature using a scanning electron microscope (model EVO LS15; Carl Zeiss, Jena, Germany) equipped with secondary electron detector.Film samples were covered with gold and images were taken at 15-20 kV with magnifi cation of 1000 and 5000×.Cross-section images were obtained by cryogenic fracture; immersing the samples into liquid nitrogen for 2 min.Film samples were fi xed to the microscope stubs using a conductive carbon double-sided tape.

Statistical analysis
Statistical analysis was accomplished with Statgraphics Centurion, v. 16.1 (Statpoint Technologies, Inc., Warrenton, VA, USA).An analysis of variance and a Tukey's multiple comparison test were performed to detect signifi cant diff erences between Z-OA and Z-OA-XG fi lm properties with 5 % signifi cance level.

Results and Discussion
Z-OA and Z-OA-XG blend fi lms were homogeneous and smooth, without visible pores and without greasy touch perception; fi lm components were visibly well integrated in the matrix.Z-OA-XG blend fi lm had glossy appearance and britt le touch perception probably due to the packed and arranged structure of the xanthan gum polysaccharides (25).

Opacity of the zein-based blend fi lms
Opacity is an indicator of the light barrier property of the fi lm, representing the capacity of the fi lm to protect the product against deterioration caused by light.The Z--OA-XG fi lm exhibited an increase in the light barrier property over 60 % compared to the Z-OA fi lm (Table 1).The addition of xanthan gum to zein-oleic acid blend fi lm promoted the reduction of light transmission through the fi lm, possibly as a consequence of the packed structure of the polysaccharide.
Blend fi lms of soya protein isolate and gelatin reported by Denavi et al. (26) exhibited lower opacity than Z-OA and Z-OA-XG blend fi lms.Besides, the Z-OA-XG blend fi lm of the current study showed higher light barrier than the same blend fi lm reported by Barbosa De Almeida et al. (17).The higher opacity displayed by the Z--OA-XG blend fi lm in this study is probably due to the diff erence in the composition of both fi lms.For instance, glycerol addition helps improving fi lm fl exibility by creating spaces in the matrix (27), although it decreases the fi lm light barrier.Thus, the lower glycerol volume fraction in the Z-OA-XG fi lm improved its opacity.Similarly, Barbosa De Almeida et al. (17) reported blend fi lms prepared with 75 % ethanol, while Z-OA-XG blend fi lms here were prepared with 95 % ethanol.Higher ethanol volume fraction improves zein solubilization, increasing intermolecular interactions between protein chains and between protein and other fi lm components; hence, creating a tight fi lm matrix that prevents light transmission (27).

Water solubility of the fi lms
Water solubility indicates the water affi nity of the fi lm, representing its water-resistance capacity.As shown in Table 1, Z-OA-XG fi lm displayed greater solubility than Z-OA fi lm, possibly due to the higher polarity of the fi lm caused by the addition of a hydrophilic compound (11,25).
Polysaccharide-based fi lms produced from chitosan, galactomannan or agar exhibited water solubility of over 22 % (20).Similarly, the water solubility of gelatin-based fi lms blended with 1 % sunfl ower oil was around 80 % (14).Based on that, Z-OA and Z-OA-XG fi lms showed greater water-resistance capacity than the fi lms mentioned above, due to the hydrophobic nature of zein as well as the high oleic acid volume fraction of the Z-OA and Z-OA-XG blend fi lms.

Gas barrier properties of the fi lms
The gas barrier properties of the Z-OA and Z-OA-XG blend fi lms that were assessed were water vapour and oxygen permeability ( (WVP) refers to the barrier property of the fi lm, indicating the degree of moisture transfer between the packaged product and the surroundings.As shown in Table 2, both blend fi lms, Z-OA and Z-OA-XG, displayed similar WVP values.Therefore, increasing the fi lm's polarity by adding xanthan gum had no eff ect on moisture transfer, which was expected, based on previously published data (11,28).This is probably a consequence of the low xanthan gum fraction added to Z-OA-XG fi lm.
The blend fi lm based on konjac glucomannan, chitosan and soya protein isolate reported by Jia et al. (29) showed higher WVP (5.18•10 -11 g/(Pa•s•m)) than Z-OA and Z-OA-XG blend fi lms.Likewise, protein-based fi lms produced from whey protein isolate (30) and blend fi lms prepared from zein and wheat gluten (31) displayed higher WVP values (66•10 -9 and 5.0•10 -11 g/(Pa•s•m), respectively) in comparison with Z-OA and Z-OA-XG fi lms.Based on that, the Z-OA and Z-OA-XG blend fi lms exhibited a bett er water vapour barrier property than other biodegradable fi lms as a consequence of their higher hydrophobicity.
Similarly to WVP, oxygen permeability (OP) indicates the oxygen transfer between the packaged product and the surroundings.This fi lm property is of great importance when the product can suff er oxidative reactions that deteriorate its composition and integrity.
As shown in Table 2, the addition of xanthan gum to zein-oleic acid blend fi lms had no impact on fi lm oxygen permeability.Both fi lms, Z-OA and Z-OA-XG, displayed similar OP values (p<0.05),likely caused by the low fraction of xanthan gum that was added.Z-OA and Z-OA-XG blend fi lms exhibited higher OP in comparison with polysaccharide-based fi lms reported by Cerqueira et al. (20) and Matt a et al. (32) which showed oxygen permeability around 1•10 -15 and 6.3•10 -17 g/(Pa•s•m), respectively.This was probably an eff ect of the high oleic acid concentration, which increased oxygen solubilization and its diff usion through the fi lm (33).Furthermore, several studies have reported bett er oxygen barrier properties of polysaccharide-based fi lms than protein-and lipid-based fi lms due to the structure and polarity of the polysaccharides (34).

Mechanical properties of the fi lms
Mechanical properties such as tensile strength (σ), elongation at break and Young's modulus (YM) represent the toughness and elasticity (η) or britt leness of the fi lm.Thus, as shown in Table 3, Z-OA-XG fi lm displayed greater toughness (higher σ) than Z-OA due to the packed and arranged structure of xanthan gum; nevertheless, polysaccharide structure increased the stiff ness and britt leness of Z-OA-XG blend fi lm (higher YM and lower η, respectively).
For instance, gelatin fi lms and 10 % zein-based fi lms with 3 % glycerol had higher tensile strength, around 22 and 10 MPa, respectively (35), in comparison with Z-OA and Z-OA-XG fi lms.The lower σ of the zein-based fi lms in this study was probably due to the higher glycerol volume fraction (10 %) added to the fi lms as well as the high oleic acid fraction.Glycerol and oleic acid are plasticizer agents that reduce the tensile strength of the fi lms (33).Moreover, protein-based fi lms produced from cowpea protein isolate with 10 % polyethylene glycol reported by Hewage and Vithanarachchi (36) had lower tensile strength (σ=6.6 MPa) compared with Z-OA and Z-OA-XG fi lms.

Blend fi lm microstructure
Scanning electron microscopy allows for the evaluation of the distribution and integration of fi lm components into the matrix.Z-OA and Z-OA-XG blend fi lms displayed homogeneous surfaces with good integration of fi lm components (Figs.1a and b).Film micrographs showed the presence of square-shaped materials on Z-OA and Z-OA-XG fi lm surfaces.As both fi lms had the same materials on the surface, we believed that these were due to contaminant substances in the reagents used for fi lm preparation.
The cross-section of Z-OA and Z-OA-XG fi lms (Figs.1c and d) showed the presence of porosity or orifi ces caused by ethanol evaporation (30).Apparently, Z-OA--XG blend fi lms were more porous as a consequence of the higher rigidity determined during the mechanical tests.

Conclusions
Performance of blend fi lms based on zein-oleic acid (Z-OA) and zein-oleic acid and xanthan gum (Z-OA-XG) were evaluated and compared.Z-OA and Z-OA-XG had homogeneous surfaces with good integration of fi lm components.Z-OA exhibited lower water solubility whereas Z-OA-XG showed greater opacity.Hydrocolloid addition had no eff ect on gas barrier properties since both blend fi lms, Z-OA and Z-OA-XG, showed similar water vapour and oxygen permeability; nonetheless, xanthan gum struc ture infl uenced mechanical properties of the fi lm, increasing the strength (higher tensile strength) and rigidity (higher Young's modulus and lower elasticity).Hence, polysaccharides such as xanthan gum should be added to zein-oleic acid blend fi lms, mainly when the food products tend to be oxidized as a consequence of the infl uence of light.

Table 1 .
Water solubility and light barrier properties of zein--based blend fi lms

Table 2 .
Water vapour and oxygen permeability properties of zein-based blend fi lms

Table 3 .
Mechanical properties of zein-based blend fi lms