International Journal of Biological Macromolecules 283 (2024) 137826

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Bioactive multilayer film based on the sage seed gum-gelatin, TiO2 and
electrospun zein fibers encapsulating pomegranate peel extract
Romina Valizadeh a , Mohsen Zandi a,* , Ali Ganjloo a, Nazila Dardmeh b
a
Department of Food Science and Engineering, Faculty of Agriculture, University of Zanjan, Zanjan 45371-38791, Iran
b
Department of Food Science and Technology, Faculty of Agriculture, Ferdowsi University of Mashhad, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: Despite the desirable features, electrospun fibers are mechanically fragile. To enhance their usability in pack­
Active packaging aging applications, these fibers need to be supported on a substrate. In this research, bio-based electrospun
Antimicrobial properties multilayered films were developed based on the strong sage seed gum-gelatin film as the outer layers and an
Composite film
electrospun zein-pomegranate flower extract (0, 20, 40, 60, and 80 g.m− 2) as the middle layer. The findings
Mechanical properties
revealed that the developed multilayer films exhibited a thickness ranging from 0.18 to 0.25 mm. Increased
electrospun layer thickness enhanced interfacial adhesion, resulting in a denser film structure, which subse­
quently improved tensile strength (from 60.8 to 70.2 MPa) and elastic modulus (from 279.9 to 379.2 MPa). These
modifications led to a reduction in water vapor and oxygen permeability by 72.4 % and 82.6 %, respectively.
Additionally, the opacity of the film >200 %. Increasing the thickness of the electrospun layer also resulted in an
increase in total phenolic content (TPC) and antioxidant activity by 899 % and 547 %, respectively. The FTIR
analysis revealed molecular interactions between the different layers of the multilayer film. Furthermore, X-ray
diffraction (XRD) analysis indicated that the multilayer film possesses a semi-crystalline structure, with an in­
crease in crystallinity observed as the thickness of the electrospun layer increased, which consequently enhanced
the thermal stability of the multilayer films. A reduction in water solubility, moisture content, and swelling ratio,
as well as an increase in antimicrobial activity, was also observed with the increasing thickness of the electrospun
layer. Release kinetics results showed that the lowest release rate occurred in the fatty food simulant. This
research advances the development of bio-based electrospun multilayered films to improve their mechanical
strength and barrier properties for packaging. It highlights the potential of these materials in sustainable
packaging and paves the way for future innovations in environmentally friendly practices.

1. Introduction hydrophilic and exhibit high water permeability. Various strategies have
been explored to overcome these challenges in the production of
Recently, there has been growing interest among researchers in biopolymer-based films ([7]; Javaid, Cheema, et al., 2024; [8,9]).
developing bioplastics using biodegradable biopolymers. These bio­ Chemical or physical modification of biopolymers, such as ozonation,
polymers, while possessing desirable characteristics such as biodegrad­ ultrasound treatment, pulsed electric fields, and microwave application,
ability, biocompatibility, safety, and edibility, result in films that are is one of the strategies employed to enhance their properties. While
prone to thermal degradation and deterioration over time, especially these techniques can improve certain characteristics of biopolymers,
when exposed to light, in comparison to petroleum-based plastics. The such as mechanical properties, they may also compromise other attri­
low tensile strength, elongation at break, and other weak mechanical butes, such as barrier properties (i.e. water vapor permeability, oxygen
properties of these films present significant challenges to their devel­ permeability and light barrier properties) [6,10–12]. The blending of
opment [1–6]. However, studies have shown that films made from a biopolymers and the production of biocomposite films is another
single biopolymer rarely offer a competitive advantage over synthetic effective technique that has garnered significant attention recently.
films, further limiting their applications. For instance, carbohydrate- Challenges associated with this method include achieving uniform
based films, such as those made from gums, mucilages, or starch, are biopolymer blending and preventing surface tension issues in the

* Corresponding author.
E-mail address: [email protected] (M. Zandi).

https://doi.org/10.1016/j.ijbiomac.2024.137826
Received 12 September 2024; Received in revised form 11 November 2024; Accepted 16 November 2024
Available online 18 November 2024
0141-8130/© 2024 Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.
R. Valizadeh et al. International Journal of Biological Macromolecules 283 (2024) 137826

resulting biocomposite. Perhaps the most effective strategy is the com­ various applications such as emulsification, foaming, stabilization, the
bination of various layers with specific properties to construct multi­ formation of biodegradable films, and encapsulation. By incorporating
layer films with enhanced characteristics. The use of multilayer films active compounds like nanoparticles, active biocomposites can be
can enhance the mechanical and barrier properties of biodegradable developed [7,13,28,29]. Titanium dioxide (TiO2), as a semiconductor
polymers. ([12–14]; Q. [15]). material, is recognized as an ideal nanomaterial due to its high stability,
Multilayer films are among the more effective methods for replacing low cost, and non-toxicity to both the environment and humans. Given
traditional food packaging materials with biopolymeric and renewable its excellent antimicrobial properties and high compatibility with most
alternatives. This approach integrates the unique properties of various biopolymers, TiO2 is an ideal filler for the production of active food
types of polymers to create packaging with improved performance in packaging films [2,30,31,55]. Some studies report the development of
terms of protection and durability. Multilayer films, used in various electrospun zein-based nanofibers [18,19,22,32,33]; however, there is
applications, consist of multiple layers of different polymers, typically currently no research on sage seed gum-gelatin composite films or
ranging from 3 to 12 layers ([14,16]; Q. [15]). The use of multilayer multilayer biocomposite films with an electrospun layer. Currently,
packaging typically extends the shelf life of products. The development there are no reports of multilayer films that include a middle layer of
of multilayer films represents a new trend in the packaging industry. electrospun zein combined with pomegranate flower extract, and outer
These films can enhance the properties of single-layer films, making layers made from a composite of sage seed gum and gelatin, both with
them more suitable for industrial applications. Due to the combination and without the addition of TiO2. Nor studies on the effects of different
of two or more different polymers, multilayer films generally exhibit electrospun zein thicknesses on the performance of TiO2 films. To
better resistance to the permeation of water vapor, oxygen, and other develop a new antimicrobial active packaging film, in this work, we
gases, which is crucial for packaging food or products sensitive to oxy­ fabricate multilayer films. The aim of this study is to investigate the
gen or moisture ([16,17]; Q. [15]). Multilayer films, through the com­ effect of varying thicknesses of the electrospun zein-pomegranate flower
bination of various polymers, become more resistant to diverse chemical extract layer (0, 20, 40, 60, and 80 g.m− 2) on the mechanical, physical,
effects, making them highly beneficial in environments containing acids, thermal, chemical, structural, visual, and release properties of the
bases, or other chemicals. Additionally, these films can offer improved designed multilayer film, ultimately leading to the development of a
mechanical and thermal stability. However, multilayer films also have final film with enhanced properties.
some drawbacks, such as the lengthy production process, which occurs
in two or more stages, leading to higher energy consumption. Moreover, 2. Material and methods
a significant challenge in producing multilayer films is ensuring
adequate adhesion between the different layers. Various techniques, 2.1. Material
such as electrospinning, have been developed to fabricate multilayer
films and address these challenges ([14]; Q. [15]). Dried pomegranate flowers and sage seeds were obtained from a
Electrospinning is one of the most innovative and extensively studied local market in Zanjan, Iran, for the production of pomegranate flower
methods for developing smart packaging materials. This technique uti­ extract and sage seed gum, respectively, following species verification.
lizes electrostatic forces to produce electrically charged jets from Gelatin was purchased from Farmand Company (Tehran, Iran). Glycerol,
viscoelastic polymer solutions. Through electrospinning, it is possible to zein, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and Tween 80 were ob­
create micro- and/or nanostructures from a wide range of polymers and tained from Sigma (USA). Ethanol (96 %), acetic acid, and hexane were
biopolymers in a controlled manner [8,18–20]. Zein, due to its favorable purchased from Mojallali Company (Tehran, Iran). Folin-Ciocalteu re­
properties such as high thermal stability, excellent oxygen barrier ca­ agent and sodium carbonate were sourced from Merck (Germany).
pabilities, and inherent hydrophobicity, is a commonly used biopolymer Other chemicals, with a purity >96 %, were also procured from Sigma
in the electrospinning process. This protein, derived from corn, can (USA).
encapsulate a broad spectrum of active compounds, including nano­
particles, essential oils, and extracts, within its structure [18,21,22]. 2.2. Preparation of sage seed gum-gelatin composite film
One of the main advantages of the electrospinning is the encapsulation
of active compounds including nanoparticles, essential oils, and extracts Sage seed gum was extracted from the seeds using the method
in the core structure that protect sensitive compounds from environ­ described by Isadi et al. [34,35]. The composite film of sage seed gum-
mental/processing conditions [19]. Pomegranate flower extract is an gelatin was prepared using the casting method. A solution of sage seed
effective active agent that possesses strong antioxidant and antimicro­ gum (1 % w/v) and gelatin (4 % w/v) was prepared and heated at 80 ◦ C
bial properties. The medicinal efficacy of pomegranate flower is attrib­ for 10 min and 70 ◦ C for 10 min, respectively. The sage seed gum so­
uted to the presence of phenolic compounds, flavonoids, and lution was allowed to hydrate overnight at 25 ◦ C. The sage seed gum and
anthocyanins. Additionally, the polyphenolic compounds are respon­ gelatin solutions were mixed in a 2:1 ratio (w/w, based on solid content).
sible for the antimicrobial activities, and the anthocyanins present in Glycerol, as a plasticizer, at a concentration of 45 % (in dry sage seed
pomegranate flowers have a high potential for neutralizing free radicals gum and gelatin weight) was added to the film solution. The mixture was
([23]; T. [24]). To leverage the properties of composite films, they can stirred at 40 ◦ C for 20 min. The resulting film solution was poured into a
be used as one or two layers within a multilayer film. Gum-protein plastic mold and dried at room temperature for 48 h. The obtained film
composite films exhibit enhanced characteristics [1,2]. was equilibrated to moisture conditions (48 h at 25 ◦ C and RH = 53 %)
A composite film is generally defined as the combination of two or before any analysis [1]. For the third layer of the multilayer film, a
more distinct constituents (Such as ac gums, proteins, nanoparticles and composite film solution of sage seed gum-gelatin containing TiO2
etc.) to obtain a material with properties that cannot be achieved by the nanoparticles was prepared using the same method; however, prior to
individual components ([25]; Javaid, Alanazi, et al., 2024). Sage (Salvia preparing the sage seed gum and gelatin solutions, TiO2 (0.5 % w/v) was
macrosiphon) seed gum, in comparison to commercial gums such as added to the water and subjected to ultrasonic treatment for 30 min.
xanthan, guar, and locust bean gum, possesses superior rheological
properties. This gum can function as a stabilizing agent, thickener, and 2.3. Preparation of electrospinning solution and fabrication of multilayer
suitable fat substitute in food, cosmetic, and pharmaceutical systems film
[1,26,27]. However, sage seed gum films have hydrophilic properties,
which result in poor barrier and mechanical properties, combining sage The pomegranate flower extract was prepared using the resistive
seed gum with proteins and nanoparticles can produce improved com­ heating method [36,37]. The electrospinning solution was prepared by
posite films. Among protein sources, gelatin is widely utilized for dissolving 20 g of zein in the 100 mL of the 80 % (v/v) aqueous ethanol

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R. Valizadeh et al. International Journal of Biological Macromolecules 283 (2024) 137826

solution and then adding 10 % (v/v) pomegranate flower extract. This

solution was stirred for 20 min at 25 ◦ C.
The electrospinning process was conducted on the surface of the sage
seed gum-gelatin composite film using an electrospinning device
(NFCO-ANVI, ANSTCO, Iran) under ambient conditions (25 ◦ C and RH
= 30 %), with an 18-gauge nozzle and a rotating collector. In this pro­
cess, the distance from the nozzle tip to the collector, the flow rate, and
the voltage were set at 10 cm, 1 mL.h− 1, and 17 kV, respectively. The
resulting bilayer film was placed in plastic molds, and the sage seed Fig. 1. Schematic representation of the trilayer of film.
gum-gelatin composite film solution containing TiO2 nanoparticles was
poured over it as the third layer (Table 1, and Fig. 1), then dried at room 2.4.5. Morphology properties
temperature for 48 h. The surface and cross-sectional morphology of the films were
examined using a field emission scanning electron microscope (FESEM,
2.4. Characteristics FE-SEM, KYKY EM8000F, China) at magnifications ranging from 1000 x
to 10,000 x. For this purpose, the fully dried film was coated with a thin
2.4.1. Thickness layer of conductive gold (8 nm), and images were captured at an
The thickness of at least 10 different points on the multilayer film accelerating voltage of 3 kV (J. [38–40]).
was measured using a digital micrometer (IP65, Mitutoyo, Tokyo,
Japan) [1]. 2.4.6. Moisture content, water solubility and swalling ratio
The multilayer film (2 × 2 cm2) was weighed (W1) and then placed
2.4.2. Mechanical properties in an oven at 105 ◦ C for 24 h, after which it was weighed again (W2).
The mechanical properties, including tensile strength, elongation at The film was then immersed in 50 mL of distilled water for 6 h (25 ◦ C,
break, and elastic modulus, were measured using a texture analyzer 100 RPM) and subsequently oven-dried again (105 ◦ C, 24 h) and
(SANTAM Co., STM5, Iran). In this analysis, the initial distance between weighed (W3). The moisture content (MC) and film solubility (WS) were
the two grips and the speed of the upper grip were set to 10 mm and 150 calculated using Eqs. 4 and 5 [1]:
mm.min− 1, respectively [1]. The elongation at break was calculated by
dividing the increase in length of the sample at the moment of rupture by MC (%) = (W2 − W1)/W1 × 100 (4)
the initial length. The tensile strength was determined by dividing the
maximum force applied at the moment of rupture by the product of the WS (%) = (W1 − W3 /W1 × 100 (5)
film's thickness and width. The fully dried film was immersed in 50 mL of distilled water to
determine the swelling ratio and was weighed at specific time intervals
2.4.3. Surface color until a constant weight was reached [1].
The surface color of the film was measured using a handheld color­
imeter (TES, 135 A, Taiwan), and the color parameters L* (lightness), a* 2.4.7. Contact angle
(red-green value), and b* (yellow-blue value) were obtained from at The contact angle was measured by placing a 5-μl droplet on the
least 10 points on three separate films. The whiteness index (WI) and the surface of the film and photographing it. The ImageJ software (version
total color difference (ΔE) were calculated using these parameters and 1.52 V) was used to determine the contact angle between the water
the following eqs. (1 and 2) [1]: droplet and the horizontal surface [19].
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
( * )2 ( )2 ( )2
ΔE = L − L*0 + a* − a*0 + b* − b*0 (1) 2.4.8. Permeability
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ Water vapor and oxygen permeability were measured using the
WI = 100 − (100 − L* )2 + (a* )2 + (b* )2 (2) method described by Ayazi et al. [1]. For the water vapor permeability
analysis, 5 g of anhydrous calcium chloride, and for the oxygen
permeability analysis, an oxidizing agent (iron powder, sodium chlo­
2.4.4. Opacity
ride, and activated carbon) were separately placed in glass vials and
The opacity of the multilayer films was measured using a spectro­
then placed at room temperature in a desiccator containing a saturated
photometer at a wavelength of 600 nm and calculated using Eq. 3. An
sodium chloride solution (the partial water vapor pressure difference
empty cuvette was used as the reference, and the analysis was repeated
across the film was approximately 1753.55 Pa). The vials containing
five times for each sample.
calcium chloride were weighed every 24 h for 7 days, and the vials
The absorption rate of wavelength 600 nm containing the oxidizing agent were weighed after 48 h (t, reaction time
opacity = (3)
Film thickness in seconds). Water vapor permeability of the film (g. m− 1.s− 1. Pa− 1) was
calculated using the weight change of the vials containing calcium
chloride, and oxygen permeability (OP) was determined using Eq 6.

Table 1 mf − mi
OP = (6)
Films formulation and codes. t×a
Films formulation Codes Where mi, mf, and a represent the weight of the vial before and after
SG-Ge film+ SG-Ge film containing TiO2 nps SG-Ge/Ze 0/ placement in the desiccator and the effective surface area of the film
2
TiO2 (m2), respectively.
SG-Ge film+20 g.m− PFE loaded zein fibers+SG-Ge film SG-Ge/Ze 2/
containing TiO2 TiO2
SG-Ge film+40 g.m− 2
PFE loaded zein fibers+SG-Ge film SG-Ge/Ze 4/ 2.4.9. Structural properties
containing TiO2 TiO2 The crystalline structure of the film was analyzed using an X-ray
2
SG-Ge film+60 g.m− PFE loaded zein fibers+SG-Ge film SG-Ge/Ze 6/ diffractometer (XRD, x'pertoPro, Netherlands) with a radiation wave­
containing TiO2 TiO2
2
length of λ = 1.5415 Å, a current of 30 mA, and a voltage of 40 kV. To
SG-Ge film+80 g.m− PFE loaded zein fibers+SG-Ge film SG-Ge/Ze 8/
containing TiO2 TiO2
better assess the degree of crystallinity, an X-ray beam (Cu Kα) was used

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R. Valizadeh et al. International Journal of Biological Macromolecules 283 (2024) 137826

under the conditions of 2θ = 5–80◦ and a step size of 0.5◦ /min. The 2.5. Statistical analysis
structural interactions of the multilayer film were examined via Fourier-
transform infrared spectroscopy (FTIR) using a tungsten/halogen source A factorial design with a completely randomized block design was
and a DTGS detector. In this analysis, the wavelength range and reso­ employed using SPSS software (V 26, USA) to analyze the effect of
lution were set at 400–4000 nm and 4 cm− 1, respectively [1,41]. electrospun layer thickness on the properties of the trilayer films.
Duncan's multiple range test was conducted at a significance level of p
2.4.10. Thermal stability < 0.05 to compare the means of the treatments, with a minimum of
Differential Scanning Calorimetry (DSC) was used to analyze the three replications. The results were presented as mean ± standard de­
thermal behavior of the multilayer film. For this purpose, film powder viation. MATLAB (R2019, USA) and Excel (V 2016, USA) software were
was placed in aluminum capsules, and the DSC analysis was conducted used for data fitting and graph plotting, respectively.
at a heating rate of 10 ◦ C.min− 1 over a temperature range of 20–400 ◦ C.
Nitrogen, at a flow rate of 50 mL.min− 1, was used as an inert gas during 3. Results and discussion
the analysis. The biodegradability rate of the films was assessed by
monitoring the average weight loss of the films buried in soil. 3.1. Thickness and mechanical properties

2.4.11. Chemical properties The results indicated that the thickness of the multilayer films
The antioxidant activity was determined using the 2,2-diphenyl-1- increased significantly (p < 0.05) with the addition of thicker electro­
picrylhydrazyl (DPPH) method. In this method, 50 mg of the film was spun layers, as expected. As shown in Fig. 2, the thickness of the film
mixed with 3 mL of an ethanolic DPPH solution (0.1 mM) and placed in containing 80 g.m− 2 of electrospun zein-pomegranate flower extract
the dark for 30 min at 25 ◦ C. The percentage of DPPH free radical (SG-Ge/Ze 8/TiO2) and the film without the electrospun zein-
scavenging activity, as an indicator of antioxidant activity, was calcu­ pomegranate flower extract (SG-Ge/Ze 0/TiO2) were 0.23 mm and
lated using Eq. 7 based on the absorbance of the sample containing the 0.17 mm, respectively. This increase in thickness is attributed to the
film (Asample) and the control sample without the film (Ablank) [1]: thicker electrospun layer, which also influences other physical and
barrier properties of the film. Wang et al. reported similar findings in the
Ablank − Asample
DPPH Scavenging activity (%) = × 100 (7) development of multilayer films composed of gelatin/dextran-propyl
Ablank
gallate/gelatin (P. [45]). The effect of varying thicknesses of electro­
The total phenolic content (TPC) was measured in terms of μg GAE. spun zein-pomegranate flower extract layers (20, 40, 60, and 80 g.m− 2)
g− 1 film using the Folin-Ciocalteu method as described by Ayazi et al. on the mechanical properties of the multilayer film is illustrated in
[1]. Figs. 2 and 3.
As observed, although the addition of zein-pomegranate flower
2.4.12. Antimicrobial activity extract fibers to the film resulted in significant changes (p < 0.05) in
. The agar disk diffusion method was employed to assess the anti­ tensile strength, elastic modulus, and elongation at break, however,
microbial activity against Escherichia coli (ATCC 25922), Staphylococcus these effects were not significant (p˃ 0.05) at lower thicknesses. Only at
aureus (ATCC 25923), Candida albicans (ATCC 10231), and Aspergillus higher thicknesses of the electrospun layer did these parameters change
niger (PTCC 5012). A film disk was placed at the center of a Mueller- significantly (p < 0.05). However, as the thickness of the electrospun
Hinton agar plate, followed by surface inoculation. The plates were zein-pomegranate flower extract layer increased, tensile strength and
then incubated at 37 ◦ C for 24 h, and the diameter of the inhibition zone elastic modulus also increased, while elongation at break decreased.
(mm) was measured [1]. This increase in mechanical strength may be attributed to the influence
of the electrospun zein-pomegranate flower extract layer containing
2.4.13. Biodegradability TiO2, which acts as a reinforcing layer within the film structure [46,47].
The oven-dried multilayer film (2 × 2 cm2, 105 ◦ C for 24 h) was The increase in the thickness of the electrospun layer strengthens
weighed and buried at a depth of 4 cm in soil. The films were weighed intermolecular bonds and increases overall thickness, ultimately
every 15 days, with the soil surface being kept consistently moist during enhancing the film's mechanical properties. This improvement is also
this period [1]. due to the increased stiffness of the film, which leads to a higher elastic
modulus [46]. The elastic modulus of the multilayer film containing 80
2.4.14. Release study g.m− 2 of electrospun zein-pomegranate flower extract (SG-Ge/Ze 8/
The experimental release data of pomegranate flower extract from TiO2) is approximately 36 % higher than that of the film without the
the multilayer film in various food simulants, including aqueous electrospun zein-pomegranate flower extract (SG-Ge/Ze 0/TiO2). The
(distilled water), oily (aqueous solution of ethanol (50 % v/v)), alcoholic decrease in elongation at break with increasing electrospun layer
(aqueous solution of ethanol (10 % v/v)), and acidic (aqueous solution of thickness indicates reduced flexibility of the film, likely due to in­
acetic acid (3 % v/v)), were fitted using the Weibull model (Eq. 8). teractions between the sage seed gum-gelatin and the electrospun layer,
Additionally, Fick's second law (Eq. 9) was employed to calculate the as well as the brittleness of the electrospun zein layer. Similar results
diffusion coefficient [40,42–44]. have been reported by other researchers [29,48].

Mt − (t− Ti )β
= 1− e α (8) 3.2. Surface color and opacity
M0
( ) Color, appearance, and opacity are critical indicators in the devel­
Mt ∑∞
8 (2π + 1)2 π 2
= 1− exp − Dt (9) opment of films for various applications [20,49]. The effect of the
M0 2 2
n=0 (2π + 1) π
4L2 thickness of the electrospun zein-pomegranate flower extract layer on
the color indices (L*, ΔE, and WI) is presented in Table 2. The results
Where Mt and M0 represent the amount of extract released at time t and showed that although increasing the thickness of the electrospun layer
at the initial time (0), respectively. Ti , β, α, and L correspond to the lag significantly (p < 0.05) reduced the lightness (L*) and WI while
time measured by the dissolution process, the parameter describing the increasing the total color difference (ΔE), these changes were minor.
shape of the dissolution curve, the time scale of the process, and half the The sage seed gum-gelatin composite film is nearly colorless or slightly
thickness of the film, respectively. tinged with yellow and red. The addition of the sage seed gum-gelatin
composite film layer containing TiO2 imparts a brighter appearance to

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R. Valizadeh et al. International Journal of Biological Macromolecules 283 (2024) 137826

Fig. 2. The effect of varying thicknesses of electrospun zein-pomegranate flower extract layers (0, 20, 40, 60, and 80 g.m− 2) on the thickness (mm) and tensile
strength (MPa) of the multilayer film. Numbers with similar lowercase or uppercase letters do not differ significantly (p˃ 0.05).

Fig. 3. The effect of varying thicknesses of electrospun zein-pomegranate flower extract layers (0, 20, 40, 60, and 80 g.m− 2) on the elongation at break (%) and
elastic modulus (MPa) of the multilayer film. Numbers with similar lowercase or uppercase letters do not differ significantly (p˃ 0.05).

the film. Despite the white color of the electrospun zein-pomegranate the electrospun zein-pomegranate flower extract layer between these
flower extract layer, increasing its thickness in the multilayer film did two layers and the increase in its thickness reduces the light trans­
not enhance the lightness (L*) and WI of the film but instead led to a mission through the film. The electrospun zein-pomegranate flower
slight decrease in these variables. These changes can be attributed to the extract layer has a fibrous, dense, and opaque structure that hinders
placement of zein-pomegranate flower extract nanofibers within the light transmission. Additionally, the presence of aromatic rings and
pores of the film and the formation of strong hydrogen bonds between unsaturated double bonds in both the free and encapsulated pome­
TiO2, the composite film, and the electrospun layer, which partially granate flower extract further prevents the passage of light photons.
reduces light refraction. Additionally, these changes indicate the proper Similar results have been reported by other researchers [29,46].
integration of the electrospun zein-pomegranate flower extract layer
between the two layers of sage seed gum-gelatin composite, with and
without TiO2 nanoparticles. 3.3. Morphology
As shown in Table 2, the opacity of the film significantly increases (p
< 0.05) with increases of the electrospun layer thickness. This change is As observed in the field emission scanning electron microscopy
such that the opacity of the multilayer film containing 80 g.m− 2 of (FESEM) images (Fig. 5), the various layers of the film are well-adhered
electrospun zein-pomegranate flower extract (SG-Ge/Ze 8/TiO2) is to each other, forming a continuous and uniform structure, which in­
nearly double that of the film without the electrospun zein-pomegranate dicates the successful formation of the multilayer film. As shown in
flower extract (SG-Ge/Ze 0/TiO2) (Fig. 4). The sage seed gum-gelatin Fig. 5, the surfaces of all the films are free of cracks and exhibit a smooth,
composite film without the electrospun layer is almost transparent, homogeneous, and uniform texture without any voids. The interaction
and the addition of the sage seed gum-gelatin layer containing TiO2 between the protein and polysaccharide may enhance biocompatibility.
nanoparticles increases its opacity. On the other hand, the inclusion of Additionally, the strong interactions between hydrogen and hydroxyl
groups contributed to the formation of a compact, soft, and even surface

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Table 2 successful integration and uniform dispersion of TiO2 nanoparticles on

Color indices (L*, WI, and ΔE) and opacity of multilayer films containing varying the film's surface, creating a micro/nano-structured surface that en­
thicknesses of electrospun zein-pomegranate flower extract (0, 20, 40, 60, and hances hydrophobic properties. These results suggest that the methods
80 g.m− 2). used were effective in achieving the desired characteristics in the
L* ΔE WI Opacity multilayer films. Similar results have been previously reported in the
SG- 81.68 ± 0.67 a
26.69 ± 1.36 a
75.20 ± 1.07 a
5.11 ± 0.82 d development of multilayer films [17,50].
Ge/
Ze 0/
TiO2 3.4. Moisture properties and contact angle
SG- 79.60 ± 0.83 b 28.18 ± 0.93 b 73.82 ± 0.86 b 6.95 ± 0.80 c
Ge/
As shown in Table 3, the moisture content in the films significantly
Ze 2/
TiO2 decreased (p < 0.05) with the increasing thickness of the electrospun
SG- 79.04 ± 0.59 b 29.32 ± 0.86 bc 73.24 ± 0.92 b 7.91 ± 1.03 bc zein-pomegranate flower extract layer, indicating the positive effect of
Ge/ the zein-pomegranate flower extract layer in reducing moisture ab­
Ze 4/
sorption. Zein is insoluble in water, and the solvent used for extracting
TiO2
SG- 78.62 ± 0.82 bc 30.10 ± 1.03 c 72.11 ± 1.11 bc 9.65 ± 0.76 ab pomegranate flower extract was also an aqueous ethanol solution (80 %
Ge/ v/v); therefore, the electrospun zein-pomegranate flower extract layer
Ze 6/ exhibits hydrophobic properties. Increasing its thickness reduces mois­
TiO2 ture absorption, thereby decreasing the moisture content of the multi­
SG- 78.02 ± 1.09 c 30.52 ± 0.85 c 70.19 ± 1.01 c 10.43 ± 1.12 a
layer film. Similar results were reported by Marcoaldi et al. [50] in the
Ge/
Ze 8/ development of multilayer films. As also shown in Table 3, the water
TiO2 solubility of the multilayer film significantly decreased (p < 0.05) with
* the increased thickness of the electrospun layer.
Numbers with similar English letters in each column do not differ signifi­
cantly (p˃ 0.05). Earlier FESEM results also indicated that increasing the thickness of
the electrospun layer led to a more complex film structure, forming a
denser, more compact, and stronger network within the film matrix.
in the sage seed gum-gelatin composite film. Similar findings were noted
This increased density could reduce water penetration into the film, thus
by Ayazi et al. [1] regarding the composite film made from sage seed
lowering its solubility. Denser and more compact structures are typically
gum and pea protein isolate, and by Davoudi et al. [2] in their work on
less permeable to water, which can prevent water from penetrating into
nanocomposite films composed of tomato seed mucilage, gelatin, and
the inner layers, thereby helping to preserve the quality of packaged
TiO2. Additionally, the increase in the thickness of the electrospun layer
food products. Additionally, the presence of hydrophilic hydroxyl and
(0, 20, 40, 60, and 80 g.m− 2) is clearly visible in the cross-sectional
amino groups in the sage seed gum-gelatin composite film, as well as the
FESEM images. As shown in Fig. 5, the increased thickness of the elec­
phenolic and hydrophobic compounds in the pomegranate flower
trospun layer enhances the interactions between the composite and
extract and zein, also contribute to reducing solubility [51]. The results
electrospun layers of the film, leading to a more complex layer structure
showed that the swelling ratio significantly decreased (p < 0.05) with
and reduced porosity. Moreover, these images demonstrate that the
the increase in the thickness of the electrospun layer. Specifically, the
electrospun zein-pomegranate flower extract layer is well-integrated
swelling ratio of the film containing 80 g.m− 2 of electrospun zein-
between the other layers, forming a continuous and uniform structure
pomegranate flower extract (SG-Ge/Ze 8/TiO2) decreased by 34.22 %
that results in multilayer structures with improved barrier properties.
compared to the film without the electrospun zein-pomegranate flower
All these changes contribute to the enhancement of the film's mechan­
extract (SG-Ge/Ze 0/TiO2). This reduction in swelling in the multilayer
ical and barrier properties (against water vapor, oxygen, light, and mi­
films is mainly due to the increased hydrophobicity and the greater
crobes). The surface FESEM image of the multilayer film shows a
density and complexity of the film network resulting from the increased
relatively smooth and crack-free surface with some surface roughness,
thickness of the electrospun zein-pomegranate flower extract layer. The
attributed to the TiO2 nanoparticles. These images also indicate the
changes in the polymer network structure and the intermolecular

Fig. 4. The effect of varying thicknesses of electrospun zein-pomegranate flower extract layers (0, 20, 40, 60, and 80 g.m− 2) on the appearance and transparency of
the multilayer film.

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R. Valizadeh et al. International Journal of Biological Macromolecules 283 (2024) 137826

Fig. 5. The effect of varying thicknesses of electrospun zein-pomegranate flower extract layers (0, 20, 40, 60, and 80 g.m− 2) on the surface and cross-sectional
morphology of the multilayer film.

pomegranate flower extract layer. The outer surface of all the multi­
Table 3
layer films was composed of the sage seed gum-gelatin composite film
Solubility, moisture content, contact angle, and swelling ratio of multilayer films
containing TiO2 nanoparticles, resulting in similar surface properties
containing varying thicknesses of electrospun zein-pomegranate flower extract
(0, 20, 40, 60, and 80 g.m− 2).
across all these films.

Moisture Water solubility Swelling Contact angle

content (MS, (WS, %) (S, %) (◦ ) 3.5. Permeability
%)

SG- 8.98 ± 0.46 e* 45.35 ± 1.37 e 365.21 ± 11.43 d 78.85 ± 1.03 a The results for water vapor permeability (WVP) and oxygen
Ge/
permeability (OP) of the multilayer films are presented in Fig. 6. As
Ze
0/
shown in the Fig., the water vapor permeability and oxygen perme­
TiO2 ability significantly decreased (p < 0.05) with the increasing thickness
SG- 8.45 ± 0.49 de
39.65 ± 1.22 d 330.23 ± 15.09 c 78.21 ± 1.11 a of the electrospun zein-pomegranate flower extract layer. This down­
Ge/ ward trend is attributed to the fact that each layer independently pos­
Ze
sesses barrier properties, and when these layers are combined, they form
2/
TiO2 a stronger multilayer barrier against water vapor penetration, resulting
SG- 7.61 ± 0.52 cd
37.83 ± 1.19 cd 290.44 ± 19.23 bc 78.93 ± 1.21 a in a reduction in overall permeability as the thickness of each layer in­
Ge/ creases [17]. It was previously noted that increasing the thickness of the
Ze
electrospun layer enhances its interaction with the polymer matrix,
4/
TiO2
thereby reinforcing the film's structure. This increased interaction also
SG- 7.03 ± 0.43 bc
35.14 ± 1.24 bc 269.43 ± 16.21 b 78.11 ± 1.19 a leads to a denser and more complex film structure, which further re­
Ge/ duces permeability. The zein-pomegranate flower extract fibers occupy
Ze the voids in the sage seed gum-gelatin composite layer, forming cross-
6/
links with the film matrix, which makes the structure more imperme­
TiO2
SG- 6.56 ± 0.54 a
33.01 ± 1.43 a 240.22 ± 10.49 a 78.22 ± 1.32 a able. Additionally, the electrospun zein-pomegranate flower extract
Ge/ layer is inherently hydrophobic, due to the hydrophobic nature of zein
Ze and, to some extent, the pomegranate flower extract.
8/ Akinalan Balik et al. [52] obtained similar results during the fabri­
TiO2
cation and evaluation of multilayer films containing electrospun pectin.
*
Numbers with similar English letters in each column do not differ signifi­ Their findings demonstrated that both water vapor permeability and
cantly (p˂0.05). oxygen permeability exhibited a downward trend with the addition of
the pectin middle layer in these films. This reduction is attributed to the
interactions with the increased thickness of the electrospun layer may presence of the electrospun pectin layer, which acts as an effective
also contribute to the reduced water absorption capacity. The FESEM barrier, reducing porosity in the film structure and decreasing perme­
results, along with the moisture content and solubility of the multilayer ability. Marcoaldi et al. [50] also observed a reduction in water vapor
films, further support these findings. Similar results for the swelling ratio and oxygen permeability in the fabrication of multilayer films, which
in multilayer films have been reported by other researchers [46]. As was due to the combination of different layers with varying barrier
shown in Table 3, the contact angle did not change significantly (p˃ properties. When these layers are combined, they enhance the overall
0.05) with the increase in the thickness of the electrospun zein- barrier properties, providing more effective protection of food against

7
R. Valizadeh et al. International Journal of Biological Macromolecules 283 (2024) 137826

Fig. 6. The effect of varying thicknesses of electrospun zein-pomegranate flower extract layers (0, 20, 40, 60, and 80 g.m− 2) on water vapor permeability (g. m− 1.
pa− 1. s− 1 × 10− 9) and oxygen permeability (g.m− 2.s− 1 × 10− 3) of the multilayer film. Numbers with similar lowercase or uppercase letters do not differ significantly
(p˃ 0.05).

moisture and oxygen infiltration. Similar findings have been reported by The amide II band in the range of 1400–1500 cm− 1 shows peaks at
other researchers as well [17,51]. wavenumbers 1454 cm− 1, 1456.2 cm− 1, and 1463 cm− 1, which corre­
spond to N–H bending and C–N stretching vibrations. Variations in
these peaks indicate differences in the interactions between the proteins
3.6. Structural properties (gelatin or zein), carbohydrates, and pomegranate flower extract. These
changes are attributed to the occupation of C– –O and N–H groups
The FTIR spectroscopy results are shown in Fig. 7. A more detailed within the film structure [13,14,26]. The increase in these peaks also
analysis of these peaks can provide further insights into the specific contributes to the formation of stronger networks within the films,
molecular interactions between the film components. As observed in the helping to stabilize the film structure. The C–O and C–N stretching
Fig., the peak in the range of 3200–3600 cm− 1 corresponds to the bands in the range of 1100–1300 cm− 1 with peaks at 1041 cm− 1, 1045.1
stretching vibrations of hydroxyl (O–H) and amide (N–H) groups in cm− 1, 1165 cm− 1, and 1245 cm− 1 may originate from carbohydrates (e.
gelatin, gum, and possibly zein. The shift of this peak to a lower wave­ g., gum) or amide groups in protein compounds. Shifts and changes in
number with increasing thickness of the electrospun zein-pomegranate these peaks across different samples could be due to variations in the
flower extract layer indicates changes in the chemical bonds within thickness of the electrospun zein-pomegranate flower extract layer and
the multilayer films, the formation of hydrogen bonds among the its interaction with the protein matrix.
different layers of the film, increased structural interactions due to the In the fingerprint region (1500–600 cm− 1), peaks at wavenumbers
formation of strong hydrogen bonds, and a reduction in the number of 859 cm− 1, 922 cm− 1, 989 cm− 1, and 1041 cm− 1 are present, corre­
free hydroxyl groups in the film. The C–H stretching vibrations in the sponding to bending and stretching vibrations of various functional
range of 2850–2950 cm− 1 observed in the FTIR spectra of all multilayer groups such as C–H, C–O, C–N, and O–H. Small shifts in the position
films correspond to the asymmetric and symmetric stretching vibrations of these peaks further indicate chemical interactions between the layers
of CH2 and CH3 groups, indicating a high organic content in the samples, and better adhesion between them. Ultimately, these analyses confirm
likely originating from the gum or zein. The amide I band in the range of the successful production process and homogeneous structure of the
1600–1700 cm− 1 shows peaks at wavenumbers 1651.9 cm− 1, 1659.4 multilayer films. The X-ray diffraction (XRD) patterns of multilayer films
cm− 1, 1660.3 cm− 1, 1667.1 cm− 1, and 1674.2 cm− 1, which are associ­ with varying thicknesses of the electrospun zein-pomegranate flower
ated with the C– –O stretching vibrations in proteins (gelatin and zein).

Fig. 7. The effect of varying thicknesses of electrospun zein-pomegranate flower extract layers (0, 20, 40, 60, and 80 g.m− 2) on the FTIR spectra of the multi­
layer film.

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R. Valizadeh et al. International Journal of Biological Macromolecules 283 (2024) 137826

extract layer (0, 20, 40, 60, and 80 g.m− 2) are shown in Fig. 8. XRD thickness of the electrospun zein-pomegranate flower extract layer. As
patterns are used in materials science and crystallography to analyze the shown in Fig. 9, the changes in melting temperature are slight with
crystalline structure of materials, identify crystalline phases, analyze increasing electrospun layer thickness, initially rising and then
crystal size, and examine changes in the degree of crystallinity [2,4]. decreasing, which may be due to the complete integration of the elec­
The peaks at 2θ = 5◦ and 2θ = 20◦ correspond to characteristic peaks of trospun layer into the film and minor changes in the crystalline structure
gelatin and Sage seed gum, respectively [11]. of the films. Finally, the thermal decomposition temperature increases
The characteristic peaks of zein appear at 2θ = 9.5◦ and 2θ = 20.5◦ with the thickness of the electrospun layer. The results of FTIR spec­
[33]. As observed in Fig. 8, the sharp peaks at 2θ = 25◦ , 2θ = 47◦ , and 2θ troscopy and X-ray diffraction pattern analyses indicated that structural
= 55◦ represent the crystalline phase of TiO2 in the film and are indic­ interactions and the degree of crystallinity of the film increase with the
ative of these nanoparticles. The appearance and increased intensity of thickness of the electrospun zein-pomegranate flower extract layer,
the peak at 2θ = 20.5◦ indicate the addition and increasing thickness of which increases the energy required to break the bonds between the film
the electrospun zein-pomegranate flower extract layer, which may result components. This increase indicates enhanced thermal stability of the
from the interaction between the electrospun zein-pomegranate flower films with increasing thickness of the electrospun layer. Similar results
extract layer and the sage seed gum-gelatin composite film. Previous were reported by Marcoaldi et al. [50].
FTIR results showed that increasing the thickness of the electrospun
zein-pomegranate flower extract layer enhances cross-linking and 3.8. Chemical and antimicrobial properties
hydrogen bonding within the film structure. These changes lead to an
increase in the degree of crystallinity and alterations in crystal size. As shown in Fig. 10, the DPPH free radical scavenging activity and
Consequently, the XRD pattern results suggest that with increasing total phenolic content of the multilayer films significantly increased (p
thicknesses of the electrospun zein-pomegranate flower extract layer, < 0.05) with the increasing thickness of the electrospun layer. The
chemical interactions between the different layers of the film can cause DPPH free radical scavenging activity and total phenolic content of the
changes in the crystalline structure and order. These changes can in­ film containing 80 g.m− 2 of electrospun zein-pomegranate flower
crease crystallinity within the nanocomposite structure, as evidenced by extract (SG-Ge/Ze 8/TiO2) increased by 547 % and 899 %, respectively,
the increased intensity or changes in the width of the XRD peaks. These compared to the film without the electrospun zein-pomegranate flower
findings can provide important insights into the improvement or alter­ extract (SG-Ge/Ze 0/TiO2). This substantial increase is attributed to the
ations in the mechanical and biological properties of these presence of phenolic compounds in the pomegranate flower extract,
nanocomposites. which have the ability to neutralize free radicals. Pomegranate flower
extract is a rich source of antioxidants and phenolic compounds ([23]; T.
3.7. Thermal properties [24]). The minimal DPPH free radical scavenging activity and phenolic
content in the film without the electrospun layer can be attributed to the
The DSC (Differential Scanning Calorimetry) thermograms of the reaction of remaining free amino groups (NH2), α-zein, peptides, and
multilayer films affected by the thickness of the electrospun layer are carotenoids in the zein structure [18,22], as well as amino groups,
shown in Fig. 9. This thermogram represents the thermal analysis of the peptides, amino acids, and -OH groups in the sage seed gum-gelatin
film, indicating changes in heat flow relative to temperature and is used composite film with free radicals. These results indicate that in the
to identify thermal transitions such as glass transition temperature (Tg), absence of pomegranate flower extract, the DPPH free radical scav­
melting temperature (Tm), and thermal decomposition temperature enging activity and phenolic content of the films are very low. Phenolic
(Td) in the multilayer film. As shown in the Fig., there are three endo­ compounds (such as Punicalagin, Ellagic acid, anthocyanins, Gallic acid,
thermic peaks in all three thermograms, corresponding to the glass and Tannins), especially polyphenols and flavonoids present in pome­
transition temperature (Tg), melting temperature (Tm), and thermal granate flower extract, directly react with and neutralize free radicals.
decomposition temperature (Td), respectively. The glass transition These compounds also help maintain the oxidative stability of the films,
temperature indicates the point at which the material transitions from a thereby enhancing their shelf life and antimicrobial properties.
glassy (hard and brittle) state to a more flexible state. The results To evaluate the antimicrobial properties of multilayer films con­
revealed that as the thickness of the electrospun layer increases, the taining different thicknesses of the electrospun layer (0, 20, 40, 60, and
glass transition temperature gradually rises. This increase indicates an 80 g.m− 2), the agar disk diffusion method was used. As shown in
improvement in the thermal resistance of the films, which is attributed Table 4, increasing the thickness of the electrospun layer significantly
to changes in the crystalline structure of the film. X-ray diffraction re­ (p˃ 0.05) enhanced the inhibitory activity of the multilayer films against
sults also demonstrated that the degree of crystallinity increases with the the Gram-positive bacterium Staphylococcus aureus, the Gram-negative

Fig. 8. The effect of varying thicknesses of electrospun zein-pomegranate flower extract layers (0, 20, 40, 60, and 80 g.m− 2) on the X-ray diffraction (XRD) pattern of
the multilayer film.

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R. Valizadeh et al. International Journal of Biological Macromolecules 283 (2024) 137826

Fig. 9. The effect of varying thicknesses of electrospun zein-pomegranate flower extract layers (0, 20, 40, 60, and 80 g.m− 2) on the thermal stability (DSC ther­
mogram) of the multilayer film.

Fig. 10. The effect of varying thicknesses of electrospun zein-pomegranate flower extract layers (0, 20, 40, 60, and 80 g.m− 2) on the antioxidant properties (DPPH)
and total phenolic content (TPC) of the multilayer film. Numbers with similar lowercase or uppercase letters do not differ significantly (p˃ 0.05).

bacterium Escherichia coli, the yeast Candida albicans, and the mold
Table 4
Aspergillus niger. The lower antimicrobial effect in films without the
Evaluation of the antimicrobial effect of multilayer films containing varying
electrospun zein-pomegranate flower extract layer indicates the anti­
thicknesses of electrospun zein-pomegranate flower extract (0, 20, 40, 60, and
80 g.m− 2).
microbial role of the pomegranate flower extract in inhibiting the
growth of microorganisms. The slight antimicrobial activity observed is
Diameter of inhibitory zone (mm)
attributed to the presence of TiO2 nanoparticles. In previous study we
E. coli S. aureus Candida Asp. Niger reported that adding PFE had an antimicrobial effect on the electrospun
PTCC-25922 PTCC- albicans PTCC-5012 fibers. Furthermore, increasing the extract's concentration enhanced the
25923 PTCC-10231
fibers' antimicrobial effect (p < 0.05). Different polyphenolic com­
SG-Ge/Ze 0/ 4.1 ± 0.2 e* 7.5 ± 0.5 e
6.1 ± 0.7 e
3.2 ± 0.6 e pounds are responsible for the antimicrobial activities of PFE [19].
TiO2
d d d d The antimicrobial effect of the extract is attributed to the diverse
SG-Ge/Ze 2/ 13.5 ± 0.4 19.1 ± 0.7 14.5 ± 0.5 10.9 ± 0.5 s
TiO2 bioactive compounds present in pomegranate flower extract, including
SG-Ge/Ze 4/ 14.9 ± 0.3 c
22.6 ± 0.5 c
15.7 ± 0.6 c
13.1 ± 0.5 c Ellagic acid, Ellagitannins, Gallic acid, anthocyanins, Piperidine alka­
TiO2 loids, Ursolic acid, Maslinic acid, Asiatic acid, and phenolics such as
b b b
SG-Ge/Ze 6/ 16.1 ± 0.6 26.2 ± 0.6 17.1 ± 0.6 15.4 ± 0.6 b Punicalagin. As the concentration of these compounds increases, the
TiO2
SG-Ge/Ze 8/ 17.6 ± 0.6 a
28.9 ± 0.6 a
18.3 ± 0.7 a
17.2 ± 0.6 a
film's ability to inhibit the growth of microorganisms is enhanced
TiO2 [51,53,54].
*
Numbers with similar English letters in each column do not differ signifi­
cantly (p˂0.05).
3.9. Biodegradability

Fig. 11 illustrates the biodegradability of multilayer films as

10
R. Valizadeh et al. International Journal of Biological Macromolecules 283 (2024) 137826

influenced by the thickness of the electrospun layer (0, 20, 40, 60, and dissolves more readily in the aqueous environment, allowing quicker
80 g.m− 2). The film without the electrospun zein-pomegranate flower access of the release medium to the electrospun layers. In the release
extract layer (SG-Ge/Ze 0/TiO2) exhibited the highest weight loss process, the simulant first penetrates the polymer matrix, followed by
among the multilayer films (up to approximately 90 % by day 50), the release of the active compound [4,7]. The highest release rate in this
indicating its high biodegradability. When 80 g.m− 2 of electrospun food simulant was observed for the film with the thickest electrospun
pomegranate flower extract (SG-Ge/Ze 8/TiO2) was added, the biode­ zein-pomegranate flower extract layer (SG-Ge/Ze 8/TiO2). The higher
gradability decreased to around 75 %. This reduction is likely due to the release rate is due to the greater driving force resulting from the higher
strong antimicrobial and antioxidant effects of the pomegranate flower concentration gradient of the pomegranate flower extract between the
extract, which prevent the rapid degradation of the film by microor­ simulant and the film at this thickness, as well as the greater amount of
ganisms and environmental factors. The slow release of the pomegranate free pomegranate flower extract on the surface of the electrospun layer.
flower extract prolongs its effectiveness in reducing the growth rate of As also shown in Fig. 12, the lowest release rate occurred in the fatty
microorganisms. Additionally, it was previously shown that increasing food simulant (50 % ethanol), where the film with 80 g.m− 2 of elec­
the thickness of the electrospun layer can enhance the film's density and trospun zein-pomegranate flower extract released about 50 % of the
strength through physical and chemical interactions with the polymer active compounds within 300 min. Since the outer layers of the multi­
matrix, such as hydrogen bonding and Van der Waals forces, leading to a layer film are sage seed gum-gelatin composite films, which have low
slower biodegradation rate. solubility in alcoholic environments, their dissolution is delayed in this
Furthermore, the increased density and reduced permeability of the environment compared to the other simulants. This delay reduces sol­
films, due to the presence of the pomegranate flower extract, may vent penetration into the film and, consequently, the release rate of the
decrease the penetration of water and degrading enzymes, further pomegranate flower extract. After the fatty food simulant, the second-
slowing the biodegradation process. Therefore, in addition to the anti­ lowest release rate was observed in the alcoholic simulant, where
microbial and antioxidant properties of the pomegranate flower extract, approximately 55 % to 65 % of the pomegranate flower extract was
the physical and chemical effects of the electrospun layer and the released after 300 min. Based on previous studies, the semi-empirical
reduced permeability of the films are logical reasons for the decreased Weibull model was used to predict the release kinetics of pomegranate
biodegradation rate with the increased thickness of the electrospun flower extract from multilayer films in various food simulants. The re­
pomegranate flower extract layer. In all multilayer films, an initial lag sults of fitting the Weibull model to the experimental release data of
phase of 10 days was observed, during which the biodegradation process pomegranate flower extract in different simulants showed that this
had not yet commenced, and weight changes were <10 %. This phase model could predict the release profile with an R2 value >98 %. By
represents the acclimation/selection period for microorganisms [16]. analyzing the Weibull model constants and based on previous studies, it
Similar results have been reported by other researchers [16]. was determined that Fickian diffusion is the dominant mechanism of
release from the multilayer films. Consequently, the diffusion coefficient
3.10. Release study (D) of pomegranate flower extract from the multilayer films was esti­
mated as a function of the type of release simulant and the thickness of
As shown in Fig. 12, the release of pomegranate flower extract from the electrospun layer (Table 5). Similar to the release profile results, the
the multilayer film containing varying thicknesses of the electrospun highest diffusion coefficient was observed for the film with the thickest
zein-pomegranate flower extract layer (20, 40, 60, and 80 g.m− 2) was electrospun zein-pomegranate flower extract layer (SG-Ge/Ze 8/TiO2)
examined in four food simulants: aqueous (distilled water), acidic (3 % in the aqueous food simulant, while the lowest was for the film with the
acetic acid), alcoholic (10 % ethanol), and fatty (50 % ethanol). The thinnest electrospun zein-pomegranate flower extract layer (SG-Ge/Ze
release profile in all environments and across all electrospun layer 2/TiO2) in the fatty food simulant. The results demonstrated that this
thicknesses occurred in two stages: an initial burst release followed by a design effectively controls the release of the antimicrobial agent and is
sustained release phase. The highest release rate was observed in the highly beneficial for extending the shelf life of packaged food products.
aqueous food simulant, where approximately 60 % to 75 % of the Similar results were reported by Vidal et al. in their study on the release
pomegranate flower extract was released after 300 min. This is due to from multilayer films [14].
the hydrophilic nature of the outer sage seed gum-gelatin layer, which

Fig. 11. The effect of varying thicknesses of electrospun zein-pomegranate flower extract layers (0, 20, 40, 60, and 80 g.m− 2) on the biodegradability (weight loss) of
the multilayer film.

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R. Valizadeh et al. International Journal of Biological Macromolecules 283 (2024) 137826

Fig. 12. Release profile of pomegranate flower extract from multilayer films containing varying thicknesses of electrospun zein-pomegranate flower extract layers
(20, 40, 60, and 80 g.m− 2).

and personal care industries. Overall, the multilayer films developed in

Table 5
this study may represent a promising new approach for creating more
Diffusion coefficient (D × 10− 10) of pomegranate flower extract from multilayer
sustainable packaging strategies. These findings could pave the way for
films containing varying thicknesses of electrospun zein-pomegranate flower
extract layers (20, 40, 60, and 80 g.m− 2).
further investigations into electrospun-laminated thin multilayer films.
Additional research is needed to explore the effects of multilayer films
SG-Ge/Ze SG-Ge/Ze SG-Ge/Ze SG-Ge/Ze
on food preservation.
2/TiO2 4/TiO2 6/TiO2 8/TiO2

Distilled water 9.68 9.57 11.94 13.45

Consent to participate
Acetic acid 3 % 5.82 5.95 6.63 7.12
(Acidic food)
Ethanol 10 % 7.73 8.16 9.79 10.65 Not applicable.
(Alcoholic Food)
Ethanol 50 % (Fatty 3.09 3.87 4.43 5.38
Consent for publication
Food)

Not applicable.
4. Conclusion
Ethics approval and consent to participate
In this study, multilayer films developed through casting and elec­
trospinning processes were examined. The integration and increased Not applicable.
thickness of the middle electrospun layer improved the film's resistance
to water and permeability. As the thickness of the electrospun layer Funding
increased, the solubility, water vapor permeability, oxygen perme­
ability, swelling ratio, and moisture content of the film all decreased. Not applicable.
However, these changes did not affect the surface properties of the
multilayer films. While the increased thickness of the electrospun layer Code availability
reduced the flexibility of the films, it also enhanced their tensile strength
and elastic modulus. The increased compactness between the layers was Not applicable.
clearly visible in the FESEM images. Additionally, the increase in
intermolecular interactions and crystallinity with the increased elec­ CRediT authorship contribution statement
trospun layer thickness was confirmed by FTIR and XRD analyses, which
also showed improved thermal stability of the multilayer films. The Romina Valizadeh: Writing – original draft, Visualization, Valida­
increase in electrospun layer thickness also altered the L*, b*, ΔE, and WI tion, Software, Methodology, Data curation. Mohsen Zandi: Writing –
indices and increased the opacity of the film. The higher concentration review & editing, Writing – original draft, Visualization, Validation,
of pomegranate flower extract in the thicker electrospun layers led to a Supervision, Software, Resources, Project administration, Methodology,
substantial enhancement of the antioxidant, antimicrobial properties, Investigation, Funding acquisition, Formal analysis, Data curation,
and total phenolic content of the films. The developed multilayer film Conceptualization. Ali Ganjloo: Writing – review & editing, Supervi­
allowed for controlled release of its active compounds, with the highest sion, Investigation. Nazila Dardmeh: Writing – review & editing, Su­
and lowest release rates occurring in aqueous and fatty food simulants, pervision, Investigation.
respectively. The novel developed active multilayer films potentially
offer environmental benefits over synthetic films, and the findings of this Declaration of competing interest
study suggest the feasibility of using these developed multilayer films in
the design of food, pharmaceutical, and medical packaging, as well as The authors confirm that there are no known conflicts of interest
potential applications in skin regeneration materials in the cosmetics (competing interests) associated with this publication and there has

12
R. Valizadeh et al. International Journal of Biological Macromolecules 283 (2024) 137826

been no significant financial support for this work that could have [22] L. Xia, L. Li, Y. Xiao, F. Xiao, W. Ji, M. Pang, H. Wang, Hydrophobic electrospun
membrane of peppermint oil loaded zein with coating of methyltriethoxysilane for
influenced its outcome.
active packaging, Food Hydrocoll. 144 (2023) 109031.
[23] A. Gościniak, A. Bazan-Woźniak, R. Pietrzak, J. Cielecka-Piontek, Pomegranate
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