Starch and whey protein isolate films including an aroma compound stabilized by nanocellulose

Abstract

Bergamot essential oil (BO) shows biological activities and is widely used as a flavoring for food products; however, its application in foods is limited because of the low stability and solubility in water. This study aimed to prepare whey protein isolate (WPI) and starch (S) films, including nanocellulose based BO nanoemulsions, potentially enhancing BO functionality. BO nanoemulsion was obtained by using a nanocellulose dispersion (2 mg/mL) including 3 mM CaCl₂ with a ratio of 2/8 (v/v, BO/nanocellulose dispersion) and then suitably dispersed (2%, w/w) in the WPI and S film-forming solutions to obtain film samples. The water vapor permeability (WVP), mechanical and optical properties and BO’s release from those obtained films were studied. The WVP (p<0.05) and tensile strength (p>0.05) of films were improved, whereas opacity increased with the addition of BO nanoemulsion (p<0.05). The release of BO from S films was faster than in WPI films. These results showed that nanocellulose could be used as carriers for essential oils such as BO to enhance its functionality within bio-polymeric matrices intended to be used as relevant carriers of aroma compounds.

Key words
Bergamot oil; nanomaterials; nanoemulsion; starch; whey protein isolate

INTRODUCTION

Essential oils, as secondary metabolites of aromatic flowers and plants, show lipophilic, volatile, and unstable characters and have been widely used as flavors, fragrances, and active agents, mainly due to their active properties such as antimicrobial, anti-inflammatory, antioxidant, and anticancer properties (Vahedikia et al. 2019). Bergamot oil (BO) is one of those mentioned valuable oils, including linalool and linalyl acetate (Mannucci et al. 2017). Many studies have demonstrated that BO possessed antimicrobial, anti-inflammatory, antiviral, and anticancer properties (Bora et al. 2020, Marchese et al. 2020). Besides, the BO flavor components are used in many food products such as Earl Grey tea (Orth et al. 2013); however, these components are often dissipated during the preparation and/or storage of food products due to their highly volatile and unstable nature (He et al. 2018). Moreover, the use of essential oils in different applications is hindered due to their low bioavailability, poor water solubility, low stability, and rapid oxidation (Gromadzki et al. 2017). Researchers have proposed different approaches such as encapsulation to improve stability of hydrophobic active agents, to enhance the solubility of essential oils, to provide efficient resistance during processing, to control the release rate, as well as for better stability and hiding unwanted flavors or enhancing flavor properties (Assadpour & Mahdi Jafari 2019, Das et al. 2021, Özogul et al. 2022, Rehman et al. 2020, Riaz et al. 2019). Such novel systems include nanoparticles, micelles, nanoemulsions, nano-hydrogels, liposomes, chelation with metals, and phospholipid complexes (Asabuwa Ngwabebhoh et al. 2018, Bahrami et al. 2020).

The nanoemulsions are composed of an aqueous phase and an oily phase in droplets with smaller diameters than 100 nm (Ashaolu 2021). These systems reduce the gravitational force due to the smaller size of the particles, thus avoiding coalescence or flocculation, interface deformation and disruption, and sedimentation during storage (McClements & Gumus 2016). Solid particles such as graphene (Wang et al. 2021), clay (Yu et al. 2021), and chitosan (Mwangi et al. 2016) have also been used as emulsion stabilizers instead of surfactants because of toxicity concerns. These emulsion systems, including solid particles, bind irreversibly at the interface producing Pickering emulsions that have been found more stable over a long period (Shah et al. 2016). Cellulose is the most abundant biopolymer in nature, and due to its unique characteristics, such as biodegradability, biocompatibility, non-toxicity, high flexibility, and mechanical strength, it has been used as drug delivery systems, biosensors, and packaging films and to obtain nanocellulose (NC) particles (Mihaly-Cozmuta et al. 2017). NC is one of the efficient stabilizers used at oil/water interfaces and has been successfully used to prepare various nanoemulsions (Asabuwa Ngwabebhoh et al. 2018, Saidane et al. 2016, Sogut 2020). It has been reported that the effectiveness of NC to stabilize the emulsions is due to its amphiphilic character, which results from hydroxyl groups on the surface and the hydrophobic face (Fujisawa et al. 2017). Thus, the stabilization of BO with NC particles to obtain a nanoemulsion can be used in aroma carrier systems for food applications.

Biodegradable polymers obtained from renewable sources such as whey protein isolate (WPI) and starch (S) have been often studied due to their biodegradability, biocompatibility, and the potential application of these materials in various industrial sectors with sustainability and environmental protection issues (Fonseca-García et al. 2021, Rodriguez Llanos et al. 2021, Seydim et al. 2020). However, the addition of essential oils into film-forming solutions has some limitations due to their low miscibility, phase separation, increasing sensitivity to environmental factors, and adverse effects on optical properties (Atarés & Chiralt 2016). Recent studies have shown that the entrapment of essential oils within a polymeric matrix via emulsions has successfully enhanced their effectiveness or controlled the release rate promoting its use during the shelf life of food products (Acevedo-Fani et al. 2015). Researchers have also reported that controlling aroma release has been possible by modifying the emulsion characteristics such as using biopolymers, changing the particle size and shape of the emulsifier (Doi et al. 2019, Ren et al. 2018). However, such systems’ design is still a challenge due to their complex structures, requiring more studies. It was assumed that the amphiphilic nature of NC may help oil droplets to a position at the interface and might be a suitable candidate as BO based emulsion stabilizer for its ability to adsorb at the emulsion interface. Polysaccharides and proteins can retain aroma compounds and be structurally modified to favor the release of aroma compounds, thus being suitable aroma carriers. Therefore, one polymer was chosen from a protein source (WPI) and another from polysaccharides (starch) to evaluate their potential as carriers for an aroma compound (BO), stabilized by NC to control its release. In this way, the impact of the polymer support characteristics contributing to flavor release may be elucidated. Besides, the interaction between aroma compounds and biopolymeric matrix and both controlling release and retention of aroma compounds are challenging points. Therefore, this study aimed to prepare nanocellulose-based bergamot essential oil nanoemulsions and then incorporate them into the polymeric matrix, whey protein isolate, and starch, potentially used as aroma transferring mediums with better miscibility. The results of this study can be used to prepare emulsion-based encapsulation/stabilization systems to control the release of volatile flavors in packaged foods during storage.

MATERIALS AND METHODS

Materials

Whey protein isolate (WPI) (90% w/w protein) was supplied by Proteinocean Gıda Inc. (Ankara, Turkey). Nanocellulose (NC) was purchased from Blue Goose Biorefineries Inc. (BGB ULTRA™, Canada) as an aqueous suspension formed a gel at 8.0 % (w/w) with a crystal length of 100 nm and crystal diameter between 9 and 14 nm.

Bergamot oil (BO) was kindly supplied from the Turkish Ministry of Food and Agriculture, Western Mediterranean Agricultural Research Institute (Antalya, Turkey). The obtained BO was analyzed and characterized by a high content of d-limonene (45.21%), α-linalool (11.51%), linalyl acetate (18.83%), c-terpinene (9.10%), and α-pinene (5.81%).

Candelilla wax was provided from Strahl and Pitsch Inc. (S&P-99, West Babylon, NY, USA). Starch (S) (from potato), glycerol, magnesium nitrate 6-hydrate, ethanol, sodium hydroxide (NaOH), and calcium chloride (CaCl₂) were all of the analytical grades and supplied from Sigma-Aldrich (St. Louis, Missouri, USA).

Formation of nanoemulsions

The BO nanoemulsion was prepared with NC aqueous suspension (2 mg NC/mL of distilled water) in the 3 mM CaCl₂ solution (continuous phase) to reduce the possible repulsive forces occurring on the surface to the charged groups. The performance of the emulsion also depends on the ratio between NC and BO. In our preliminary studies, lower NC concentrations (less than 0.5 mg/mL) caused a coalescence of oil droplets, while higher concentrations than 2 mg/mL resulted in less effective NC accumulation. Therefore, the concentration of NC in the aqueous suspension containing 3 mM CaCl₂ was maintained at 2 mg/ml, which is sufficient to cover the interfacial area. BO was then added to the NC suspension with a weight ratio of 2/8 (BO/NC aqueous suspension), and the pH of this mixture was adjusted to 5.0, followed by homogenization at 15000 rpm for 15 min (Kasiri & Fathi 2018). The film materials used components show over a broad pH range, the pH of the dispersion was adjusted to study at a stable pH. The weight ratio of 2/8 was selected after the preliminary studies measuring the zeta potential of prepared emulsions in different ratios (1/9, 2/8, 3/7) with a nanoparticle analyzer (Nanopartica, SZ-100V2, Horiba Scientifica, Germany). The selected ratio presented a zeta potential lower than −30 mV, which are accepted as stable systems to be sufficient for ensuring the physical stability of nanoemulsion (Gurpreet & Singh 2018). The success of the prepared nanoemulsion was also controlled by particle size distribution analysis, and the average particle size was found as 95±7 nm.

Film preparation

Whey protein isolate (WPI) films were prepared according to the method described by Seydim & Sarikus (2006). Briefly, WPI at 5% w/w was dissolved in distilled water, including glycerol at 50% w/w (based on the dry weight of WPI), and the pH of the film solution was adjusted to 8 with NaOH (2N). Then, the solutions were heated to 90 °C while adding candelilla wax at 0.8% w/w and stirring continuously for 25 min. The film-forming solution was filtered three times and cooled before adding nanoemulsion and casting.

Starch (S) films were also prepared by the casting method. S at 5% was dissolved in distilled water, and glycerol at 30% (based on the dry weight of S) was added. The S film-forming solution was obtained by heating at 85-90 °C for 15 min and then cooled for further applications.

BO nanoemulsion was added to WPI and S film-forming solutions with a final BO concentration of 2% (based on WPI and S content) and homogenized at 10000 rpm for 5 min before removing air bubbles. In the preliminary studies, it was discovered that higher BO concentrations caused the loss of film integrity. Thus, the threshold concentration of BO inside the film-forming solutions was selected as 2%. Film-forming solutions were cast onto Teflon® coated Petri-plates (Ø=15 cm) with controlled weight to adjust the thickness and dried at 25 °C. The solid content per cm² of petri dishes was adjusted for film samples and the pouring amount of each film-forming solution was measured based on their solid content.

WPI and S films, including BO nanoemulsion, were coded as WPI-BO and S-BO, respectively. Final film samples were conditioned at 25 °C and 53% relative humidity (RH) for 1 week before the physical characterization analyses. At least 6 random points were selected to measure each film’s thickness using a digital micrometer (Digimatic Micrometer, Mitutoyo, Japan).

Characterization of film samples

ASTM standard method (D882) (ASTM 2018) was used to determine the elastic modulus (EM), tensile strength (TS), and elongation at break (E, %) values of film samples. Film samples (2.5 cm wide and 5 cm long) were stretched at 50 mm/min until break by a universal testing machine (Lloyd LR5, AMETEK, Inc, UK), and the corresponding values were determined from strain stress curves, estimated from force-distance data. At least ten replicates were obtained from each sample.

ASTM E96/E96M-16 gravimetric method (ASTM 2016) (cup method) was used to measure water vapor permeability (WVP) values of film samples. A 5 mL of distilled water was placed in permeability cups (3.5 cm in diameter), and then the cups were transferred to a desiccator having 53% RH (including oversaturated magnesium nitrate) at 25 °C. During the drying process, the air contact side of the film sample was exposed to 53% RH, and the other side was exposed to 100% RH at 25 °C. The decrease in permeability cups’ weight was recorded (every 1.5 h) to calculate the permeability rates using linear regression.

The opacity of film samples was determined by a spectrophotometer (Shimadzu, UV-1601, Japan). Films were cut into 1×4 cm size, and the opacity (absorbance units per film thickness, AU nm/mm) was calculated from the absorption spectrum of films obtained between 400-800 nm. The color values of film samples were determined with Minolta Chroma Meter (CR-400, Konica Minolta, Inc., Japan) using a white standard calibration plate (Y=92.7, x=0.3160, y=0.3321) as a background. Results were expressed as CIE L* (lightness), a* (red-green), and b* (yellow-blue) coordinates in the color space.

Estimation of BO release from films

The food simulant D1 (50 %, v/v ethanol) was selected as a release medium for film samples (European Commission 2011). BO is more soluble in ethanol than water, and an increase in ethanol concentration will increase the solubility of BO. Our preliminary study revealed that an increase in ethanol concentration limited the hydration of the film samples in ethanol, and BO diffusion from the film matrix has become difficult due to the polymer network’s attenuating effect. Therefore, to determine the highest release of BO from the film samples, D1 simulant was chosen as the release medium in this study. Film samples (0.1 g) were immersed into 10 mL of simulant and stirred at 25 °C for 48 h. The absorbance of the medium was recorded after various exposure times of released BO concentration measurements by using the corresponding calibration curve. The calibration curve of d-limonene obtained by the analysis of standard solutions prepared in different concentrations was used to measure the concentration of BO at the determined time interval for release studies.

The following equation proposed by Peleg (1988) was used to model the release kinetics of BO in the D1 simulant.

$$M_t=\frac{t}{k_1+k_2}$$ (1)

where M_t is the amount of BO released at each time, and the kinetic constants, k₁ and k₂, are the inverse of the initial release rate and the asymptotic release value, respectively.

Statistical analysis

Each experiment was replicated two times with at least three observations for each sample. The differences between samples were determined with an analysis of variance (ANOVA) and Tukey’s multiple comparison tests at a 95 % confidence level. The statistical analysis was performed using Minitab 17 software (Minitab Inc., Brandon, UK).

RESULTS AND DISCUSSION

Physical properties of film samples

The effect of BO nanoemulsion on the thickness, WVP, and mechanical properties of film samples was evaluated, and the results are shown in Table I. BO nanoemulsion incorporated S film showed higher thickness values. In contrast, the addition of nanoemulsion caused a decrease in the thickness of WPI film compared to corresponding neat films (p<0.05). The thickness of films is related to the interaction between compounds and polymer chains; thus, a decrease in the thickness might be related to the better interaction and compatibility between WPI and BO nanoemulsion. On the other hand, the differences in water uptake behaviors and holding capacities of WPI and S films might have caused different thickness values, affecting the water permeability and mechanical stability. Kalateh-Seifari et al. (2021) reported that the addition of nettle essential oil nanoemulsion caused an increase in the thickness of starch/chitosan films.

Table I
WVP, thickness, and mechanical properties of film samples.

Sample	Thickness (μm)	EM (MPa)	TS (MPa)	E (%)	WVP (g mm/kPa h m2)
WPI	151±9a	172.3±16.1a	13.1±1.6a	18.8±3.8a	60.49±0.45ab
S	140±1ab	146.6±30.7a	7.9±2.9a	2.7±0.4b	86.87±16.86a
WPI-BO	129±6b	190.2±10.7a	14.1±1.5a	16.3±2.2a	21.57±2.14c
S-BO	146±2a	204.3±32.5a	10.1±1.1a	1.8±0.3b	43.73±5.76bc

• ^a–^c Different letters in the same column indicate significant differences among the film samples (p < 0.05) (Mean ± SD).

The addition of BO nanoemulsion resulted in an increase in EM and TS values (p>0.05) with a concomitant decrease in % E (p<0.05), leading to more rigid and less flexible films. These results suggest an adequate adhesion was obtained through the interaction between functional groups of polymer matrices and BO nanoemulsion at the interface (Zhu et al. 2018). Similarly, de Oliveira Filho et al. (2021) reported improved mechanical properties for starch-based films when incorporated with carnauba wax nanoemulsion, cellulose nanocrystals, and essential oils. Kong et al. (2020) have also stated an increase in EM and TS values of starch/polyvinyl alcohol films when incorporated with carvacrol nanoemulsions. On the other hand, Norcino et al. (2020) observed a decrease in EM and TS with an increase in % E values of pectin films due to the plasticizing effect of copaiba oil nanoemulsions leading to weakening the intermolecular interactions between polymer chains. The incorporation of BO nanoemulsion into S films resulted in a 27-40% increase in TS and EM values, whereas approximately 10% of the increase was observed in WPI films for EM and TS values. The lowest % E values were found in S-based films with a 32% reduction in elasticity upon the addition of BO nanoemulsion, while only a 14% decrease was observed in BO nanoemulsion added WPI films. Elongation is a capacity of film flexibility desired for easy handling of films, while the elastic modulus and tensile strength are related to the resistance required to maintain the structural integrity of films. Nanoemulsion droplets might provoke hydrogen bonding of polymer and increase the rigidity and resistance while decreasing flexibility and mobility in polymer chains (Almasi et al. 2020). A carrier film should maintain the integrity of itself and the added emulsion during the handling and processing. It can be concluded that the incorporation of BO nanoemulsion had a more pronounced effect on the mechanical properties of S based films than those obtained for WPI based films.

In contrast to the results obtained in this study, Hasheminya & Dehghannya (2021), Li et al. (2020), and Lee et al. (2019) reported a decrease in strength and an increase in elongation values for nanoemulsion included film samples. Lee et al. (2019) attributed the decrease in mechanical properties of hydroxypropyl methylcellulose films when incorporated with oregano nanoemulsion to the replacement of polymer-polymer interaction with polymer-nanoemulsion interactions causing discontinuities in the polymer. Besides, Li et al. (2020) stated that thymol nanoemulsion addition resulted in weakened hydrogen bonds and decreased internal network and cohesiveness of the gelatin film.

An improvement in WVP of film samples was observed with BO nanoemulsion. The permeability of water molecules through polymer depends on the number of polar groups present in the structure, such as hydroxyl groups, which affect intermolecular attraction (Hasheminya & Dehghannya 2021). BO nanoemulsion caused a decrease between 50-65%, according to the relative neat film. The decrease in WVP values upon the addition of BO nanoemulsion might be due to the contribution of nanocellulose based emulsion to the establishment of a tortuous pathway for water molecules. The nanoemulsion droplets could increase certain tortuosity and reduce the diffusivity of water molecules due to the amorphous regions of the polymer matrix that have hydrophilic and hydrophobic molecules. The ratio between the hydrophobic and hydrophilic parts of the film determines the diffusion of water molecules through the film matrix. The homogeneous distribution of nanoemulsions with hydrophobic nature might lower the adsorption and diffusion of water molecules within the film by increasing the hydrophobicity of films. Besides, the polymer chains could become less mobile, reducing the diffusion of water molecules at the interface (Almasi et al. 2020). The lower WVP values observed for WPI films compared to S films can be explained by the differences in components and concentration used for plasticizing due to the different requirements of films. These results agree with the studies reported by Shen et al. (2021), who studied the properties of clove essential oil loaded nanoemulsion and Pickering emulsion activated pullulan-gelatin based edible film. Norcino et al. (2020) studied the potential use of copaiba oil nanoemulsion in pectin films and reported a decrease in WVP. The improvement in the water barrier of WPI and S-based carrier films could avoid deterioration arising from water activity for the nanoemulsion as the water-resistance increased by strengthening the interaction between polymer chains while reducing the chain mobility by filling the free spaces in the polymer matrix (Zhu et al. 2018). Similarly, Oliveira Filho et al. (2020) have found a reduction in WVP values of arrowroot starch films, including carnauba wax, due to its high-water repellent properties and high fatty alcohol contents and reported that carnauba wax nanoemulsions showed a higher reduction in WVP when compared to their macro emulsion forms. The decrease of WVP upon the addition of a nanoemulsion into WPI-based films was also confirmed by Ghadetaj et al. (2018).

The optical properties of film samples are shown in Table II. WPI based films had lower opacity values than those obtained for S based films (p<0.05). The addition of BO nanoemulsion significantly increased the opacity of film samples (p<0.05). These results might be attributed to the light absorption characteristics of phenolics found in BO, especially at lower wavelengths (Chen et al. 2016). The increase in opacity could also be related to the increase in the light scattering throughout the film with the oil droplets in the film structure. With the help of the decrease in light transmittance, the light barrier effect might show a protective impact on the oxidative reactions of BO. The addition of nanoemulsion might scatter the light and block the light passage depending on the concentration and volume fractions of the lipid phase of the emulsion (Hasheminya et al. 2019).

Table II
The optical properties of film samples.

Sample	T (%)	Opacity (AU nm/mm)	L*	a*	b*
WPI	74.7±0.6a	198.6±22.2c	95.91±0.32a	-0.18±0.06a	4.36±0.18b
S	62.5±0.5b	352.7±12.5b	95.68±0.18a	-0.35±0.02b	3.00±0.07c
WPI-BO	64.6±3.3b	414.9±22.9b	94.81±0.03a	-0.58±0.06c	7.33±0.36a
S-BO	52.1±1.1c	632.7±104.3a	95.76±1.27a	-0.45±0.02b	3.22±0.12c

• ^a–^c Different letters in the same column indicate significant differences among the film samples (p < 0.05) (Mean ± SD).

Besides, the addition of BO nanoemulsion into films caused a decrease in lightness (p>0.05) and a* values (p<0.05) while increasing the b* values (p<0.05). The changes observed in a* and b* values depend on the nature of the essential oil used to form nanoemulsions, as confirmed by Kalateh-Seifari et al. (2021). Similarly, Hasheminya & Dehghannya (2021) reported a decrease in lightness and light transmittance values of Cordia dichotoma gum-based films when incorporated with Salvia mirzayanii essential oil nanoemulsion. The color values of S films did not change significantly upon the addition of BO nanoemulsion. Almasi et al. (2020) also reported that the addition of nanoemulsion did not significantly change the color of pectin films due to the smaller droplet size of the emulsion. The differences observed for the color values between WPI and S films can be explained by the differences in the light absorption behavior of each film, the differences in surface roughness of film samples, or differences in compatibility and homogeneity of the constituents within different polymer matrices (Acevedo-Fani et al. 2015). The color film samples also depend on the phenolic composition, natural color, and concentration of added essential oil in the nanoemulsion formulation. Similar results have been reported by Lee et al. (2019) and Agudelo-Cuartas et al. (2020) for hydroxypropyl methylcellulose and WPI films when enhanced with oregano essential oil nanoemulsions and α-tocopherol nanoemulsions, respectively.

The estimation of BO release from film samples

A moiety is released from a polymeric matrix by a three-stage process: i) solvent penetration through the polymer, ii) swelling of the polymer, and iii) diffusion of a constituent from swollen polymer (Zhu et al. 2018). The release of BO from WPI and S films was examined, and the related kinetics with release profiles are shown in Figure 1 and Table III.

Figure 1
Cumulative release rates of film samples.

Table III
Peleg’s model parameters of film samples.

Sample	1/k1 (wt.%/min)	1/k2 (mg/g film)	R2
WPI-BO	0.048±0.002b	27.38±3.63b	0.93
S-BO	0.267±0.024a	46.59±2.76a	0.98

• ^a–^b Different letters in the same column indicate significant differences among the film samples (p < 0.05) (Mean ± SD).
  k₁ is related to the release rate at the beginning of the process and k ₂ is related to the asymptotic value, which is obtained from the inverse of M∞, the amount of active agents released at equilibrium.

The release rate of BO from S films was higher than WPI films. The interaction between BO nanoemulsion and S film matrix might be weaker than in WPI films, leading to a more open structure and more relaxed polymer matrix related to the polymer solubility (Chen et al. 2016). Similarly, Sogut (2020) reported an initial faster release and subsequent slower release rates for nanocellulose stabilized BO from WPI based films. Chu et al. (2020) reported that the retention of cinnamon essential oil inside pullulan-based films had been increased by nanoemulsion formation. The cumulative release rates obtained after 48 h were 29.28±0.17% for WPI-BO and 44.91±2.61% for S-BO films. The presence of nanocellulose within the polymer matrix might also slow down the release of BO due to the interaction at the interface (Sogut & Seydim 2018). An initial fast release followed by a slower release rate of nanoemulsion of eugenol from chitosan was demonstrated by Das et al. (2021), and the authors stated that the hydrophilic nature of chitosan and possible weak interactions might affect the release behavior of eugenol, resulting in rapid nanoparticle hydration. Sheorain et al. (2019) also reported a fast dissolution followed by a sustained release of thymol from chitosan-tragacanth nanoparticles with biphasic release kinetics. The obtained cumulative release rates for BO from WPI and S after 48 h showed a potential opportunity in the controlled release of bioactive components such as aroma compounds, suggesting the potential of selected polymers as wall matrix for controlling aroma release.

As shown in Table III, the absorbance data and measured cumulative release rates were fitted to the selected model equation to calculate the release kinetics. The release behavior of BO from films to the selected food simulant exhibited a good fit with the model proposed by Peleg (1988) (R² > 0.93). The initial release rate (1/k₁) and the equilibrium/asymptotic value (1/k₂) of WPI-BO and S-BO films were obtained by this equation and are shown in Table III. The release rate of an active agent such as flavors from film matrix is significant for its potential use in food applications, active films, or coatings. The higher 1/k₁ and 1/k₂ (M∞) values were observed in S-BO films (p<0.05). The total BO released from WPI films was lower than S-BO films, which coincided with the lower 1/k₂ values obtained for WPI-BO films (p<0.05). Peleg (1988) proposed that the lower concentrations reached equilibrium were related to the higher k₂ values.

CONCLUSION

BO is one of the widely used flavors in the food industry; however, its volatile nature limits its use. The stabilization of BO using polymeric matrices and nanoparticles for packaging applications may provide a broader application of BO. Thus, in this study, BO nanoemulsion was prepared with NC before adding into WPI and S film-forming solutions and aimed to test the potential of selected film samples to be used as carriers of BO, which stabilized with NC. The film samples had improved mechanical and water vapor barrier properties when incorporated with BO nanoemulsion. The release rate of BO from S films was faster than WPI films, which might be due to the partial solubility of S films in the selected food simulant, as confirmed with WVP values. This study showed that essential oils could be added to biopolymeric film solutions via nanoemulsion formation without phase separation, miscibility problems. Besides, these results can be used to control the release of aroma compounds commonly used in food products such as BO. However, further studies are needed to understand the compatibility between components and their effectiveness in packaged foods.

ACKNOWLEDGMENTS

This study was conducted at Suleyman Demirel University Food Engineering Department Laboratories. Special thanks to Oguz Sogut for nanoemulsion formulation and characterization.

REFERENCES

• ACEVEDO-FANI A, SALVIA-TRUJILLO L, ROJAS-GRAÜ MA & MARTÍN-BELLOSO O. 2015. Edible films from essential-oil-loaded nanoemulsions: Physicochemical characterization and antimicrobial properties. Food Hydrocoll 47: 168-177.
• AGUDELO-CUARTAS C, GRANDA-RESTREPO D, SOBRAL PJA, HERNANDEZ H & CASTRO W. 2020. Characterization of whey protein-based films incorporated with natamycin and nanoemulsion of α-tocopherol. Heliyon 6(4): e03809.
• ALMASI H, AZIZI S & AMJADI S. 2020. Development and characterization of pectin films activated by nanoemulsion and Pickering emulsion stabilized marjoram (Origanum majorana L.) essential oil. Food Hydrocoll 99: 105338.
• ASABUWA NGWABEBHOH F, ILKAR ERDAGI S & YILDIZ U. 2018. Pickering emulsions stabilized nanocellulosic-based nanoparticles for coumarin and curcumin nanoencapsulations: In vitro release, anticancer and antimicrobial activities. Carbohydr Polym 201: 317-328.
• ASHAOLU TJ. 2021. Nanoemulsions for health, food, and cosmetics: a review. Environ Chem Lett 19(4): 3381-3395.
• ASSADPOUR E & MAHDI JAFARI S. 2019. A systematic review on nanoencapsulation of food bioactive ingredients and nutraceuticals by various nanocarriers. Crit Rev Food Sci Nutr 59(19): 3129-3151.
• ASTM (AMERICAN SOCIETY FOR TESTING AND MATERIALS STANDARD). 2016. Standard test methods for water vapor transmission of materials: E96/E96M- 16. Annual book of American society for testing and materials standards. Annu B Am Stand Test Methods 04.06: 14.
• ASTM (AMERICAN SOCIETY FOR TESTING AND MATERIALS STANDARD). 2018. Standard Test Method for Tensile Properties of Thin Plastic Sheeting: D882-02. ASTM International. Annual book of American society for testing and materials standards. Anu B Am Stand Test Methods 2002: 1-12.
• ATARÉS L & CHIRALT A. 2016. Essential oils as additives in biodegradable films and coatings for active food packaging. Trends Food Sci Technol 48: 51-62.
• BAHRAMI A, DELSHADI R, ASSADPOUR E, JAFARI SM & WILLIAMS L. 2020. Antimicrobial-loaded nanocarriers for food packaging applications. Adv Colloid Interface Sci 278: 102140.
• BORA H, KAMLE M, MAHATO DK, TIWARI P & KUMAR P. 2020. Citrus essential oils (CEOs) and their applications in food: An overview. Plants 9(3): 357.
• CHEN H, HU X, CHEN E, WU S, MCCLEMENTS DJ, LIU S, LI B & LI Y. 2016. Preparation, characterization, and properties of chitosan films with cinnamaldehyde nanoemulsions. Food Hydrocoll 61: 662-671.
• CHU Y, CHENG W, FENG X, GAO C, WU D, MENG L, ZHANG Y & TANG X. 2020. Fabrication, structure and properties of pullulan-based active films incorporated with ultrasound-assisted cinnamon essential oil nanoemulsions. Food Packag Shelf Life 25: 100547.
• DAS S, SINGH VK, DWIVEDY AK, CHAUDHARI AK, DEEPIKA & DUBEY NK. 2021. Eugenol loaded chitosan nanoemulsion for food protection and inhibition of Aflatoxin B1 synthesizing genes based on molecular docking. Carbohydr Polym 255: 117339.
• DE OLIVEIRA FILHO JG, ALBIERO BR, CIPRIANO L, DE OLIVEIRA NOBRE BEZERRA CC, OLDONI FCA, EGEA MB, DE AZEREDO HMC & FERREIRA MD. 2021. Arrowroot starch-based films incorporated with a carnauba wax nanoemulsion, cellulose nanocrystals, and essential oils: a new functional material for food packaging applications. Cellulose 28(10): 6499-6511.
• DOI T, WANG M & MCCLEMENTS DJ. 2019. Emulsion-based control of flavor release profiles: Impact of oil droplet characteristics on garlic aroma release during simulated cooking. Food Res Int 116: 1-11.
• EUROPEAN COMMISSION. 2011. No 10/2011 on plastic materials and articles intended to come into contact with food. Off J Eur Union 15(1): 12-88.
• FONSECA-GARCÍA A, JIMÉNEZ-REGALADO EJ & AGUIRRE-LOREDO RY. 2021. Preparation of a novel biodegradable packaging film based on corn starch-chitosan and poloxamers. Carbohydr Polym 251: 117009.
• FUJISAWA S, TOGAWA E & KURODA K. 2017. Nanocellulose-stabilized Pickering emulsions and their applications. Sci Technol Adv Mater 18(1): 959-971.
• GHADETAJ A, ALMASI H & MEHRYAR L. 2018. Development and characterization of whey protein isolate active films containing nanoemulsions of Grammosciadium ptrocarpum Bioss. essential oil. Food Packag Shelf Life 16: 31-40.
• GROMADZKI D, TZANKOVA V, KONDEVA M, GORINOVA C, RYCHTER P, LIBERA M, MOMEKOV G, MARIĆ M & MOMEKOVA D. 2017. Amphiphilic core-shell nanoparticles with dimer fatty acid-based aliphatic polyester core and zwitterionic poly(sulfobetaine) shell for controlled delivery of curcumin. Int J Polym Mater Polym Biomater 66(18): 915-925.
• GURPREET K & SINGH SK. 2018. Review of nanoemulsion formulation and characterization techniques. Indian J Pharm Sci 80(5): 781-789.
• HASHEMINYA SM & DEHGHANNYA J. 2021. Development and characterization of novel edible films based on Cordia dichotoma gum incorporated with Salvia mirzayanii essential oil nanoemulsion. Carbohydr Polym 257: 117606.
• HASHEMINYA SM, MOKARRAM RR, GHANBARZADEH B, HAMISHEKAR H, KAFIL HS & DEHGHANNYA J. 2019. Development and characterization of biocomposite films made from kefiran, carboxymethyl cellulose and Satureja Khuzestanica essential oil. Food Chem 289: 443-452.
• HE L, HU J & DENG W. 2018. Preparation and application of flavor and fragrance capsules. Polym Chem 9(40): 4926-4946.
• KALATEH-SEIFARI F, YOUSEFI S, AHARI H & HOSSEINI SH. 2021. Corn starch-chitosan nanocomposite film containing nettle essential oil nanoemulsions and starch nanocrystals: Optimization and characterization. Polymers 13(13): 2113.
• KASIRI N & FATHI M. 2018. Entrapment of peppermint oil using cellulose nanocrystals. Cellulose 25(1): 319-329.
• KONG R, WANG J, CHENG M, LU W, CHEN M, ZHANG R & WANG X. 2020. Development and characterization of corn starch/PVA active films incorporated with carvacrol nanoemulsions. Int J Biol Macromol 164: 1631-1639.
• LEE JY, GARCIA CV, SHIN GH & KIM JT. 2019. Antibacterial and antioxidant properties of hydroxypropyl methylcellulose-based active composite films incorporating oregano essential oil nanoemulsions. LWT 106: 164-171.
• LI X, YANG X, DENG H, GUO Y & XUE J. 2020. Gelatin films incorporated with thymol nanoemulsions: Physical properties and antimicrobial activities. Int J Biol Macromol 150: 161-168.
• MANNUCCI C, NAVARRA M, CALAPAI F, SQUERI R, GANGEMI S & CALAPAI G. 2017. Clinical Pharmacology of Citrus bergamia: A Systematic Review. Phyther Res 31(1): 27-39.
• MARCHESE E, D’ONOFRIO N, BALESTRIERI ML, CASTALDO D, FERRARI G & DONSÌ F. 2020. Bergamot essential oil nanoemulsions: antimicrobial and cytotoxic activity. Zeitschrift fur Naturforsch - Sect C J Biosci 75(7-8): 279-290.
• MCCLEMENTS DJ & GUMUS CE. 2016. Natural emulsifiers — Biosurfactants, phospholipids, biopolymers, and colloidal particles: Molecular and physicochemical basis of functional performance. Adv Colloid Interface Sci 234: 3-26.
• MIHALY-COZMUTA A, PETER A, CRACIUN G, FALUP A, MIHALY-COZMUTA L, NICULA C, VULPOI A & BAIA M. 2017. Preparation and characterization of active cellulose-based papers modified with TiO2, Ag and zeolite nanocomposites for bread packaging application. Cellulose. 24(9): 3911-3928.
• MWANGI WW, HO KW, TEY BT & CHAN ES. 2016. Effects of environmental factors on the physical stability of pickering-emulsions stabilized by chitosan particles. Food Hydrocoll 60: 543-550.
• NORCINO LB, MENDES JF, NATARELLI CVL, MANRICH A, OLIVEIRA JE & MATTOSO LHC. 2020. Pectin films loaded with copaiba oil nanoemulsions for potential use as bio-based active packaging. Food Hydrocoll 106: 105862.
• OLIVEIRA FILHO JG DE, BEZERRA CC DE ON, ALBIERO BR, OLDONI FCA, MIRANDA M, EGEA MB, AZEREDO HMC DE & FERREIRA MD. 2020. New approach in the development of edible films: The use of carnauba wax micro- or nanoemulsions in arrowroot starch-based films. Food Packag Shelf Life 26: 100589.
• ORTH AM, YU L & ENGEL KH. 2013. Assessment of dietary exposure to flavouring substances via consumption of flavoured teas. Part 1: occurrence and contents of monoterpenes in Earl Grey teas marketed in the European Union. Food Addit Contam - Part A Chem Anal Control Expo Risk Assess 30(10): 1701-1714.
• ÖZOGUL Y, EL ABED N & ÖZOGUL F. 2022. Antimicrobial effect of laurel essential oil nanoemulsion on food-borne pathogens and fish spoilage bacteria. Food Chem 368: 130831.
• PELEG M. 1988. An Empirical Model for the Description of Moisture Sorption Curves. J Food Sci 53(4): 1216-1217.
• REHMAN A, JAFARI SM, AADIL RM, ASSADPOUR E, RANDHAWA MA & MAHMOOD S. 2020. Development of active food packaging via incorporation of biopolymeric nanocarriers containing essential oils. Trends Food Sci Technol 101: 106-121.
• REN JN, DONG M, HOU YY, FAN G & PAN SY. 2018. Effect of olive oil on the preparation of nanoemulsions and its effect on aroma release. J Food Sci Technol 55(10): 4223-4231.
• RIAZ T, IQBAL MW, SAEED M, YASMIN I, HASSANIN HAM, MAHMOOD S & REHMAN A. 2019. In vitro survival of Bifidobacterium bifidum microencapsulated in zein-coated alginate hydrogel microbeads. J Microencapsul 36(2): 192-203.
• RODRIGUEZ LLANOS JH, TADINI CC & GASTALDI E. 2021. New strategies to fabricate starch/chitosan-based composites by extrusion. J Food Eng 290: 110224.
• SAIDANE D, PERRIN E, CHERHAL F, GUELLEC F & CAPRON I. 2016. Some modification of cellulose nanocrystals for functional Pickering emulsions. Philos Trans R Soc A Math Phys Eng Sci 374(2072): 20150139.
• SEYDIM AC & SARIKUS G. 2006. Antimicrobial activity of whey protein based edible films incorporated with oregano, rosemary and garlic essential oils. Food Res Int 39(5): 639-644.
• SEYDIM AC, SARIKUS-TUTAL G & SOGUT E. 2020. Effect of whey protein edible films containing plant essential oils on microbial inactivation of sliced Kasar cheese. Food Packag Shelf Life 26: 100567.
• SHAH BR, LI Y, JIN W, AN Y, HE L, LI Z, XU W & LI B. 2016. Preparation and optimization of Pickering emulsion stabilized by chitosan-tripolyphosphate nanoparticles for curcumin encapsulation. Food Hydrocoll 52: 369-377.
• SHEN Y, NI ZJ, THAKUR K, ZHANG JG, HU F & WEI ZJ. 2021. Preparation and characterization of clove essential oil loaded nanoemulsion and pickering emulsion activated pullulan-gelatin based edible film. Int J Biol Macromol 181: 528-539.
• SHEORAIN J, MEHRA M, THAKUR R, GREWAL S & KUMARI S. 2019. In vitro anti-inflammatory and antioxidant potential of thymol loaded bipolymeric (tragacanth gum/chitosan) nanocarrier. Int J Biol Macromol 125: 1069-1074.
• SOGUT E & SEYDIM AC. 2018. Development of Chitosan and Polycaprolactone based active bilayer films enhanced with nanocellulose and grape seed extract. Carbohydr Polym 195: 180-188.
• SOGUT E. 2020. Active whey protein isolate films including bergamot oil emulsion stabilized by nanocellulose. Food Packag Shelf Life 23: 100430.
• VAHEDIKIA N, GARAVAND F, TAJEDDIN B, CACCIOTTI I, JAFARI SM, OMIDI T & ZAHEDI Z. 2019. Biodegradable zein film composites reinforced with chitosan nanoparticles and cinnamon essential oil: Physical, mechanical, structural and antimicrobial attributes. Colloids Surfaces B Biointerfaces 177: 25-32.
• WANG X, HE J, MA L, YAN B, SHI L & RAN R. 2021. Self-assembling graphene oxide/modified amphipathic hydroxyethyl cellulose hybrid stabilized Pickering emulsion polymerization for functional hydrogel. Colloids Surfaces A Physicochem Eng Asp 610: 125742.
• YU L, LI S, STUBBS LP & LAU HC. 2021. Characterization of clay-stabilized, oil-in-water Pickering emulsion for potential conformance control in high-salinity, high-temperature reservoirs. Appl Clay Sci 213: 106246.
• ZHU JY, TANG CH, YIN SW & YANG XQ. 2018. Development and characterization of novel antimicrobial bilayer films based on Polylactic acid (PLA)/Pickering emulsions. Carbohydr Polym 181: 727-735.

Publication Dates

• Publication in this collection
  21 Nov 2022
• Date of issue
  2022

History

• Received
  5 Sept 2021
• Accepted
  20 Nov 2021