Active whey protein isolate films including bergamot oil emulsion stabilized by nanocellulose

Food Packaging and Shelf Life 23 (2020) 100430

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Active whey protein isolate films including bergamot oil emulsion stabilized by nanocellulose

T

Ece Sogut Suleyman Demirel University, Food Engineering Department, Isparta, 32260, Turkey

A R T I C LE I N FO

A B S T R A C T

Keywords: Nanocellulose-stabilized emulsion Bergamot oil Whey protein isolate Active film

The aim of this study was to develop whey protein isolate (WPI) films incorporated with bergamot oil (BO) in the form of nanoemulsion stabilized with nanocellulose (NC). WPI films were prepared by incorporating different concentrations of BO and NC-stabilized BO (0–40%, w/w). The mechanical properties of neat WPI films were improved with the inclusion of NC-stabilized BO emulsion (p < 0.05). Water vapor permeability of WPI films decreased with the increase in BO content up to 20 % concentration (p < 0.05) and then started to increase. The increase in BO content also caused a decrease in transmittance and lightness confirming higher opacity values (p < 0.05). Scanning electron microscopy images showed that films including NC presented more homogeneous surfaces. The release rate of films including NC-stabilized BO was slower than WPI films directly incorporated with BO. The incorporation of BO with or without NC provide a noticeable antimicrobial and antioxidant activity to WPI films.

1. Introduction Essential oils are natural aromatic products, which are known for their functional properties such as antimicrobial activity, oxygen scavenging properties, analgesic, and anti-inflammatory effects (Xing et al., 2019). However, the direct use of essential oils in foods have some major drawbacks, due to being sensitive to light, moisture, heat, being highly volatile, which results in instability during the shelf life, and being easily oxidized by oxygen or ultraviolet light. In recent years, the utilization of emulsions to improve the stability, dispersion capacity, and efficacy of such systems including essential oils has gained attention. Emulsions, which show thermodynamic instability and phase separation when heat or centrifugation applied, consist of nano- or micron-ordered droplets of one immiscible liquid dispersed in another (Dammak & do Amaral Sobral, 2018). The complex structure of an emulsion and the interactions between emulsions and other components present within a food system influence its stability (Dammak, de Carvalho, Trindade, Lourenço, & do Amaral Sobral, 2017). Thus, amphiphilic surfactants, which adsorb at immiscible liquid/liquid interfaces, are incorporated to stabilize the system (Saidane, Perrin, Cherhal, Guellec, & Capron, 2016). However, solid fine particles have more potential to stabilize the emulsion when compared to surfactants (Fujisawa, Togawa, & Kuroda, 2017). The high adsorption energy of solid fine particles provide more stable emulsion systems due to their irreversible adsorbing behavior at liquid/liquid interfaces (Dickinson,

2012). Recently, different stabilizers for these emulsions such as silica nanoparticles (Binks & Whitby, 2005), montmorillonite (Bon & Colver, 2007), and polymer particles (Gautier et al., 2007) have been studied by researchers. The encapsulation of essential oils or direct addition of these compounds in the form of stabilized micro- or nanoemulsions are the most powerful tools to overcome these drawbacks (Kasiri & Fathi, 2018). Nanocellulose (NC) particles have been accepted as an efficient stabilizer at oil/water interfaces and have been studied to stabilize various nanoemulsions (Saidane et al., 2016). The amphiphilic surface nature of NC, which results from the presence of large amounts of hydroxyl groups on the surface and the hydrophobic face, strengthens the stabilizing effect of NC (Kalashnikova, Bizot, Cathala, & Capron, 2011). Furthermore, the higher elastic modulus and surface modification of NC give opportunity to form structurally stabilized interfaces and to tailor the wettability at oil/water interfaces (Fujisawa et al., 2017). The hydrophobic edge plane of NC with an amphiphilic nature help them to efficiently position at the interface. Thus, NC will be used as emulsion stabilizer for its ability to adsorb at the emulsion interface, despite not being surface active. Among the recent studies, entrapment of active compounds inside a bio-based packaging material via emulsions have been investigated to increase their effectiveness or to control the release rate promoting its use during the shelf life of food product (Acevedo-Fani, Salvia-Trujillo, Rojas-Graü, & Martín-Belloso, 2015). Whey protein isolate (WPI) has

E-mail address: [email protected]. https://doi.org/10.1016/j.fpsl.2019.100430 Received 17 June 2019; Received in revised form 20 October 2019; Accepted 21 October 2019 2214-2894/ © 2019 Elsevier Ltd. All rights reserved.

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been one of the promising bio-based polymer due to its functional properties such as desirable film-forming ability, excellent gas barrier properties, foaming, and the amphiphilic character allowing them to interact with other components such as anionic polysaccharides (Lam & Nickerson, 2014). However, the direct incorporation of broad range of hydrophobic compounds into WPI to develop active films is limited due to the hydrophilic nature of WPI. Thus, the development of active films including encapsulated hydrophobic compounds such as essential oils in the form of nanoemulsions have great potential to form stable films. Moreover, whey proteins have been used in emulsions due to its ability to provide physical stability, which depends on the interfacial properties of oil and water, in emulsions by controlling the ratio of hydrophobic and hydrophilic aminoacids (Rahalli, Chobert, Haertle, & Gueguen, 2000). Thus, WPI was chosen as polymer matrix for produced nanoemulsions to form a dense viscoelastic/interconnected network with high elastic modulus. Recently, the combination of whey proteins and nanoemulsions including cumin seed oil (Farshi et al., 2019), βcarotene (Zhou et al., 2018), curcumin (Ma et al., 2018), and cinnamaldehyde (Chen et al., 2018) have been studied by different researchers. Bergamot oil (BO) is a valuable compound including essential components such as limonene, linalool, and linalyl acetate that have various beneficial effects (Xing et al., 2019). BO was chosen as active agent to produce active WPI films because BO has been widely studied due to its antimicrobial activity against serious food spoilage bacteria such as Escherichia coli, Salmonella spp., Staphylococcus aureus, Bacillus subtilis, etc. (Chi et al., 2019; Froiio et al., 2019; Kollanoor-Johny et al., 2012; Tao, Liu, & Zhang, 2009; Guo et al., 2018). However, the poor solubility of BO limits its application and bioavailability in water-soluble polymers like WPI (Oliveira et al., 2013). Therefore, to enhance the dispersivity of BO, while maintaining its stability and release, BO was stabilized with NC and then incorporated into WPI film solutions in the form of nanoemulsions. The aim of this study is to fabricate active WPI films incorporated with BO having potential to maintain its stability with the help of NC based emulsion.

Table 1 Particle size, polidispersity index (PDI), and zeta potential of BO-nanoemulsions prepared in different NaCl concentrations.*. NaCl concentration (mM)*

Average particle size (nm)

PDI

0 25 50 100

342.6 ± 2.6b 241.4 ± 1.4c 119.4 ± 0.9d 424 ± 10a

0.46 0.42 0.37 0.65

Zeta potential (mV) ± ± ± ±

0.01b 0.02c 0.01d 0.03a

−84.1 −54.0 −37.8 −33.2

± ± ± ±

4.0c 4.1b 0.8a 0.3a

a–d

Different letters in the same column indicate significant differences among the film samples (p < 0.05). * Continuous phase includes 2 mg NC/mL of distilled water with varying NaCl concentrations with a weight ratio of 2:8 (BO:NC aqueous suspension).

10,000 rpm for 15 min to obtain a creamy emulsion in white color. The zeta potential, polydispersity index (PDI), and average droplet diameter of these systems were measured with a nanoparticle analyzer (Horiba Scientifica, Nanopartica, SZ-100V2). The results for BO nanoemulsions prepared with NC aqueous suspension in NaCl (0–100 mM) are shown in Table 1. All emulsion systems showed a zeta potential lower than −30 mV, which are accepted as stable systems to be sufficient for ensuring physical stability of nanoemulsion (Gurpreet & Singh, 2018). As the salt concentration increased, zeta potential of nanoemulsions increased (p < 0.05) but stayed within acceptable limits ( ± 30 mV), showing all tested salt concentrations could be used. Thus, the average diameter of the obtained nanoemulsions and polydispersity index values were used as other parameters to ensure the success of the nanoemulsion production. Nanoemulsion prepared with 50 mM NaCl showed the lowest average particle size (119.4 ± 0.9 nm) with a PDI of 0.37. PDI values between 0.3 and 0.5 are typical for nanoemulsions and nanoemulsions are considered destabilized when PDI is larger than 0.3 (Zdrali, Okur, & Roke, 2019). Thus, the emulsion to add into film forming solutions was prepared with NC aqueous suspension in 50 mM NaCl (continuous phase). The particle size and zeta potential of NC based emulsion in 50 mM NaCl were also measured after one-week storage to observe the stability of emulsions and any significant differences was not observed. The shape of oil droplets, emulsion ratio, stability of emulsions, and retention ratio were only evaluated for BOnanoemulsion prepared with NC aqueous solution in 50 mM NaCl. The shape of oil droplets was visualized by a Carl Zeiss inverted microscope (Primo Vert, Germany) using a digital camera (Primo Vert HDcam, Germany). A single droplet of diluted emulsion was placed on a petri and then images were taken with Labscope 2.0 (Carl Zeiss, Germany) video acquisition software. Emulsion ratio is defined as the volume of emulsion over the volume of system (Li et al., 2018). This ratio was determined by measuring the volume of emulsion phase after keeping the samples for 24 h and oneweek at 25 °C. This ratio was evaluated after centrifugation (after a physical treatment) to control the oil phase separation/coalescence. The stability of emulsions was also tested by centrifugation for 5 min at 4100 rpm and then by taking the images of emulsion. The retention ratio (RR) of BO was determined according to the method described by Dammak and do Amaral Sobral (2018). Briefly, 1 mL of emulsion was dissolved in methanol (20 mL) followed by ultrasonication for 20 min. The methanolic extract was then centrifuged at 2000 rpm for 15 min to collect the supernatant. The absorbance of supernatant was read at 226 nm using UV–vis spectrophotometer (Shimadzu, UV-1601, Japan) after diluting with methanol. The concentration of BO stabilized by NC was measured by using suitable calibration curves. Then RR was determined by following equation:

2. Materials and methods 2.1. Materials Whey protein isolate (WPI) (90 % w/w protein) was supplied by Davisco Foods International Inc. (BiPRO, Le Sueur, MN, USA). Nanocellulose (NC) was obtained from Blue Goose Biorefineries Inc. (BGB ULTRA™, Canada) in 8.0 % (w/w) suspension of type I cellulose nanocrystals (the crystallinity index of 80 %, crystal length of 100 nm, crystal diameter between 9 and 14 nm, zeta potential of −35 mV, and carboxyl content of 0.15 mmol/g). Bergamot oil (BO) was kindly obtained from the Western Mediterranean Agricultural Research Institute (Antalya, Turkey). 1,1-diphenyl-2-picrylhydrazyl (DPPH), glycerol, methanol, magnesium nitrate 6-hydrate, ethanol, sodium chloride (NaCl) and sodium hydroxide were all of analytical grade and supplied from Sigma-Aldrich (St. Louis, Missouri, USA). 2.2. Preparation and characterization of emulsions The nanoemulsions were prepared with NC aqueous suspension in different concentration of NaCl solution (continuous phase). The reason for choosing a solution with an ionic strength is to limit the repulsive forces due to the charged groups that may be present on the surface. Salt concentration was determined in preliminary studies by preparing BO emulsions in NC aqueous suspension with different concentrations of NaCl (0–100 mM). Briefly, NC was dispersed in NaCl (0–100 mM) solutions at pre-determined concentration (continuous phase) (2 mg NC/mL of distilled water). Then, BO was dispersed in NC aqueous suspension (dispersed phase: BO in water) with a weight ratio of 2:8 (BO:NC aqueous suspension). The mixture was homogenized at

RR = (Ct/C0) × 100

(1)

where C0 is initial concentration and Ct is the concentration of sample at time t.

2

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color measurement of the films. Results were expressed as CIE L* (lightness), a* (red–green) and b* (yellow–blue) coordinates in the color space.

2.3. Preparation of film samples Whey protein isolate (WPI) films were prepared by casting method. Briefly, WPI at 5% w/w was dissolved in distilled water and glycerol was added at 35 % w/w (based on the dry weight of WPI). Then, the solutions were heated to 90 °C while being stirred continuously for 25 min. Film forming solution was cooled for further applications. The effect of emulsion incorporation was studied with different concentrations of BO stabilized with NC (prepared emulsion) and BO alone by adding to WPI film forming solution. To compare the results, the final concentration of BO was maintained at equivalent concentrations of 5, 10, 20, and 40 % BO (based on WPI powder, g oil/100 g WPI powder). Briefly, films including equivalent concentrations of BO at 5, 10, 20, and 40 % (based on WPI powder) included 2.5, 5, 10, and 20 mg/ml essential oils (0.25–2 %, w/w), respectively. In the preliminary studies, lower concentrations than 2.5 mg/mL showed poor antimicrobial activity while at higher oil concentrations than 20 mg/mL, film samples could not maintain their integrity. Therefore, these concentrations were selected as limit concentrations, which presented required activity without loss of film structure. All film-forming solutions were homogenized at 10,000 rpm for 5 min before removing air bubbles. WPI solutions directly incorporated with BO were coded as WPI-5, WPI-10, WPI-20 and WPI-40 while films including NC-stabilized BO emulsion were coded as WPI-5NC, WPI-10NC, WPI-20NC and WPI-40NC. Particle size and zeta potential of film forming solutions including nanoemulsions and pure WPI solutions prepared with different NaCl concentrations (0–100 mM) were analyzed with a nanoparticle analyzer (Horiba Scientifica, Nanopartica, SZ-100V2) at 25 °C. Suitable dilutions of film forming solutions were used to measure the surface charge at the interface of oil as well as reporting the average droplet size. The final film solutions (5 mg solid/cm2) were cast onto Teflon coated plates (Ø = 15 cm) followed by drying at 25 °C. All films were conditioned at 25 °C and 50 % relative humidity (RH) for 1 week before their characterization. The thickness of the conditioned films was measured at six random positions with a digital electronic micrometer (Digimatic Micrometer, Mitutoyo, Japan).

2.7. Scanning electron microscopy (SEM) The microstructure of film samples were obtained by a scanning electron microscope (Quanta 250 FEG, Oregon, USA) with an accelerating voltage of 10 kV in a low vacuum environment. Each film sample was scanned through a magnification of 500–2000 times. 2.8. Fourier transform spectroscopy (FTIR) The Fourier transform infrared (FTIR) spectra were recorded using Spectrum Two FTIR spectrometer (Perkin Elmer, USA) equipped with a horizontal attenuated total reflectance (ATR). All spectra were the average of scans at a resolution of 4 cm−1, from 500 cm−1 to 4000 cm−1 which were recorded at 25 °C. 2.9. Release behavior of film samples The release of BO from the film samples into food simulant D1 (50 %, v/v ethanol) was tested (Commission Regulation, (EC), 2016 EU No: 2016/1416). For the release experiment, film samples (100 mg) were immersed into simulant (10 mL) while stirring throughout the experiment. The concentration of released component after various exposure times was evaluated by absorbance measurements using the corresponding calibration curve. The model proposed by Ritger and Peppas (1987) (Eq. 2) was applied to the data to estimate the release kinetics. Mt/M∞ = Ktn

(2)

where Mt/M∞ is the ratio of released component at time (t), K is the rate constant (a characteristic of the matrix related to the diffusion process) and n is the diffusion exponent (characteristic of the release mechanism). Mt/M∞ gives the amount of BO released at time t with respect to the amount released at equilibrium. The total amount of active compound in film samples were determined by solvent extraction. Briefly, Mt is the amount of active compound released at time t and M∞ is defined as the amount of active compound released as time approaches infinity (equilibrium). Peleg’s model, (Eq. 3), was also used to predict the amount of BO released at each time (Mt) (PELEG, 1988).

2.4. Mechanical properties of film samples Mechanical properties of film samples were determined by the ASTM standard method D882 (ASTM, 2018). Films were cut into 2.5 × 5 cm size and analyzed by stretching at 50 mm/min with extension grips of the universal testing machine (Lloyd LR5, AMETEK, Inc, UK). Mechanical properties, namely tensile strength (TS), elastic modulus (EM), and elongation-at-break (E, %) were measured from strainstress curves.

Mt = t / (k1 + k2t)

(3)

where the kinetic constants, k1 and k2 are the inverse of the initial release rate, and the inverse of the asymptotic release, respectively. 1/ k2 is the amount of active compound released at equilibrium (M∞) while 1/k1 is defined as the active compound release rate.

2.5. Water vapor permeability (WVP) of film samples The WVP of films was determined according to the E96/E96M-16 gravimetric method (ASTM, 2016). The permeability cups in the controlled atmosphere (53 ± 2% RH inside the desiccator) were periodically weighted at 25 °C by exposing the film samples to 100% RH to measure the WVP values.

2.10. Antioxidant activity of film samples The potential antioxidant activity of film samples were measured with regard to radical scavenging activity using the stable radical DPPH (Sánchez‐Moreno, Larrauri, & Saura‐Calixto, 1998). Briefly, films were exposed to the mixture of ethanol and water (50:50, v/v) during one week. At different time intervals, extracts were treated with a DPPH solution (0.1 mM) before absorbance measurements at 517 nm. The total activity of each sample was expressed as the percentage reduction of DPPH.

2.6. Optical properties of film samples The transparency of the films was determined by measuring the percent transmittance at a wavelength of 450 nm using a UV–vis spectrophotometer (Shimadzu, UV-1601, Japan). The absorption spectrum of film samples (1 × 4 cm) were taken in the range of 400–800 nm (Shimadzu, UV-1601, Japan) to determine the opacity values. Film opacity was expressed as absorbance units per thickness (AU nm/mm). The color measurements were performed with a Minolta Chroma Meter (CR-400, Konica Minolta, Inc., Japan). A white standard calibration plate (Y = 92.7, x = 0.3160, y = 0.3321) was used as a background for

2.11. Antimicrobial activity of film samples The antimicrobial effects of film samples were tested against Escherichia coli (ATCC 26922), Listeria monocytogenes (ATCC 19115), 3

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emulsion ratio of this system was measured after centrifugation and was found as 0.46 ± 0.01 after 24 h and one-week storage at 25 °C (no change). The emulsion system without NC was also prepared, however, large interfacial tension between BO and aqueous system did not allow the formation of emulsion. The incorporation of NC to the aqueous solution provided a stable oil in water emulsion system with the help of irreversible adsorption of nanoparticles on the interface reaching the required minimum coverage (Li et al., 2018). The emulsification performance in such systems depends on particle/oil mass ratio related to energy input. Generally, low solid content results in insufficient cover on interfacial area while high particle/oil ratios caused excessive particles within the matrix (Zhu, Tang, Yin, & Yang, 2018). Thus, the concentration of NC in the aqueous suspension containing 50 mM NaCl was maintained at 2 mg/mL. According to our preliminary studies, lower concentrations of NC were not effective as selected concentration to obtain an emulsion system. The oil droplets started to coalescence when concentrations of NC lower than 0.5 mg/ mL were used. Therefore, a separation between oil and water phases was observed and then emulsion broke. The images of optical microscopy were taken to further understand the stability of emulsions (Fig. 1). It was observed that BO droplets were dispersed within NC-based aqueous dispersion without aggregation. This is probably due to the effective emulsification of all oil droplets within the matrix, which resulted in a resistance against coalescence (Kalashnikova, Bizot, Bertoncini, Cathala, & Capron, 2013). The formation of stable emulsions with the addition of NC depends on its type, concentration, surface charge, aspect ratio, and crystalline structure (Kalashnikova et al., 2013).

Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853) with a zone of inhibition assay on solid media. These microorganisms are some of the main bacteria responsible for food spoilage (both Gram negative and Gram positive bacteria). Thus, WPI films were tested whether having a potential to be an active food packaging film. Brain heart infusion (BHI) solid media was inoculated with selected cultures (with colony counts from 106 to 108 CFU/mL) after incubation at suitable temperatures. Film samples (Ø = 10 mm) were placed on Petri dishes inoculated with bacterial strains. The plates were incubated at 37 °C for 24 h and were then examined for antimicrobial activity. 2.12. Statistical analysis An analysis of variance (ANOVA) and Tukey’s multiple comparison tests were used to compare the different treatments at a 95 % confidence level. The statistical analysis was performed using Minitab 17 software (Minitab Inc., Brandon, UK). Each experiment was replicated three times with three observations for each sample. 3. Results and discussion 3.1. Characterization of emulsions The NC based BO emulsion and stability of emulsion after 24 h and 1 week are shown in Fig. 1. The obtained NC stabilized BO emulsion was stored at 25 °C before and after centrifugation to measure the emulsion rate during one-week storage. The emulsions were also stable after 24 h (Fig. 1-b) and one-week storage at 25 °C (Fig. 1b-1). The

Fig. 1. Appearance (b, b1, stabilized emulsion; c, c1 stabilized emulsion after centrifugation) and optical micrographs (a, a1) of stabilized emulsions after 24 h (a, b, c) and one week (a1, b1, c1) of storage. 4

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In other studies, Li et al. (2018) reported that higher concentrations than 0.3–0.5 g/L of crystalline nanocellulose (type I-II) were able to promote the emulsion stability and higher NC concentrations resulted in better emulsions whereas Kalashnikova et al. (2013) showed that emulsion droplets aggregated to clusters when higher concentrations of NC were used. This is probably due to the different aspect ratios of nanocellulose sources used in those experiments. Emulsions showed whitish color and good flowing ability immediately after preparation and maintained their physical appearance. NC-stabilized BO emulsion exhibited 48.8 ± 0.1 % retention (RR) after 1 week of storage at 25 °C. The stability of active agent like BO within the NC-based emulsion is one of the most important parameters. On the other hand, Dammak and do Amaral Sobral (2018) recorded 0.84 mg/ mL and 0.72 mg/mL of retention for hesperidin from oil in water emulsions containing chitosan nanoparticles after 30 days of storage at 20 and 40 °C, respectively. These studies suggested that the adsorption nanoparticles onto the oil droplets might prevent active compounds inside the oil from degradation such as oxidation confirming higher retention rates.

Fig. 2. Size distribution of WPI film forming solutions including nanoemulsions ; WPI-10NC, ; WPI-20NC, ; WPI-40NC, ). (WPI-5NC,

3.2. Properties of film forming solutions including nanoemulsions

solution might be nanocellulose particles, which were not adsorbed at the interface (Rao & McClements, 2012). WPI-5NC film forming solution presented the highest peak before 100 nm region followed by WPI10NC film forming solution. As the nanoemulsion concentration increased, the peak length decreased and expanded through higher particle sizes, which means that higher BO droplets were obtained in WPI20NC and WPI-40NC film forming solutions. Besides, the multimodal size distribution of WPI-40NC film forming solution could be due to recoalescence of oil droplets, leading instability and polydisperse behavior (Atares, Bonilla, & Chiralt, 2010).

The average particle size, PDI, and zeta potentials of pure WPI film forming solutions with different salt concentrations and WPI film solutions including nanoemulsion (0–40%) prepared in 50 mM NaCl are presented in Table 2. In the absence of NaCl, the zeta potential tended to increase (p < 0.05). The zeta potential of pure WPI film forming solution was found as −33.5 ± 0.5, while WPI film forming solutions including salt showed higher zeta potentials than −30 mV, which is out of stable region (Gurpreet & Singh, 2018). However, the zeta potentials of WPI film forming solutions including nanoemulsion prepared with 50 mM NaCl presented zeta potentials lower than −30 mV, which are accepted as stable systems. The lowest zeta potential was found in WPI40NC film forming solution (p < 0.05), while there was no significant differences between other film forming solutions including nanoemulsions. On the other hand, WPI-5NC film forming solution showed the lowest average particle size. When compared with other film forming solutions, WPI-40NC had higher particle size with lower PDI (p < 0.05). Besides, WPI film forming solutions including nanoemulsions presented more stable systems when compared with pure WPI and WPI film solutions including NaCl. These results indicated that the addition of BO emulsions provided greater electrostatic repulsion. Similar results were reported by Qui et al. (2017) who studied active polysaccharide loaded maltodextrin nanoparticles. The droplet size distribution of WPI film forming solutions including nanoemulsion are shown in Fig. 2. WPI-40NC film forming solution presented two major peaks around 30 nm and 300 nm. One major peak around 20–100 nm was observed in WPI-5NC, WPI-10NC, and WPI20NC film forming solutions. These peaks were considered as BO droplets. The peak around 25 nm obtained for WPI-40NC film forming

3.3. Mechanical properties of film samples The thickness, elastic modulus (EM), tensile strength (TS) and elongation at break (E) values of film samples are shown in Table 3. Films were obtained by casting the same amount of final film solutions (5 mg solid/cm2) onto Teflon coated plates (Ø = 15 cm) to get similar thickness. However, film samples including higher amount of BO showed higher thickness values (p < 0.05). The thickness of film samples depends on the interactions between the polymer chains and other components included in the film matrix as well as due the differences in homogeneity inside the film matrix. Thus, the same thickness for all films was not obtained. The EM, TS, and E (%) values of neat WPI films were 135.8 ± 32.0 MPa, 19.8 ± 2.5 MPa, and 14.7 ± 2.7 %, respectively. The incorporation of nanoemulsions improved the mechanical resistance of neat films probably due to the better interaction at the interface and strong adhesion through the interaction between functional groups of WPI and nanoemulsion (Zhu et al., 2018). However, the increase of BO content in NC-based emulsions up to 10 % resulted in an

Table 2 Size, polidispersity index, and zeta potential of film forming solutions.

Table 3 Mechanical properties of film samples.

Sample

Average particle size (nm)

PDI

Zeta potential (mV)

WPI WPI-25mM WPI-50mM WPI-100mM WPI-5NC* WPI-10NC WPI-20NC WPI-40NC

– – – – 62.8 ± 0.8d 67.5 ± 0.5c 85.2 ± 0.2b 143.0 ± 3.1a

– – – – 0.74 0.79 0.79 0.53

−33.5 ± 0.5 −26.3 ± 0.3y −24.7 ± 0.7z −22.6 ± 0.6t −39.05 ± 0.07a −38.30 ± 0.42a −38.05 ± 0.07a −40.80 ± 0.28b

Film sample

x

± ± ± ±

0.03a 0.06a 0.02a 0.01b

WPI WPI-5 WPI-5NC WPI-10 WPI-10NC WPI-20 WPI-20NC WPI-40 WPI-40NC

Thickness (μm) 103.5 104.0 123.0 103.5 126.5 108.0 125.0 127.0 128.0

± ± ± ± ± ± ± ± ±

b

2.1 1.4b 1.4a 0.7b 7.8a 2.8b 7.1a 4.2a 7.8a

EM (MPa) 135.8 161.1 215.2 170.5 180.7 135.6 149.3 134.2 154.3

± ± ± ± ± ± ± ± ±

TS (MPa) b

32.0 8.9b 57.2a 31.2b 20.7b 34.1b 12.4b 7.5b 3.5b

19.8 22.0 24.9 29.5 29.4 16.8 17.0 12.9 14.9

± ± ± ± ± ± ± ± ±

E (%) ab

2.5 2.8ab 3.1ab 3.0a 1.4a 0.4ab 2.4ab 1.2b 0.8b

14.7 ± 2.7bc 26.7 ± 6.1ab 25.9 ± 4.5ab 35.1 ± 6.5a 26.3 ± 8.9ab 31.6 ± 7.5ab 9.8 ± 0.3c 15.0 ± 5.0bc 8.1 ± 2.1c

x−t, a−d

Different letters in the same column indicate significant differences among the film samples (p < 0.05). * Nanoemulsions were prepared with 50 mM NaCl.

a−c

Different letters in the same column indicate significant differences among the film samples (p < 0.05).

5

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3.5. Optical properties of film samples

increase in EM, TS, and E values while the increment in BO content more than 10 % did not further enhance the mechanical resistance of film samples. Film samples containing BO in the form of nanoemulsion always presented higher EM and TS values and lower elasticity when compared to films containing BO without nanocellulose. The presence of droplets might decrease the entanglement of the polymer chains leading to an increase in their molecular mobility (Dammak, Lourenço, & do Amaral Sobral, 2019). Results showed that films including 20 and 40 % BO produced with or without nanocellulose, showed the values of EM were closer to control film, while the values of TS and E were the lowest for WPI-20NC, WPI-40 and WPI-40NC films. The presence of high concentration of oil droplets inside the film matrix could avoid the formation of intermolecular interactions, and so reduced the strength and stiffness of polymer chains. As BO concentration increased inside film with or without nanocellulose, the polymer network might become more heterogeneous, thus leading decrease in chain-chain interactions and mechanical strength. Besides, the decrease in TS values of WPI20NC and WPI-40NC films might be due to the insufficient dispersion of nanoemulsions within the polymer network. Similar results were reported for alginate-based films including thyme, lemongrass or sage oil nanoemulsions (Acevedo-Fani et al., 2015) and pectin-based films including cinnamaldehyde nanoemulsions (Otoni et al., 2014).

The optical properties of film samples are shown in Table 4. Generally, the direct addition of oils into the film forming solutions cause free oil droplets to be sensed on the surface of films after removal of solvent from the film solution. This behavior can be explained by the weak stability of oil within the film solution leading to coalescence due to destabilization (Wang et al., 2013). On the other hand, the incorporation of oil stabilized with nanocellulose does not follow the same trend largely due to the high resistance to coalescence (Zhu et al., 2018). Visually all film samples showed homogeneous structure without oil deposition onto the film surface. Films were in different transmittance profiles where WPI films including BO without stabilization presented higher transmittance, whereas the combination of WPI and NC-based emulsions caused a sharp decrease in the transmittance of the films. The direct inclusion of BO up to 40 % into film samples did not significantly change the transmittance values (T) while the increase in the concentration of NC-stabilized BO within the film matrix resulted in significant decrease in T values. Therefore, WPI films prepared with NC-based nanoemulsions may be used as barrier against to light being a potential preservative to oxidation induced by light. Similar to transmittance, the highest opacity values were observed in WPI-20NC and WPI-40NC films (p < 0.05). The L* values decreased with the increased concentrations of BO with or without NC, however a significant difference was observed for higher concentrations than 20 %. There is no significant differences between T and L* values of WPI and WPI films directly incorporated with BO, except WPI-40 films. It is expected that there is a correlation between transmittance and lightness, however the surface differences (roughness or homogeneity) and the light absorption behavior of constituents at different wavelength might cause different results (Acevedo-Fani et al., 2015). WPI-10 films showed higher T value and lower L* value than WPI (p > 0.05). This might be due to the differences in surface, leading to a different light scattering behavior. Similarly, Acevedo-Fani et al. (2015) reported that films including lemongrass essential oil emulsions had unexpectedly higher opacity values even these films had the highest L* values. The a* values did not change significantly while b* values significantly increased upon the addition of BO with or without NC-based emulsions. Similar results were reported by Chen et al. (2016) who studied films with cinnamaldehyde nanoemulsions.

3.4. Water vapor permeability (WVP) of film samples The WVP values of film samples are shown in Fig. 3. The addition of BO and NC-stabilized BO emulsion into films significantly decreased the WVP values (p < 0.05), except film with 40 %. WPI films including NC-stabilized BO emulsion showed lower WVP values than relative WPI films, which might be due to that emulsions droplet could provoke certain tortuosity, contributing to reducing diffusivity. In this type of system, diffusion occurs by molecular diffusion in the amorphous regions of the matrix that have hydrophilic and hydrophobic molecules. Similarly, a significant decrease in WVP was reported in other studies including alginate films with nanoelmulsions of thyme, lemongrass or sage oil (Acevedo-Fani et al., 2015), and hydroxypropyl methylcellulose films prepared with nisin in the form of nanoliposomes (Imran et al., 2012). WPI films directly incorporated with BO also showed lower WVP values than neat WPI films. This behavior can be explained by the hydrophobic nature of oil droplets leading to decrease in the permeability. However, WPI-40 film samples showed the highest WVP values and the WVP values of WPI-40NC films were not significantly different from WPI films. The excessive concentrations of essential oils could disrupt the internal network of films causing an increase in WVP. Higher oil concentrations might also cause to the formation of porous structure, leading water molecules to pass through the film matrix easily. Similarly, Kavoosi, Rahmatollahi, Dadfar, and Purfard (2014)) reported an increase in WVP values of gelatin films with the increase in essential oil concentration.

3.6. Scanning electron microscopy (SEM) The surface morphology of film samples were analyzed and the SEM images of film samples are shown in Fig. 4. The neat WPI films showed more homogeneous, dense and smooth structure when compared to films incorporated with BO and NC-stabilized BO emulsions. Generally, films prepared with nanoemulsions showed a few small sized droplets within the matrix. The surfaces of WPI-5NC and WPI-10NC were also homogeneous and smooth however, higher concentrations of BO nanoemulsions resulted in slightly heterogeneous surfaces. The relatively homogeneous surface of films including NC-stabilized BO emulsions might be largely related to the strong stabilizing ability of NC against coalescence. On the other hand, the direct incorporation of BO into WPI films produced more heterogeneous surfaces. This might be due to lower physical stability and lower resistance to flocculation and coalescence of oil droplets within the polymer matrix (Wang et al., 2013). Nanoemulsion films exhibited high resistance to coalescence and deposition of colloidal particles on the surface of lipid droplets where the film produces firm interface layers. Similar SEM images were obtained by other studies for chitosan/zein films including high concentrations of nanoemulsions (Shi et al., 2016). Besides, Dammak et al. (2019) prepared gelatin films with higher concentrations of emulsions stabilized with chitosan nanoparticle to obtain highly viscous continuous phase with more homogeneous network.

Fig. 3. WVP values of film samples. a–b Different letters indicate significant differences among the film samples (p < 0.05) 6

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Table 4 Optical properties of film samples. Film sample

Transmittance (%)

WPI WPI-5 WPI-5NC WPI-10 WPI-10NC WPI-20 WPI-20NC WPI-40 WPI-40NC

77.0 77.3 60.2 78.1 59.9 75.4 30.8 70.6 13.7

a−d

± ± ± ± ± ± ± ± ±

0.4a 1.8a 1.4c 0.6a 0.7c 1.3a 0.3cd 3.3b 0.1d

Opacity (AU nm/mm)

L*

315.9 ± 0.6b 308.2 ± 3.8b 199 ± 8b 304.0 ± 6.6b 522.5 ± 31.0b 321.0 ± 2.4b 1496.5 ± 84.4a 367.2 ± 27.2b 1592.6 ± 322.5a

95.6 95.6 95.4 95.4 94.5 95.6 94.0 94.7 92.3

a*

± ± ± ± ± ± ± ± ±

0.1a 0.4a 0.9a 0.3a 0.4ab 0.1a 0.4b 0.1ab 0.9c

−0.42 −0.37 −0.42 −0.33 −0.44 −0.35 −0.45 −0.52 −0.48

b*

± ± ± ± ± ± ± ± ±

0.09a 0.08a 0.11a 0.07a 0.07a 0.04a 0.02a 0.10a 0.03a

6.3 ± 0.6c 6.5 ± 0.6bc 7.4 ± 1.9bc 6.3 ± 0.7bc 7.6 ± 0.9bc 6.9 ± 0.2bc 8.7 ± 0.7ab 7.5 ± 0.6bc 11.0 ± 0.7a

Different letters in the same column indicate significant differences among the film samples (p < 0.05).

3.7. Fourier transform spectroscopy (FTIR)

functional groups of WPI and BO.

FTIR analysis were carried out to determine the potential chemical interactions between WPI and BO in the absence or presence of nanoemulsion while evaluating the interactions at the oil-water interface. The spectrums of film samples are shown in Fig. 5. The broad peak in the range of 3000–3700 cm−1 is associated with the free and bound OeH (stretching) and NeH groups while the small peaks at 2800–3000 cm−1 are attributed to CeH stretching vibrations of alkane groups in the film chains (Ramos et al., 2013). The peaks in the range of 2800–3600 cm−1 became less intense with the incorporation of BO, indicating the change of hydrogen bonds (−OH) among the WPI matrix (Dammak et al., 2019). The peaks observed in the range of 1600–1700 cm−1, 1400–1550 cm−1, and 1050-1100 cm−1 are assigned to the stretching vibration of C]O and C–N groups (amide I), NeH bending (amide II), and NeH in plane bending with CeN stretching vibrations (amide ІІІ), respectively (Piccirilli, Soazo, Pérez, Delorenzi, & Verdini, 2019). The absorption peaks in the range of 1600-1700 cm−1 (amide I) became intense in the presence of NC-stabilized BO, confirming an interaction occurred intervening amide groups (Lagaron, Fernandez-Saiz, & Ocio, 2007). On the contrary, the intensity of the band at 1630 cm−1 decreased when WPI films were directly incorporated with BO, which might be attributed to an interaction via hydrogen bonds (Gilbert et al., 2005). The band observed at 1630 cm−1 is characteristic of amide groups involved in the extended β-sheet structures, which lead amino acids to be engaged in β-sheet aggregation (Jiang, Li, Chai, & Leng, 2010). The higher content of β-sheet structures is commonly associated with aggregated proteins in whey protein isolates that result in the formation of intermolecular antiparallel β-sheets. Thus, this behavior increases the gel strength and the development of intermolecular antiparallel β-sheets (Lefèvre, Subirade, & Pézolet, 2005). Furthermore, it might improve interactions between whey proteins and other compounds and make possible intermolecular bonds during the aggregation of whey. Similar to amide I region, the intensity of peak at 1450 cm−1 (amide ІІ) decreased in the WPI films directly incorporated with BO, which means an interaction with NH groups (Oliveira et al., 2013). Peaks at 1200–1350 cm−1 are associated with combination of NeH in-plane bending with CeN stretching vibrations (amide III) while the peaks covering the range between 1200 and 1700 cm−1 are stretching vibrations of amide bonds governed by the protein network (Piccirilli et al., 2019). The band at 1038 cm−1 is associated with CeC and CeO stretching vibrations (Piccirilli et al., 2019). As compared to the spectrum of WPI films directly incorporated with BO, more intense peaks in the range of 900–1150 cm−1 appeared in the presence of NC-stabilized BO, which could be a proof for interaction of WPI and BO. The band located in the spectral range between 800 and 1150 cm−1 is also attributed to glycerol, which was used as a plasticizer in the film forming solution (Guerrero, Retegi, Gabilondo, & De La Caba, 2010). These results indicated that some interactions have occurred between

3.8. Release behavior of film samples The release behavior of BO from WPI films was analyzed to examine how nanoemulsion affected its diffusion to food systems. The release of an agent from a polymeric network is a three-stage process, which starts with the penetration of solvent through the polymeric matrix and then continues with the relaxation due to the swelling of matrix that leads the diffusion of agent through the swollen network until the agent reaches equilibrium (Zhu et al., 2018). Fig. 6 shows the release profiles of BO from film samples. The release rate of BO increased with the increase of concentration, yielding the lowest release rate for WPI-5 and WPI-5NC films. Initially a faster release and a subsequent slower trend were observed. Generally, the film samples including nanoemulsions showed slower release behavior. The interaction between WPI and BO might be favored in the presence of nanocellulose thus leading to a slower release of BO with a more close structure. In the WPI films directly incorporated with BO, a faster release ratio was found probably due to the weaker interactions of BO with the polymer matrix. The release rate might be also promoted because of a more relaxed polymer matrix or high solubility of essential oils in the ethanol solution. These results are in agreement with the study of Chen et al. (2016) who reported that the release rate initially increased with nanoemulsion content and then decreased which might be due to the changes in microstructure with high levels of essential oil. Release kinetics were calculated for films exposed to the selected food simulant by fitting Eq.s 2–3 to experimental data. Table 5 shows the Ritger-Peppas and Peleg’s parameters for BO release into simulant. Film samples including NC-stabilized BO emulsions, except WPI-5NC films, had n values lower than 0.5 where the behavior was non-Fickian. WPI films directly incorporated with BO showed n values between 0.5 and 1.0, which defines the release as anomalous transport behavior (Siepmann & Peppas, 2011). The lower values than 0.5 for n might be related to the partial solubility of films in the solvent. This behavior can be explained by a quasi-Fickian diffusion mechanism, which means that BO diffuses partially through swollen film matrix as a result of structural rearrangements in the polymer support. This means that NC-stabilized BO partially diffuse from matrix; the process is still in evolution, leading a release for a longer time. The n values of film samples including NC-stabilized BO emulsions were lower than WPI films directly incorporated with BO. Moreover, as the BO concentration increased, n values gradually decreased (p < 0.05). The lowest values were observed in films prepared with 40 % of BO. The higher diffusion rate constants (K) were found in WPI-5 and WPI-5NC films while showing a decreasing trend with increasing BO content (p < 0.05). However, these model parameters obtained from Ritger-Peppas model are valid for the first 60 % of compound released. Thus, Peleg’s model parameters were measured to determine the release behavior at equilibrium when time tends to infinity. Table 5 also presents Peleg’s model parameters where k1 is related to the inverse of the initial release rate, and 7

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Fig. 4. SEM images of film samples.

incorporated with BO, the lower k1 (higher 1/k1) values were observed while the highest M∞ (1/k2) occurred in WPI-20 and WPI-40 films (p < 0.05). Besides, the higher k1 (lower 1/k1) values were found in WPI films including nanoemulsions as an indication of slower initial release velocity. The total BO released at equilibrium (M∞) from WPI

k2 is the inverse of the total amount of released compounds at equilibrium. The release of BO to selected medium exhibited a good fit with Peleg’s model (R2 > 0.97). Film formulations exhibited a significant influence on the initial release rate of BO from film samples due to the presence of NC inside the polymeric matrix. In WPI films directly 8

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Fig. 5. FTIR spectrums of film samples from 700-1800 cm−1 (a) and 1800-4000 cm−1 (b) (9); WPI-5, (4); WPI-10, (WPI, (3); WPI-20, (1); WPI-40, (5); WPI(7); WPI-10 N, (6); WPI-20 N, 5 N, (2); WPI-40 N, (8)).

nanoemulsion form affected the antioxidant activity of film samples (p < 0.05) confirming NC protected BO from evaporation and facilitated a controlled release under selected test conditions. WPI films including NC-stabilized BO emulsions had higher antioxidant activity values at the end of storage period probably due to the gradual release of active agents in the presence of NC (Mikulcová, Bordes, & Kašpárková, 2016). The incorporation of NC-stabilized BO emulsions led to a significant increase in the antioxidant activity of films, which was more noticeable for the film samples containing 10 and 20% BO emulsion. However, WPI-5NC films had the lowest values, consistent with the lowest release rate. The antioxidant activity values significantly increased within 300 min (p < 0.05) and after this point, the increase in the activity of film samples were very slow indicating all samples reached equilibrium. These results were similar to those reported by Dammak et al. (2017) for gelatin-based films incorporated with rutin-loaded nanoemulsions, and Zhu et al. (2018) for polylactic acid films incorporated with zein/ chitosan particles and maize germ oil based Pickering emulsion.

films including nanoemulsions were lower than other film samples. Thus, 1/k2 values were lower in WPI films including nanoemulsions (p < 0.05). The higher k2 values correspond to the lower concentrations reached at equilibrium (PELEG, 1988). The obtained values for the total BO released at equilibrium coincide with experimental values (Fig. 6). Peleg’s model was applied to estimate long range of values from data obtained from relatively short duration due to the ability of model to predict the released amount at equilibrium when time tends to infinity. 3.9. Antioxidant activity of film samples DPPH radical scavenging activity tests were carried out to determine whether the antioxidant ability of film samples are retained during one-week storage at 25 °C. The antioxidant activity values of film samples through one week are shown in Fig. 7. As expected, films showed concentration dependent DPPH radical scavenging activities (p < 0.05). As the BO ratios increased to 40 %, the DPPH scavenging activity increased up to 42.73 ± 2.40 %, confirming BO inside the film matrix retained its antioxidant properties. The addition of BO in 9

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Fig. 6. Release profiles of BO from WPI-5 ( ), WPI-10 ( ), WPI-20 ( ), WPI-40 ( ), WPI-5NC ( ), WPI-10NC ( ), WPI20NC ( ), and WPI-40NC ( ) with fitted lines.

3.10. Antimicrobial activity of film samples

antimicrobial effectiveness of bergamot oil included poly(lactic acid) films with or without nanoparticles and reported that films with bergamot oil suppressed the microbial load of mangoes stored at room temperature. The antimicrobial activity had a concentration dependent trend that as the concentration increased, the inhibition zones were found higher. This dose-dependent manner is in agreement with Chen et al. (2016) who observed that the growth of fungus was progressively inhibited with higher cinnamaldehyde content. Moreover, the inhibition zones of film samples including NC-stabilized nanoemulsions were larger than that of WPI films directly incorporated with BO, reflecting that nano-sized particles have much larger surface areas, which increased the accessibility of the dispersed compounds (Huang, Yu, & Ru, 2010). Similarly, Otoni et al. (2014), who studied pectin/papaya puree/ cinnamaldehyde nanoemulsion films, reported that smaller droplets

The inhibition zones of film samples against E. coli, L. monocytogenes, S. aureus, and P. aeruginosa are shown in Table 6. The agar diffusion method was conducted to measure the inhibition capacity based on the clear zone surrounding the hole. Generally, the inhibitory activity of film samples were higher for E. coli and L. monocytogenes than those obtained for S. aureus and P. aeruginosa. However, there was no significant differences between the antimicrobial activity of film samples (p > 0.05). Mancuso et al. (2019) reported that bergamot oil and terpene showed antimicrobial activity against wide variety of bacterial pathogenic strains and some Candida sp. strains. Different from this study, the authors found that S. aureus was more sensitive to bergamot oil than E. coli. Besides, Chi et al. (2019) studied the 10

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Table 5 Parameters of Ritger-Peppas model and Peleg’s model. Film sample

Ritger-Peppas model

Peleg’s model

n

WPI-5 WPI-5NC WPI-10 WPI-10NC WPI-20 WPI-20NC WPI-40 WPI-40NC a–e

0.58 0.51 0.56 0.43 0.53 0.31 0.57 0.37

K

± ± ± ± ± ± ± ±

0.01a 0.01a 0.04a 0.01b 0.01a 0.01c 0.02a 0.02bc

R

1/k1 (μg/min g film)

0.95 0.97 0.88 0.82 0.85 0.95 0.87 0.90

299.6 341.9 287.7 170.1 995.1 241.2 935.9 449.5

2

2.41 2.53 1.76 2.20 2.15 1.86 2.19 2.05

± ± ± ± ± ± ± ±

0.01b 0.02a 0.01d 0.01c 0.01c 0.03d 0.01c 0.06d

± ± ± ± ± ± ± ±

1.2c 3.4bc 15.5c 18.3d 5.1a 16.1cd 4.3a 2.1b

1/k2 (g/100 g film) 1.01 3.08 4.48 3.87 6.28 4.12 7.88 6.25

± ± ± ± ± ± ± ±

0.01e 0.01d 0.07c 0.26cd 0.02b 0.13c 0.02a 0.62b

R2

0.98 0.96 0.99 0.99 0.99 0.99 0.98 0.97

Different letters in the same column indicate significant differences among the film samples (p < 0.05).

and degradation. Similarly, Froiio et al. (2019) reported an antimicrobial activity around 90% for encapsulated bergamot oil independently from bergamot oil concentration. 4. Conclusion WPI films were prepared by the direct incorporation of BO or in the form of NC-stabilized BO emulsions. The combination of NC-stabilized BO with WPI films enhanced the mechanical resistance and WVP of WPI films. Higher concentrations of BO showed a plasticizer effect, which increases the elongation at break; however, films including BO nanoemulsions higher than 20 % were less resistant to tension. Moreover, the WVP of films decreased with the increase in oil content probably due to the high aspect ratio of NC and the hydrophobic nature of oil droplets. WPI films incorporated with NC-stabilized BO emulsions resulted in lower transmittance and lightness values while showing higher opacity. SEM results showed that incorporation of NC-stabilized BO emulsions resulted in more homogeneous structure, confirming the microstructural stabilization of active compound inside the polymer matrix. FTIR analysis presented that intermolecular interactions might exist between NC-stabilized BO and polymer matrix. The release of BO from films was slower when stabilized with NC. NC stabilization also provided sustained antioxidant and antimicrobial activity. These results suggested that the combination of WPI films with NC-stabilized BO could produce a better film structure such as higher mechanical and water resistance, slower release, etc. when compared to films directly incorporated with BO. As a conclusion, WPI-based packaging films including BO nanoemulsions up to 20% can be prepared without any adverse effect on physico-mechanical properties. Among the film samples WPI-10NC presented promising advantages such as providing enhanced mechanical and water vapor permeability while promoting the antimicrobial and antioxidant activities at the same time. Thus, these results indicated that the application of WPI films including BO nanoemulsions could have promising effect in the food packaging applications such as for foods susceptible to microbial spoilage and oxidation. However, further studies are needed to investigate these effects on real foods.

Fig. 7. DPPH radical scavenging activity of film samples during one week (WPI; WPI-10, ; WPI-20, ; WPI-40, ; WPI-5NC, ; 5, ; WPI-20NC, ; WPI-40NC, ). WPI-10NC, Table 6 Inhibition zones (mm) of film samples against selected bacteria. Film sample

E. coli

L. monocytogenes

S. aureus

P. aeruginosa

WPI-5 WPI-5NC WPI-10 WPI-10NC WPI-20 WPI-20NC WPI-40 WPI-40NC

12.5 ND 13.5 15.0 15.0 20.5 15.5 21.0

13.5 13.0 14.0 17.5 18.5 20.0 22.0 21.0

ND ND 15.5 14.0 16.0 12.5 16.5 15.0

ND ND ND ND 17.0 17.5 19.4 19.5

± 3.0 ± ± ± ± ± ±

3.0 1.1 1.1 0.7 1.8 1.4

± ± ± ± ± ± ± ±

3.0 2.2 2.7 0.7 2.1 2.8 1.4 2.8

± ± ± ± ± ±

2.8 2.7 2.5 1.5 1.2 1.1

± ± ± ±

1.4 0.7 0.9 0.7

ND: Not detected any clear zone against selected bacteria.

delivered greater antimicrobial properties indicating greater inhibition zones. Farshi et al. (2019) also showed that WPI-guar gum stabilized with cumin seed oil nanoemulsions were more effective to inhibit the selected microorganisms. Ghadetaj, Almasi, and Mehryar (2018)) reported that WPI films including nanoemulsions had higher antimicrobial activity due to their higher release rates. Different from this study, WPI films including BO nanoemulsions presented lower release rates and higher antimicrobial activity. The differences between these studies might be due to the use of different release mediums. Besides, the higher antimicrobial activity of BO-nanoemulsions, independently from the release rate, might be due to the combined effect of the cationic character of the nanoparticles and essential oil. The weak interactions between free BO and WPI might also cause a rapid evaporation

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