zein nanocomposite film using pH-driven method

Industrial Crops & Products 130 (2019) 71–80

Contents lists available at ScienceDirect

Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop

Preparation and characterization of curcumin loaded caseinate/zein nanocomposite film using pH-driven method Lei Wanga, Jia Xueb, Yue Zhanga, a b

T

⁎

Department of Food Science and Technology, University of Nebraska-Lincoln, 1901 North 21st Street, Lincoln, Nebraska, 68588, United States College of Food Engineering and Nutritional Science, Shaanxi Normal University, No.620. West Chang’an Avenue, Xi’an, Shanxi, 710119, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Zein Sodium caseinate (NaCas) Curcumin-loaded nanoparticles Antioxidant film pH-driven method

Functional edible films manufactured from crop by-products is promising for food industry. In this study, antioxidant films prepared from self-assembled curcumin-loaded sodium caseinate (NaCas)-zein composite nanoparticles was investigated. Nanocomposite film-forming dispersions were obtained by dissolving curcumin into polymer mixtures with NaCas/zein ratios of 100:0, 75:25 and 50:50 at pH 12.0, and then neutralized back to pH 7.0. The colloidal particles showed an average particle size and ζ-potential around 131–184 nm and -40 mv, respectively. Regardless of the NaCas/zein ratio in film-forming dispersions, the curcumin presence enabled NaCas/zein film to exhibit red-yellow color and antioxidant properties as evaluated by DPPH radical scavenging activity. Surface properties of films were further characterized by contact-angle measurement and scanning electron microscopy. An increasing proportion of zein could significantly improve the tensile strength (from 3.12 to 7.85 MPa) and constrain moisture transfer as indicated by decreased water vapor permeability from 0.87 to 3.56 × 10−10 g·Pa·m-1·s-1. However, the elongation resistance of NaCas-based films was compromised from 50.5% to 30.8%. When the zein proportion in the formulation reached 50:50, curcumin showed the best stability under elevated temperatures and long-time storage. The underlying mechanism of this alcohol-free process to prepare zein-based nanocomposite film is also proposed and discussed.

1. Introduction Biopolymer-based edible films or coatings are promising as alternatives to diminish the use of commercial nondegradable and nonrenewable food packaging materials to enhance food quality and extend shelf life. Among the various natural biopolymers, proteins, especially those isolated from food processing by-products, are very attractive as film-forming materials due to their excellent nutritional values, numerous functional properties and generally recognized as safe (GRAS) nature. Films, made from soy protein, whey, zein and wheat gluten, were proved to possess excellent mechanical and organoleptic properties, as well as gas barrier characteristics (Cho et al., 2002; Li et al., 2012; Seydim and Sarikus, 2006). For example, various functional components like flavorings, colorings, antioxidants and antibacterial agents have been incorporated into protein films to improve the organoleptic properties of packed foods (Bourtoom, 2008). Although the tensile strength of the soy protein isolate (SPI)-based film was still lower than that of the commercially available synthetic film, a comparable EAB% value (178.2% of SPI-based vs. 179.2% of nylon-metalocene catalyzed linear low-density polyethylene films) was observed

⁎

(Cho et al., 2010). In another study, the tensile strength of rapeseed protein-gelatin blend film reached as high as 53.45 MPa, which was much better that low-density polyethylene films with TS value around 16–18 MPa (Jang et al., 2011). Casein, is the principal protein in bovine milk with the supramolecular structures known as “casein micelles” (Atarés et al., 2010). Casein shows flexible random-coil nature and possesses the ability to cross-link with each other by the extensive non-chemical intermolecular forces. Thus, the utilization of casein or its commercial forms such as sodium caseinate (NaCas) as food wrapping materials has been extensively reviewed (Atarés et al., 2010; Schou et al., 2005; Tomasula et al., 2003). Without further treatment, NaCas films could be readily formulated after dehydration and showed impressive transparency, flexibility, insipidity, and good barrier property for oxygen and other nonpolar molecules (Atarés et al., 2010; Tomasula et al., 2003). However, like other hydrophilic polysaccharide- or protein-based films, NaCas films displayed poor moisture barriers and mechanical stress that limited their applications (Schou et al., 2005). Several approaches were explored to lessen its water vapor transmission, including lamination of an additional hydrophobic layer, incorporation of lipophilic

Corresponding author. E-mail address: [email protected] (Y. Zhang).

https://doi.org/10.1016/j.indcrop.2018.12.072 Received 6 August 2018; Received in revised form 19 December 2018; Accepted 21 December 2018 0926-6690/ © 2018 Published by Elsevier B.V.

Industrial Crops & Products 130 (2019) 71–80

L. Wang et al.

1.5:0.5, and 1:1, respectively. For curcumin-loaded nanodispersions, curcumin was added into film-forming dispersions at a concentration ranging from 0 to 2.0 mg/mL. After stirring for 30 min, the pH was neutralized back to 7.0 using 50 or 500 g/L citric acid, followed by addition of glycerol as a plasticizer to achieve a final concentration of 0.3 g/g protein. After another 1 h stirring, nanodispersions were subjected to the removal of insoluble solids and air bubbles using filter paper (Whatman No. 1) and degassed vacuum.

components, and reinforcement with biopolymer-based nanoparticles into film-forming dispersions (Yin et al., 2014). Zein, the major storage protein in corn, is the main by-product from industrial corn starch and ethanol processing (Gilbert et al., 2017). It has been documented as an efficient co-material to improve the moisture barrier properties of NaCas-based films, reduce the loss, as well as perform the sustained release of incorporated bioactive compounds (Li et al., 2012). However, these improvements were obtained at the expense of losing the transparency and extensibility (Cho et al., 2002). Also, alcohol or other organic solvents are required to prepare films composed of zein and dissolve the incorporated hydrophobic bioactive compounds such as curcumin. Although these solvents could be removed in the subsequent drying-process, a preparation process without conventional organic solvents would be more desirable for “green consumption” to reduce environmental waste and hazard (Wang and Zhang, 2017). In our previous study, an organic solvent-free method, called “pH-driven” emulsification process, had been developed to prepare eugenol incorporated NaCas-zein nanoemulsions (Wang and Zhang, 2017). Thanks to the pH-dependent solubility of eugenol and zein as well as the self-assembly properties of NaCas, the three components were readily dispersed in hot alkaline solutions, while the following neutralization facilitated oil to be encapsulated into the NaCas/zein complex through the hydrophobic interaction, electrostatic attraction and hydrogen bonding, which are also main forces reuquired for film formations (Bourtoom, 2008; Moradi et al., 2016; Wang and Zhang, 2017). To retard or prevent the food oxidative deterioration, including the nutritional losses, color and flavor changes, and prolong food shelf-life, films incorporated with antioxidants especially natural ones have become a focus of interest (Byun et al., 2010). Curcumin, extracted from the rhizomes of Curcuma longa, is a representative lipophilic phytochemical that has been found to possess attractive antioxidant activity and pH-dependent solubility (Pan et al., 2014). At the alkaline condition within a short period, it was deprotonated with increased watersolubility but negligible degradation. Followed by a neutronization, curcumin was protonated again and simultaneously incorporated into the biopolymer matrix driven by hydrophobic forces and hydrogen bonds (Pan et al., 2014). So, it is conceivable that curcumin could be encapsulated into NaCas/zein nanocomposite particles using the pHdriven self-assembly method. Thus, the objective of current work was to manufacture NaCas/zein nanocomposite films with or without curcumin incorporated by this pH-driven self-assembly method. This is the first study to utilize the innovative method to prepare curcumin loaded NaCas/zein nanocomposite films without the utilization of alcohol or other organic solvents. The formation pathway of zein/NaCas nanoparticle-based films was proposed. Additionally, the mechanical, physical properties and antioxidant activity of resultant films were characterized.

2.3. Film formation Films were prepared by casting 50 mL of film-forming solutions on Fisher glass petri dishes (diameter = 15 cm) and drying at ambient conditions (21 °C) for 36 h. The dried films were peeled and then conditioned in a desiccator with 50 ± 1% relative humidity (RH) controlled by a saturated solution of magnesium nitrate for at least 48 h before measurement. Films were prepared in triplicate. 2.4. Encapsulation efficiency (EE) and turbidity The EE of curcumin in the nanodispersions was determined based on a previously reported method (Patel et al., 2013). Briefly, one mL of freshly prepared nanodispersions was partitioned three times with an equal volume of ethyl acetate and vortexed to completely extract the entrapped curcumin. The upper phase was transferred to a quartz cuvette, and then the absorbance at 425 nm was taken after appropriate dilutions using the Evolution 201 UV–vis spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The absorbance of zein/ NaCas without curcumin was also measured as blank. Curcumin concentration was quantified based on a pre-established standard curve of curcumin (0–6 μg/mL) in ethyl acetate (R2 = 0.9999), and EE was then calculated by Eq. (1):

EE (%) =

Curcumin in the nanodispersions (mg / mL) × 100% Total amount of added curcumin (mg / mL)

(1)

The turbidity of freshly-prepared dispersions at a final concentration of 0.5% (w/v) protein was determined at 600 nm using the aforementioned spectrophotometer. All the measurements were carried out in triplicate. 2.5. Hydrodynamic diameter and ζ-potential of nanocomposite dispersions The particle diameter and ζ-potential of film-forming dispersions were characterized by dynamic light scattering (DLS) technique using Nano − Zetasizer (Malvern Instruments Ltd., UK) at a fixed scattering angle of 173° and 25 °C. The dispersions were diluted by Milli − Q water (pH 7.0) to a final protein concentration of 0.01% (w/v) to avoid multiple scattering effects. The weight-volume diameter (d4,3) was calculated as Eq. (2):

2. Materials and methods

∑ ni di4 ∑ ni di3

2.1. Materials

d4,3 =

NaCas was purchased from American Casein Company (Burlington, NJ). Zein (> 99%) and curcumin (> 98%) were obtained from Acros Organics (Morris Plains, NJ, USA). Other chemicals were purchased from Fisher Scientific Inc. (Pittsburgh, PA, USA) and of analytical grade.

where ni is the particle number in the determined size class, and di is the hydrodynamic diameter value determined by DLS directly. The d4,3 represents the size class around which most of the mass of the particles lies and is sensitive to the presence of small amounts of large particles (Kaci et al., 2014).

2.2. Preparation of NaCas/zein nanocomposite dispersions

2.6. Film characterization

Two grams of NaCas or zein was hydrated in 100 mL Milli − Q water at 21 °C, respectively. The pH of dispersions was brought up to 12.0 using 3 M NaOH under magnetic stirring at 600 rpm. Film-forming dispersions with a final protein concentration at 2% (w/v) were prepared by mixing stock NaCas and zein solutions at a mass ratio of 2:0,

2.6.1. Film thickness Film thickness was determined using a digital microcaliper (Mitutoyo Corp., Kawasaki, Japan) with 0.001 mm precision. Measurements were taken at five random locations for each film sample. Mean values from ten replicates were reported and used to 72

(2)

Industrial Crops & Products 130 (2019) 71–80

L. Wang et al.

calculate the mechanical and barrier properties of the film.

where the slope is obtained from the linear regression plot of weight loss vs. time in g/(m2·s), d is the thickness of film specimens in m, A was the exposed area of film in m2, and Δp is the actual difference of water vapor pressure across the film specimens in Pa.

2.6.2. Color measurement The color of films was measured using a portable meter (CR-300 Chroma Meter, Minolta Camera Co., Osaka, Japan). Film specimens were placed on the surface of a standard white plate (L = 94.47, a = -0.33, and b = -0.20) and Hunter L, a, and b color values were measured. Color differences (ΔE) were defined as follows:

(L − L′)2 + (a − a′)2 + (b − b′)2

ΔE =

2.6.7. Contact angle The contact angle of films was evaluated by the sessile drop method with a DSA10 goniometer (KRÜSS GmbH., Hamburg, Germany) (Chevalier et al., 2018). A droplet (2 μL) of Milli − Q water gently dripped onto the film surface using a micro-syringe (Becton, Dickinson and Company, NJ, USA). The contact angle, as well as kinetic sorption from 10 to 60 s, was measured at the room temperature (21 °C). For each film formula, five replicates were performed, and the average was taken. Side-view images of droplets were captured and used to compare the water sorption of films over time.

(3)

where Lʹ (95.79), aʹ (0.05) and bʹ (1.73) are Hunter color values of standard low-density polyethylene (LDPE) film measured by the same method (Benavides et al., 2012). 2.6.3. Transparency (TR) Film transparency was determined according to a previously published method (Ramos et al., 2013). In brief, film samples were cut into strips (1 cm × 4 cm) and placed on the internal side of a plastic cuvette. The percent TR was determined by measuring absorbance at 600 nm using the aforementioned spectrophotometer while empty cuvette was treated as blank, according to Eq. (4):

TR =

Absorbance600 nm Film thickness (mm)

2.6.8. Mechanical properties The tensile strength (TS, MPa) and elongation at break (EAB, %) of films were measured according to the ASTM D882 method using TA-XTplus texture analyzer (Stable Micro Systems, London, UK) equipped with a tension grip accessory (ASTM International, 2002). Films equilibrated in 50% RH (21 °C) were cut into 2.5 cm × 8 cm rectangular strips. The initial gap and cross-head test speed were 50 mm and 50 mm/min, respectively. All the mechanical measurements were examined in triplicate.

(4)

2.6.4. Field emission scanning electron microscopy (FE-SEM) The surface morphology of films was characterized using a Hitachi S-4700 FESEM (Hitachi, Ltd. Tokyo, Japan). The freshly-prepared films were adhered to the conductive carbon tape mounting onto the specimen stubs and dried at 50 °C for 24 h, followed by being coated with a conductive gold layer (< 0.5 nm) using a Desk V TSC sputter-coater (Denton Vacuum, LLC, Moorestown, NJ), and stored in a vacuum oven (3 kPa) at 21 °C before taking images.

2.7. Antioxidant activity and stability of curcumin Antioxidant capacity of the curcumin encapsulated films was expressed as (2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity/mg film, using a previous method with slight modifications (Li et al., 2012). Briefly, 50 mg of film specimen was vigorously mixed with 25 mL methanol for 5 min and incubated in the dark (21 °C) for 3 h. The mixture was then centrifuged (Allegra X-I5R, Beckman Coulter Inc., CA, USA) at 4000 g at 10 °C for 10 min, resulting in two separate phases. The upper phase was 10 times diluted with methanol, and then 1 mL of the diluted solution was transferred into a 10 mL tube containing 2 mL of 0.1 mM DPPH solution. The mixture was vortex thoroughly and allowed to stand in the dark (21 °C) for 30 min. The absorbance at 517 nm of each solution was measured to determine the remaining amount of DPPH. The DPPH radical scavenging activity was calculated by Eq. (6):

2.6.5. Film solubility and moisture content (MC) The aqueous solubility of films was gravimetrically measured according to a method by Floros et al. after modification (Ozdemir and Floros, 2008). Briefly, film samples were cut into square pieces (2.5 cm × 2.5 cm) and placed in a desiccator containing phosphorus pentoxide (0% RH) for 72 h. Then, the dried samples were individually weighed and placed in 15 mL screw-topped centrifuge tubes with 10 mL of Milli − Q water. After being magnetically stirred at 4 °C for 2 min, followed by centrifugation (Allegra X-I5R, Beckman Coulter Inc., CA, USA) at 3000 g for 20 min and removal of the supernatant, tubes with insoluble components were dried at 50 °C for 6 h. The solubility was calculated from the initial and final dry weight of tubes with film samples. The moisture content (MC) of film specimens was determined by drying at oven (105 ± 1 °C) for 12 h and expressed as the percentage of weight loss by initial film weight.

DPPH scavenging activity (%) =

Slope × d A × Δp

Abscontrol

× 100%

(6)

where the DPPH ethanol solution alone was used as the control. Stability of curcumin in the form of dispersions and the resultant films was evaluated by heating the dispersions at 50 °C for 100 min or exposing the film to natural sunlight (21 °C, RH = 50%) for 15 days, respectively. Control the curcumin solution at 2 mg/mL was prepared by dispersing the stock ethanol solution of curcumin with 10 mg/mL into water. The amount of curcumin after treatment was determined using spectrophotometry as described above.

2.6.6. Water vapor permeability (WVP) WVP of films was determined by using poly(methyl methacrylate) permeability cups (BYK-Gardner, Geretsried, Germany) with 25 cm2 area according to the standard gravimetric method of ASTM E96-95 (1995c) with modifications (Li et al., 2012). Briefly, freshly-prepared films without physical defects (i.e., cracks and pinholes) were sealed to cups containing 15 mL of double-distilled water to provide 100% RH. Then, the cups were placed in a desiccator equilibrated at 75% RH created by a saturated solution of NaCl. The weight loss of cups was recorded at every 2 h interval using an analytical balance to the nearest 0.0001 g in duplicate. The WVP value was calculated from Eq. (5):

WVP =

Abscontrol − Abssample

2.8. Statistical analysis Data are recorded in at least duplicates and reported as the mean ± standard deviation (SD). Analysis of variance (ANOVA) was processed using the Statistical Analysis Software (version 9.4, SAS Institute, Cary, NC, USA). Tukey test was used to compare the differences between pairs of means. The significance level was defined as Pvalue < 0.05.

(5) 73

Industrial Crops & Products 130 (2019) 71–80

L. Wang et al.

Fig. 1. Schematic illustration of the film formation based on curcumin-loaded NaCas/zein colloid nanoparticles.

and hence more heterogeneous fibrous network of the resultant films. To confirm our hypothesis on the formation mechanism of nanocomposite films and further explore their properties and potential applications, the film-forming nanodispersions, as well as resultant films, were characterized by various techniques as discussed below.

3. Results and discussion 3.1. Mechanism of nanocomposite film dispersion formation As discussed in our previous study (Wang and Zhang, 2017), the dissociation of NaCas and dissolution of zein occurred at high alkaline pH (> 11.0), allowing the inward diffusion of zein in the opened NaCas network. Moreover, in the subsequent neutralization to pH 7.0, NaCas micelles was re-associated with in situ folded zein, thus producing stable colloidal particles when NaCas/zein mass ratio was equal to or greater than 1:1. This organic solvent free method can be utilized to develop self-assembled curcumin-loaded NaCas/zein nanoparticles and the resultant nanocomposite films. The schematic principle of film formation is proposed in Fig. 1. At pH 12.0, curcumin was dissolved due to deprotonation, allowing the access of curcumin molecules into opened NaCas network, and interactions with unfolded NaCas and zein proteins. When the pH of the dispersion was slowly adjusted back to 7.0, the solubility of curcumin molecules in dispersion gradually decreased, and more free curcumin molecules tended to bind with the proteins via non-covalent interactions, while core-shell zein nanoparticles stabilized by re-associated NaCas were self-assembled at the same time. A few curcumin molecules could bound with the flexible NaCas in dispersion, or the surface of NaCas coated zein nanoparticles via hydrogen bonds or van der Waals. However, since the environment became a bad solvent for both zein and curcumin, most curcumin molecules migrated into the hydrophobic core of zein nanoparticles in an amorphous form (Hu et al., 2015). Thanks to the repulsive electrostatic and steric interactions provided by sufficient hydrophilic NaCas, the curcumin loaded nanoparticles could be well dispersed in the aqueous phase. As shown in Fig. 1, the lack of NaCas could cause unstable zein aggregates, which may weaken the compactness and homogeneity of the protein network of resultant films (Li et al., 2012). To avoid this undesirable phenomenon, formulations with NaCas/zein mass ratio ≥ 1:1 were hereby chosen as the film-forming dispersions. In the subsequent film-drying process, the evaporation of water led to an increase in protein concentration, which promoted the interaction between the protein chains, migration of NaCas molecules themselves and their deposition onto the surface of zein nanoparticles, and eventually the self-assembly into films. Since zein molecules were self-folded into intact colloidal nanoparticles surrounded by sufficient NaCas, the film matrix was mainly built by NaCas with zein nanoparticles embedded in the network. Curcumin was preferably remained in the hardcore of zein nanoparticles and thus may not affect the microstructure of films. The retention of its antioxidant activity could be maximized after this encapsulation. However, with higher zein contents, the intensification of depletion phenomena with time was expected to take place, leading to the formation of bigger zein aggregates,

3.2. Characterization of film-forming nanodispersions 3.2.1. Turbidity and encapsulation efficiency (EE) The turbidity (absorbance at 600 nm) and appearance of filmforming nanodispersions with and without curcumin are shown in Fig. 2A. As expected, all the three formulations at 2% (w/v) protein retained a homogenous and translucent appearance with no visible precipitates (Fig. 2A inset: upper panel). However, the turbidity was significantly (P < 0.05) enhanced with increasing zein ratio, e.g., 0.21 vs. 0.52 in the formulation of NaCas/zein at 100:0 and 50:50, respectively. The turbidity may be affected by several factors, including number, density, and dimension (Giroux et al., 2010). As shown in Table 1, the particle dimension of nanodispersions measured by DLS method was in the following order: 100:0 > 75:25 = 50:50. The turbidity tendency at the same protein concentration was not in accordance with the particle dimension, which suggested that the increased turbidity by increasing zein concentration may be contributed by an enhanced particle density and more compact structure of zein nanoparticles. To prepare antioxidant dispersions, curcumin was codissolved in the polymer solutions at alkaline pH. Irrespective of NaCas/zein ratios, all dispersions exhibited dark yellow color (Fig. 2A: inset). However, the turbidity of dispersions showed no significant difference with increasing amount of curcumin (P < 0.05), indicating curcumin was successfully encapsulated within the polymer matrix as proposed above. The EE of nanodispersions as affected by curcumin concentration and NaCas/zein ratio is presented in Fig. 2B. A gradual decrease in EE with increasing content of curcumin and zein could be observed. When the curcumin concentration was enhanced from 0.5 to 2.0 mg/mL, the EE of curcumin in pure NaCas dispersions significantly decreased (P < 0.05) from 95.5% to 76.3%, followed by that prepared by NaCas/ zein at 75:25 (from 87.1% to 67.9%) and 50:50 (from 76.2% to 57.1%). These results were in accordance with other related studies (Pan et al., 2014; Sun et al., 2017). As reported (Pan et al., 2014), the EE of curcumin in NaCas nanoparticles by the pH-cycle process was around 70–100%. In another study, curcumin loaded zein nanoparticles showed an EE ranging from 82.7% to 42.5% using antisolvent co-precipitation method (Sun et al., 2017). With increasing zein mass, a lower EE of curcumin and more turbid appearance of nanodispersions could be obtained, which was consistent with others using antisolvent preparation method (Sun et al., 2017). This observation could be ascribed 74

Industrial Crops & Products 130 (2019) 71–80

L. Wang et al.

3.2.2. Particle size and ζ-potential The size of nanoparticles can affect the microstructure of films, which plays a crucial role in films characteristics, such as mechanical and barrier properties (Seydim and Sarikus, 2006). During the drying process, the destabilization of nanoparticles caused by solvent evaporation was also in direct proportion to size (Fabra et al., 2012). As shown in Table 1, for the film-forming dispersions without curcumin, the weight mean diameter (d4,3) of NaCas was 183.1 nm and exhibited a significant decrease (P < 0.05) to 117.1–120.9 nm with the addition of zein. No visible precipitates could be observed indicating a new interface was created as a result of pH-cycle treatment to protect hydrophobic zein against aggregation in water. The reduction of d4,3 was not significantly affected by the NaCas/zein ratio, suggesting that amount of casein molecules absorbed onto the surface of zein nanoparticles was sufficient in the currently tested concentrations. Similarly, a reduction of d4,3 from 183.1 nm to 139.3 nm was also observed when curcumin was entrapped solely by NaCas nanoparticles. These observations confirmed the re-assembly of caseins during pH-cycle treatment (Pan et al., 2014). As stated in Fig. 1, during the neutralization, re-associated caseins entrapped the gradually insoluble curcumin or zein within their biopolymeric network, and the hydrophobic attraction between caseins and curcumin/zein intensified the packing of casein molecules, thus leading to a remarkable decrease in particle size (P < 0.05). This phenomenon has been corroborated by other previous studies (Pan et al., 2014; Pan and Zhong, 2016). But for the NaCas coated zein nanoparticles, a larger d4,3 was obtained with increasing amount of curcumin, which was in accordance with those prepared by the antisolvent method (Patel and Velikov, 2014). The enhanced curcumin amount within particle interior increased the hydrophobic character of particles, resulting in bigger value of d4,3 accompanied by lower encapsulation efficacy as observed in Fig. 2B. The ζ-potential of all film-forming dispersions is also shown in Table 1. It is known that colloidal particles with a ζ-potential magnitude no less than 30 mV could typically prevent the aggregation tendency by strong electrostatic repulsion (Wang and Zhang, 2017). Regardless of the dispersion formulas, the mean value of ζ-potential for all samples was about −40 mV, confirming the excellent stability of all dispersions at room temperature. Our results were also in accordance with Sun et al., who reported that the curcumin content did not significantly influence the ζ-potential of curcumin-loaded particles (Sun et al., 2017). To simplify the analysis and maintain a relatively high EE, filmforming nanodispersions with/without 1 mg/mL of curcumin were used to manufacture films for the rest of this study.

Fig. 2. Turbidity (A) and encapsulation efficiency (B) of film-forming nanodispersions with various curcumin concentrations and NaCas/zein ratios. Inset: appearance of dispersions prepared at a curcumin concentration of 1 mg/mL. Different letters within NaCas/zein ratio indicate significant differences (P < 0.05).

3.3. Characterization of nanocomposite films

to the insufficiency of NaCas, which acted as the electrostatic stabilizer on the surface of zein/curcumin nanoparticles, to prevent spontaneous aggregate formation and facilitate better stability of colloidal particles.

3.3.1. Optical properties Optical properties of films are fundamental for packaging purpose, which bring a significant contribution to the customers’ consumption willingness. In this study, transparency of films was investigated by

Table 1 Particle distribution, ζ-potential of the film-forming nanodispersions at various mass ratios of NaCas/zein. Curcumin (mg/mL)

NaCas/zein ratio 100:0

75:25 ζ-potential (mv)

d4,3 (nm) 0 0.5 1.0 1.5 2.0

183.1 156.9 148.3 143.3 139.3

± ± ± ± ±

6.6a 9.7b 8.2b 9.5b 6.9b

−40.9 −41.3 −41.6 −41.0 −42.0

± ± ± ± ±

50:50 ζ-potential (mv)

d4,3 (nm) 2.0a 1.5a 0.8a 1.7a 0.8a

117.1 115.6 119.8 127.2 130.7

± ± ± ± ±

5.9b 8.1b 7.6b 8.9a 9.3a

*Numbers are mean ± standard deviation (n = 3). *Different superscript letters within the same column indicate significant differences (P < 0.05). 75

−41.7 −43.0 −42.8 −42.0 −41.3

± ± ± ± ±

ζ-potential (mv)

d4,3 (nm) 1.9a 1.0a 1.1a 1.5a 1.3a

120.9 128.9 137.6 141.1 146.8

± ± ± ± ±

8.9b 6.7b 5.8ab 8.2a 7.9a

−40.7 −41.0 −41.5 −40.9 −41.0

± ± ± ± ±

0.6a 1.2a 1.5a 0.9a 2.3a

Industrial Crops & Products 130 (2019) 71–80

L. Wang et al.

Table 2 Optical properties of nanocomposite films at various NaCas/zein ratios. Mass ratio (NaCas/zein)

Curcumin (mg/mL)

Abs600

L 100:0 75:25 50:50

0 1 0 1 0 1

2.3 2.3 2.8 2.9 3.1 3.2

± ± ± ± ± ±

ΔE

Color values

nm

c

*

94.3 45.3 91.5 52.3 89.5 48.1

0.15 0.17c 0.21b 0.10b 0.09a 0.23a

a ± ± ± ± ± ±

b

−1.2 ± 0.1 35.6 ± 1.3a −4.6 ± 0.3d 30.0 ± 2.9a −4.9 ± 0.2d 27.5 ± 1.5b

a

c

2.9 3.0b 1.4a 2.0b 3.6a 3.9b

7.4 ± 0.4e 13.1 ± 1.2d 27.8 ± 1.9b 18.9 ± 1.8c 40.4 ± 1.2a 29.1 ± 1.8b

5.9 ± 0.4d 63.0 ± 1.3a 26.8 ± 3.8d 55.7 ± 1.9b 39.5 ± 1.6c 61.5 ± 2.4a

*L – Lightness, a – Redness (positive or Greenness (negative), b – Yellowness (positive) or Blueness (negative). *Numbers are mean ± standard deviation (n = 10). *Different superscript letters within the same column indicate significant differences (P < 0.05). Table 3 Physical properties of nanocomposite films prepared with different NaCas/zein ratios. Mass ratio (NaCas/zein)

Curcumin (mg/mL)

Thickness (μm)

100:0

0 1 0 1 0 1

157.3 158.5 159.8 159.2 160.1 160.6

75:25 50:50

± ± ± ± ± ±

MC

5.3a 3.8a 7.1a 8.1a 9.3a 9.9a

19.6 19.3 13.9 14.0 11.2 11.0

± ± ± ± ± ±

1.3c 0.9c 1.2b 1.0b 0.9a 1.6a

Solubility (%)

WVP (g·Pa−1·m−1·s−1) × 10-10

99.9a 99.9a 99.1a 98.9a 98.8a 99.3a

35.6 36.0 18.1 19.1 12.5 13.2

± ± ± ± ± ±

1.3a 3.1a 1.0c 0.9c 1.5b 2.6b

*Numbers are mean ± standard deviation (n = 5). *Different superscript letters within the same column indicate significant differences (P < 0.05).

(Fig. 3D-F), especially for the films with higher zein proportion in the initial dispersions. This result could be contributed to the presence of particles with a larger size (Table 1) and heterogeneity as indicated by increasing polydispersibility index (PDI) from 0.16 to 0.29 with the decreasing NaCas/zein ratio from 100:0 to 50:50 (data not shown). A higher PDI value suggested that particles in the dispersion would be less stable and agglomerate more quickly during storage (Acevedo-Fani et al., 2015). In the subsequent film-drying process, the magnitude of depletion forces would become much larger (Matsakidou et al., 2013). The higher amount of curcumin and zein in the nanodispersions induced a greater extent of aggregation because of their hydrophobic nature, thus inducing the irregular appearance of film microstructure.

UV–vis absorption at 600 nm, and a higher value for the films indicated a lower transparency. The films showed similar thickness that would not affect the transparency measurement (Table 3). The color parameters including L (lightness), a (greenness), b (yellowness), and total color difference (ΔE) of films were also determined as shown in Table 2. Compared with the opaque films fabricated from laminating zein layer onto NaCas film (Cho et al., 2002), all films in this study were transparent, and easy to peel off with no apparent bubbles. The transparency slightly decreased with the increasing zein proportion in the formulations (P < 0.05), while incorporation of curcumin exhibited negligible impact on the light scattering, confirming the fact that the microstructure of films was mainly affected by zein nanoparticles. In other words, films prepared with higher zein content could be promising barriers to reduce light-induced lipid oxidation when used to coat fat-rich products. No difference was detected in lightness (L) values of films without curcumin, while the yellowness and greenness increased with increasing concentration of zein. Correspondingly, the ΔE was significantly higher with zein than that of NaCas film, which could be attributed to the original yellow color of zein powder. With incorporation of curcumin, the color of films was evidently changed to a darker red-yellow, as confirmed by the drastically increase of a and b values. Generally speaking, the ΔE values of films were mainly dominated by the curcumin concentrations rather than NaCas/zein ratios.

3.3.3. Physicochemical properties The physicochemical properties of films including thickness, moisture content, solubility, WVP and contact angle are respectively summarized in Table 3 and Fig. 4. With an increasing zein proportion, the film thickness showed minor change (P > 0.05), but progressive decrease in moisture content (Table 3), probably because the hydrophobic character of zein induced a marked reduction of film ability to absorb water. Meanwhile, curcumin showed no impact on both thickness and moisture content, which may attribute to the relatively low concentration of curcumin incorporated in the formulation. Solubility, as an essential property of edible films, was measured to evaluate their potential use in food packaging (Pérez‐Gago et al., 1999). As shown in Table 3, all film samples could be easily dissolved in cold water (4 °C). The resultant colloidal dispersions were stable with no observable aggregates, further confirming that zein particles were still distributed within NaCas network after the film-drying process. Some studies reported the addition of alcohol-dissolved zein in film formulation caused drastically decrease in solubility of hydrophilic casein film although it could cause increase of TS (Wihodo and Moraru, 2013). The WVP of films, a parameter to show the moisture transfer between the packaging materials and the food, is a critical capacity to be considered for the storage and distribution of specific products (Pérez‐Gago et al., 1999; Seydim and Sarikus, 2006). Table 3 showed the WVP values as a function of NaCas/zein ratio with or without

3.3.2. Morphology The typical topographies of films were obtained by SEM as shown in Fig. 3. For the films without curcumin (Fig. 3A-C), specimens with NaCas/zein at 100:0 and 75:25 were smoother than that of NaCas/zein at 50:50. It was also clearly observed that the presence of curcumin greatly affected film morphology by causing more wrinkles on the film surface, especially for those with a high concentration of zein. As discussed above, with the protein concentration increased in the filmforming dispersions as a result of the removal of water, the mergence of zein nanoparticles were expected to take place on account of depletion mechanism, and thus the film surface occasionally altered by the presence of prominent aggregates (Li et al., 2012). The encapsulation of curcumin gave rise to a more heterogeneous surface appearance 76

Industrial Crops & Products 130 (2019) 71–80

L. Wang et al.

Fig. 3. SEM images of films prepared at NaCas/zein ratio of 100:0 (A and D), 75:25 (B and E), and 50:50 (C and F) without (upper panel) and with (lower panel) 1 mg/mL curcumin. The images were taken at 20,000× magnification.

curcumin. When zein proportion increased from 0% to 50%, WVP values of films significantly decreased from 35.6 to 12.2 × 10−10 g·Pa-1 m1 -1 ·s (P < 0.05), showing the remarkable improvement of water vapor barrier by zein particles within NaCas network. As previously reported, the transmission of water vapor is a balance of several aspects, including the hydrophobic/hydrophilic ratio of film components, film crystallinity, pathway tortuosity, and presence of structural defects (Cho et al., 2002). It was known that hydrophobic components in film matrix increased the tortuosity factor of the diffusion pathway of water vapor through films. This improvement by hydrophobic zein was in consistency with the observed irregularities on the film surface as shown in SEM micrographs. For NaCas/zein nanocomposite films, NaCas was used for both film matrix formation and stabilizer of zein. During air-drying, there would be a counteraction that more caseins were absorbed onto the merged zein aggregates, which brought about a reduction in caseins available for hydrophilic matrix formation. Curcumin appeared to have a slight but insignificant effect on the decrease of WVP rates (P > 0.05) for all films when compared at the same NaCas/zein ratio. A similar observation was also reported that the water barrier properties of NaCas films were not significantly influenced by the incorporation of alpha-tocopherol, a commonly used hydrophobic antioxidant (Fabra et al., 2011). Since curcumin with a relatively low tested concentration was mainly entrapped in the core of nanoparticles, WVP of all nanocomposite films was mainly dominated by the NaCas/zein ratio. Contact angle (θ), the angle between the film surface and droplet of water, was measured to quantify the surface hydrophobicity of films (Fig. 4A). In general, quantitative differentiation between hydrophobic or hydrophilic surface is dependent on whether θ > 90° or < 90°. The contact angle values of all tested samples were smaller than 90°, indicating a hydrophilic surface nature of all films. Moreover, this also confirmed our hypothesis that indicating all films were formulated based on NaCas matrix (as proposed in Fig. 1). The film hydrophobicity gradually increased from 58.8° to 65.2° when the NaCas/zein ratio changed from 100:0 to 50:50, which was in accordance with the WVP measurement. The above observations agreed with a previous study reported by Matsakidou et al. that films fabricated from NaCas solutions

Fig. 4. (A) Effect of NaCas/zein mass ratio and curcumin on instant water contact angles of films. (B) Pictures of water droplet sorption on film surface over 60 s.

77

Industrial Crops & Products 130 (2019) 71–80

L. Wang et al.

after emulsifying 5% to 25% oil showed a concomitant increase in the film hydrophobicity, but their surfaces were still typically hydrophilic (Matsakidou et al., 2013). The contact angle was insignificantly increased by adding curcumin into the formulation, conforming that the film nature was mainly determined as a function of NaCas/zein ratio. This observation was consistent with the WVP value measurement, and further confirmed the hypothesis that curcumin molecules were mainly entrapped within the nanoparticles rather than deposited on the protein surface. Kinetic sorption of water over time was also monitored to imply the interaction between the film surface and water droplet (Fig. 4B). The decreasing contact angle could be attributed to the hydrated and swelling behavior of film-forming materials (Rhim and Ng, 2007). The NaCas film was highly sensitive to water, with water molecules absorbed quickly, which was greatly in accordance with previous studies (Chevalier et al., 2018). As expected, after 60 s, the water adsorption of films was better restricted by higher zein concentration thanks to the barrier properties of zein particles in the film matrix. The incorporation of curcumin caused no noticeable impact on the water sorption behavior of film. The abovementioned results were comparable to the study by Li et al. for NaCas/film prepared by antisolvent process with the utilization of alcohol (Li et al., 2012). 3.3.4. Mechanical properties The mechanical parameters of films including tensile strength (TS) and elongation at break (EAB) are shown in Fig. 5. TS or EAB is the ability of film to resist or withstand extensibility under external stress, of which both depended on the polymer strength and surface area, as well as the strength of internal bonding (Gilbert et al., 2017). As shown in Fig. 5A, the TS of films was enhanced with increasing zein concentration and reached the maximum value of 7.4 MPa at a NaCas/zein ratio of 50:50, indicating that the film became stronger. It was suggested that the strong hydrophobic interactions kept zein molecules

Fig. 6. (A) Thermal stability of curcumin in film-forming nanodispersions as compared to free curcumin in aqueous ethanol at 50 °C. (B) Storage stability of curcumin in nanocomposite films with different NaCas/zein ratios at 21 °C.

together which may reinforce the internal network to maintain film integrity. The similar tendency of TS with increasing zein concentration was also observed in films with NaCas/zein nanoparticles fabricated by antisolvent technique (Li et al., 2012). With the incorporation of curcumin, an insignificant decrease of TS (P > 0.05) could be observed. However, on the other hand, the insignificant difference between films with and without the presence of curcumin indicated the film strength was also dominated by the interactions of NaCas with zein particles. Concomitantly, the EAB decreased from 50.5% to 30.8% with increasing zein proportion (Fig. 5B), suggesting films became stiffer and less stretchable regardless of the curcumin presence. Overall, these results demonstrated that the incorporation of zein made NaCas-based films more resistant to stress but less flexible and extensible, while the encapsulation of curcumin into zein nanoparticles may postpone this impact but not significant at the current concentration. 3.4. Stability and antioxidant capacity of curcumin Curcumin is easily degraded especially when exposed to certain environmental conditions, such as light, oxygen, and high temperature (Pan et al., 2014). Thus, the encapsulated curcumin with high stability would guarantee a film with little compromising functional properties

Fig. 5. Mean values of tensile strength (A) and elongation at break (B) of films at various NaCas/zein ratios with and without 1 mg/mL curcumin. Different letters indicate significant differences (P < 0.05). 78

Industrial Crops & Products 130 (2019) 71–80

L. Wang et al.

nanocomposite film without using alcohol or other organic solvents. And curcumin incorporated functional film showed great potential to be applied as edible food packaging material with retained antioxidant activity. Conflict of interest The authors declare no competing financial interest. Acknowledgements The project was financially supported by the University of Nebraska-Lincoln. References

Fig. 7. DPPH radical scavenging activities of curcumin-loaded films as a function of NaCas/zein ratios.

Acevedo-Fani, A., Salvia-Trujillo, L., Rojas-Graü, M.A., Martín-Belloso, O., 2015. Edible films from essential-oil-loaded nanoemulsions: physicochemical characterization and antimicrobial properties. Food Hydrocoll. 47, 168–177. ASTM International, 2002. Annual Book of ASTM Standards. ASTM International, West Conshohocken, PA. Atarés, L., Bonilla, J., Chiralt, A., 2010. Characterization of sodium caseinate-based edible films incorporated with cinnamon or ginger essential oils. J. Food Eng. 100 (4), 678–687. Benavides, S., Villalobos-Carvajal, R., Reyes, J.E., 2012. Physical, mechanical and antibacterial properties of alginate film: effect of the crosslinking degree and oregano essential oil concentration. J. Food Eng. 110 (2), 232–239. Bourtoom, T., 2008. Edible films and coatings: characteristics and properties. Int. Food Res. J. 15 (3), 237–248. Byun, Y., Kim, Y.T., Whiteside, S., 2010. Characterization of an antioxidant polylactic acid (PLA) film prepared with α-tocopherol, BHT and polyethylene glycol using film cast extruder. J. Food Eng. 100 (2), 239–244. Chevalier, E., Assezat, G., Prochazka, F., Oulahal, N., 2018. Development and characterization of a novel edible extruded sheet based on different casein sources and influence of the glycerol concentration. Food Hydrocoll. 75, 182–191. Cho, S.Y., Park, J.W., Rhee, C., 2002. Properties of laminated films from whey powder and sodium caseinate mixtures and zein layers. LWT-Food Sci. Technol. 35 (2), 135–139. Cho, S.Y., Lee, S.Y., Rhee, C., 2010. Edible oxygen barrier bilayer film pouches from corn zein and soy protein isolate for olive oil packaging. LWT-Food Sci. Technol. 43 (8), 1234–1239. Fabra, M., Hambleton, A., Talens, P., Debeaufort, F., Chiralt, A., 2011. Effect of ferulic acid and α-tocopherol antioxidants on properties of sodium caseinate edible films. Food Hydrocoll. 25, 1441–1447. Fabra, M.J., Talens, P., Gavara, R., Chiralt, A., 2012. Barrier properties of sodium caseinate films as affected by lipid composition and moisture content. J. Food Eng. 109 (3), 372–379. Gilbert, J., Cheng, C.J., Jones, O.G., 2017. Vapor barrier properties and mechanical behaviors of composite hydroxypropyl methylcelluose/zein nanoparticle films. Food Biophys. 13 (1), 1–12. Giroux, H.J., Houde, J., Britten, M., 2010. Preparation of nanoparticles from denatured whey protein by pH-cycling treatment. Food Hydrocoll. 24, 341–346. Hu, K., Huang, X., Gao, Y., Huang, X., Xiao, H., McClements, D.J., 2015. Core–shell biopolymer nanoparticle delivery systems: synthesis and characterization of curcumin fortified zein–pectin nanoparticles. Food Chem. 182, 275–281. Jang, S.A., Lim, G.O., Song, K.B., 2011. Preparation and mechanical properties of edible rapeseed protein films. J. Food Sci. 76 (2), C218–C223. Kaci, M., Meziani, S., Arab-Tehrany, E., Gillet, G., Desjardins-Lavisse, I., Desobry, S., 2014. Emulsification by high frequency ultrasound using piezoelectric transducer: formation and stability of emulsifier free emulsion. Ultrason. Sonochem. 21 (3), 1010–1017. Li, K.K., Yin, S.W., Yang, X.Q., Tang, C.H., Wei, Z.H., 2012. Fabrication and characterization of novel antimicrobial films derived from thymol-loaded zein–sodium caseinate (SC) nanoparticles. J. Agric. Food Chem. 60 (46), 11592–11600. Matsakidou, A., Biliaderis, C.G., Kiosseoglou, V., 2013. Preparation and characterization of composite sodium caseinate edible films incorporating naturally emulsified oil bodies. Food Hydrocoll. 30 (1), 232–240. Moradi, M., Tajik, H., Rohani, S.M.R., Mahmoudian, A., 2016. Antioxidant and antimicrobial effects of zein edible film impregnated with Zataria multiflora boiss. Essential oil and monolaurin. LWT-Food Sci. Technol. 72, 37–43. Ozdemir, M., Floros, J.D., 2008. Optimization of edible whey protein films containing preservatives for water vapor permeability, water solubility and sensory characteristics. J. Food Eng. 86 (2), 215–224. Pan, K., Luo, Y., Gan, Y., Baek, S.J., Zhong, Q., 2014. pH-driven encapsulation of curcumin in self-assembled casein nanoparticles for enhanced dispersibility and bioactivity. Small 10, 6820–6830. Pan, K., Zhong, Q., 2016. Low energy, organic solvent-free co-assembly of zein and caseinate to prepare stable dispersions. Food Hydrocoll. 52, 600–606. Patel, A.R., Velikov, K.P., 2014. Zein as a source of functional colloidal nano-and microstructures. Curr. Opin. Colloid Interface Sci. 19 (5), 450–458. Patel, A., Heussen, P., Dorst, E., Hazekamp, J., Velikov, K.P., 2013. Colloidal approach to

during shelf-life storage. Fig. 6A represented the thermal resistance (50 °C) of curcumin in different film-forming dispersions, while the stability of curcumin in resultant films during storage with exposure to sunlight at room temperature was given in Fig. 6B. In general, the stability of curcumin was greatly improved after encapsulation in all three colloidal particles (P < 0.05). As shown, about 83% of curcumin degraded after 1.5 h heat treatment, while the encapsulated curcumin revealed a residual activity of 39–71%. The highest retention of curcumin was achieved in the formulation at a NaCas/zein ratio of 50:50, showing increased thermal stability with increasing zein proportion in the film-forming dispersions. The effectiveness of zein as an encapsulant to enhance curcumin stability and other instable components was proved by other researchers. For example, a colorant blend containing curcumin and indigocarmine was stabilized by zein-based colloidal particles and showed enhanced stability against photodegradation (Patel et al., 2013). Hydrophobic zein showed higher attraction force compared with the hydrophilic NaCas, and hence readily entrapped curcumin within its biopolymeric matrix. This was in agreement with a previous study that a sustainable release of thymol could be better achieved within NaCas/zein films matrices compared with that of NaCas (Li et al., 2012). Our study further confirmed that zein could provide better protection for targeted bioactive compounds. The antioxidant capacity of films with and without encapsulated curcumin was evaluated using the DPPH test as shown in Fig. 7. Overall, the antioxidant activity of encapsulated curcumin within films was concentration-dependent and comparable to that of the theoretical dose of curcumin contained in films (i.e., in agreement with others) (Pan et al., 2014). It was also observed that the DPPH radical scavenging activity decreased from 55.9% to 41.1% when zein-to-NaCas increased from 0:100 to 50:50. This result may be due to the EE difference as observed in Fig. 2B. Furthermore, films containing no curcumin showed around 7% of DPPH scavenging activity, which might be associated with some antioxidant peptides existing in NaCas as reported previously (Li et al., 2012). 4. Conclusions In summary, antioxidant nanocomposite films were fabricated based on curcumin encapsulated NaCas/zein nanoparticles using the novel self-assembling “pH-driven” method. The moisture barrier property and tensile strength of nanocomposite films were gradually improved with the increasing zein proportion in the polymer formulations. Incorporation of curcumin could alter the optical properties of films but show no significant on their physical and mechanical properties. Curcumin showed maximum heat stability in film at a NaCas/ zein mass ratio of 50:50 without compromising antioxidant activities. Our work demonstrated that this pH-driven self-assembly method could be an alternative “green” method to prepare NaCas/zein 79

Industrial Crops & Products 130 (2019) 71–80

L. Wang et al.

incorporated with oregano, rosemary and garlic essential oils. Food Res. Int. 39 (5), 639–644. Sun, C., Xu, C., Mao, L., Wang, D., Yang, J., Gao, Y., 2017. Preparation, characterization and stability of curcumin-loaded zein-shellac composite colloidal particles. Food Chem. 228, 656–667. Tomasula, P.M., Yee, W.C., Parris, N., 2003. Oxygen permeability of films made from CO2-precipitated casein and modified casein. J. Agric. Food Chem. 51 (3), 634–639. Wang, L., Zhang, Y., 2017. Eugenol nanoemulsion stabilized with zein and sodium caseinate by self-assembly. J. Agric. Food Chem. 65 (14), 2990–2998. Wihodo, M., Moraru, C.I., 2013. Physical and chemical methods used to enhance the structure and mechanical properties of protein films: A review. J. Food Eng. 114 (3), 292–302. Yin, Y.C., Yin, S.W., Yang, X.Q., Tang, C.H., Wen, S.H., Chen, Z., Xiao, B.J., Wu, L.Y., 2014. Surface modification of sodium caseinate films by zein coatings. Food Hydrocoll. 36, 1–8.

prepare colour blends from colourants with different solubility profiles. Food Chem. 141 (2), 1466–1471. Pérez‐Gago, M., Nadaud, P., Krochta, J.M., 1999. Water vapor permeability, solubility, and tensile properties of heat‐denatured versus native whey protein films. J. Food Sci. 64 (6), 1034–1037. Ramos, Ó.L., Reinas, I., Silva, S.I., Fernandes, J.C., Cerqueira, M.A., Pereira, R.N., Vicente, A.A., Pocas, M.F., Pintado, M.E., Malcata, F.X., 2013. Effect of whey protein purity and glycerol content upon physical properties of edible films manufactured therefrom. Food Hydrocoll. 30 (1), 110–122. Rhim, J.W., Ng, P.K., 2007. Natural biopolymer-based nanocomposite films for packaging applications. Crit. Rev. Food Sci. Nutr. 47 (4), 411–433. Schou, M., Longares, A., Montesinos-Herrero, C., Monahan, F., O’Riordan, D., O’sullivan, M., 2005. Properties of edible sodium caseinate films and their application as food wrapping. LWT-Food Sci. Technol. 38 (6), 605–610. Seydim, A., Sarikus, G., 2006. Antimicrobial activity of whey protein based edible films

80