Food Hydrocolloids 99 (2020) 105338

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Development and characterization of pectin films activated by T

nanoemulsion and Pickering emulsion stabilized marjoram (Origanum
majorana L.) essential oil
Hadi Almasi∗, Saeedeh Azizi, Sajed Amjadi
Department of Food Science and Technology, Faculty of Agriculture, Urmia University, Urmia, P.O. Box 57561-51818, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: The aim of this study was to prepare the pectin films activated by marjoram essential oil-loaded nanoemulsions
Active packaging (NE) and Pickering emulsions (PE). Low molecular surfactant of Tween 80 and blend of whey protein isolate
Controlled release (WPI) and inulin were used for preparation of NE and PE, respectively. The size, zeta potential, encapsulation
Marjoram essential oil efficiency, surface morphology, and antibacterial activity of fabricated NE and PE were investigated. The droplet
Pectin
size of MEO-loaded NE and PE were 97.5 and 233.3 nm, respectively. The obtained nanocarriers were in-
Pickering emulsions
corporated into the pectin film formulation at three levels (2.5, 5, 7.5 %wt.) and the morphological, physical and
functional properties of active films were investigated. The FT-IR, XRD and FE-SEM results exhibited the
compatibility between pectin and loaded nanocarriers. The films containing PE had good mechanical and water
barrier properties due to their high dense and less permeable structure. The antioxidant activity of the films
containing PE were significantly (p < 0.05) lower than its value in the NE incorporated films. The en-
capsulation of essential oil with PE provided significantly slower release profile in the film samples compared to
the NE. In conclusion, the fabricated active pectin film containing Pickering emulsion showed great potential for
active food packaging.

1. Introduction
Nowadays the environmental concerns raised from the use of non-
biodegradable petroleum-based synthetic polymers have been caused to
grow interest in the development of new packaging materials based on
naturally accruing polymers. Proteins, polysaccharides and their com-
binations are the used biopolymers for production of biodegradable
packaging materials (Ghanbarzadeh & Almasi, 2013). Pectin, naturally
found on plant cell walls as a by-product of agriculture and food in-
dustry is a good candidate for film forming applications (Espitia, Du,
Avena-Bustillos, Soares, & McHugh, 2014). Pectin is made up of a
polymer of α-1→4-linked D-galacturonic acid units, some of which are
partially esterified with methanol at the C-6 carboxyl group and may be
esterified with acetyl groups at C-2 or C-3 (Mishra, Banthia, & Majeed,
2012). According to degree of methyl-esterification (DM), pectin is
classified as high-methoxyl (HMP) or low-methoxyl (LMP) pectin. One
of the most promising classes of biodegradable food packaging is active
packaging. Active releasing packaging is a type of novel food packaging
systems in which a preservative such as antioxidant or antimicrobial
agent is loaded into the package instead of being added directly into the
food (Benbettaïeb, Chambin, & Karbowiak, 2016). Recent studies have
been explored the potential of pectin films for the preparation of active
packaging of foods (Bayarri, Oulahal, Degraeve, & Gharsallaoui, 2014;
Eça, Machado, Hubinger, & Menegalli, 2015; Mendes et al., 2019; Nisar
et al., 2018).
Recently, essential oils (EOs) extracted from plants and recognized
as GRAS food additives, have received much attention to use as active
agents in biodegradable active films especially due to their safety and
simultaneous antioxidant and antimicrobial activities (Ribeiro-Santos,
de Melo, Andrade, & Sanches-Silva, 2017). Marjoram (Origanum ma-
jorana L.) is a herbaceous, perennial plant grown in many parts of the
world that it is used for flavoring food, in cosmetics and in traditional
medicine (Hajlaoui, Mighri, Aouni, Gharsallah, & Kadri, 2016). Mar-
joram essential oil (MEO) has been indicated to possess antioxidant,
antimicrobial, anticancer and anti-inflammatory activities (Arranz,
Jaime, López de las Hazas, Reglero, & Santoyo, 2015). The main
compounds of MEO are terpinen-4-ol (> 20%), (+)-cis-sabinene hy-
drate (3–18%), α- and γ-terpinene and terpinolene, thymol and carva-
crol (Vági, Simándi, Suhajda, & Héthelyi, 2005). MEO has been used as
food preservative and flavoring agent and its effect on shelf life

∗
Corresponding author.
E-mail address: h.almasi@urmia.ac.ir (H. Almasi).

https://doi.org/10.1016/j.foodhyd.2019.105338
Received 20 June 2019; Received in revised form 24 August 2019; Accepted 24 August 2019
Available online 26 August 2019
0268-005X/ © 2019 Elsevier Ltd. All rights reserved.
H. Almasi, et al.
extension of some foods has been confirmed (Busatta et al., 2008;
Mohamed & Mansour, 2012). The only research on the using of MEO in
the fabrication of active packaging is the report of Alboofetileh, Rezaei,
Hosseini, and Abdollahi (2014) who prepared alginate/clay nano-
composite films enriched with clove, coriander, caraway, marjoram,
cinnamon and cumin EOs. According to their results, the MEO in-
corporated films exhibited the highest antimicrobial activity.
Incorporation of EOs into the film forming solutions has some major
challenges. Poor miscibility and phase separation during the film-
forming process, adverse effect on transparency of film and sensitivity
of bioactive compounds against environmental factors (e.g., oxygen,
light, temperature, moisture, and pH) are the most common short-
comings of the direct addition of bioactive compounds into biode-
gradable active films (Atarés & Chiralt, 2016). The second problem of
free EOs loaded active packaging is rapid migration of active com-
pounds and thus decreasing the activity of film during shelf life of food
(Ribeiro-Santos et al., 2017).
The encapsulation of EOs is an alternative method to overcome the
problems related to the direct application of EOs in food and/or active
packaging. The most suitable nano-scale carrier materials for food ap-
plications are lipid based nanocarriers and biopolymer based nano- or
microcapsules (Fathi, Martín, & McClements, 2014; Fathi, Mozafari, &
Mohebbi, 2012). Oil-in-water nanoemulsions (NE) consist on lipid nano
droplets (between 10 and 100 nm diameter) dispersed in an aqueous
solution. More transparency, enhanced physicochemical properties,
more stability, improved controlled release and improved biological
activity of EO is the main advantages of NE formation (McClements &
Rao, 2011). The incorporation of lipid based nanoencapsulated EOs
especially in the form of nanoliposomes (Almasi, Zandi, Beigzadeh,
Haghju, & Mehrnow, 2016; Aziz & Almasi, 2018; Haghju, Beigzadeh,
Almasi, & Hamishehkar, 2016; Jiménez, Sánchez-González, Desobry,
Chiralt, & Tehrany, 2014) and nanoemulsions (Acevedo-Fani, Salvia-
Trujillo, Rojas-Graü, & Martín-Belloso, 2015; Ghadetaj, Almasi, &
Mehryar, 2018; Noori, Zeynali, & Almasi, 2018; Otoni, Avena-Bustillos,
Olsen, Bilbao-Sáinz, & McHugh, 2016; Pérez-Córdoba et al., 2018;
Robledo et al., 2018) in various biopolymer-based active films and
coatings has been described. Pectin film has also been studied recently
for incorporation of cinnamaldehyde nanoemulsion (Otoni et al., 2014)
and orange oil microemulsion (Jantrawut et al., 2018).
Biopolymer-stabilized emulsions, also known as Pickering emul-
sions (PE) are another interesting sustained delivery system of bioactive
compounds. Unlike surfactants, a thicker interface layers can be formed
at the interface of oil-water phase when solid particles are adsorbed.
Higher coalescence stability, higher loading capacity, stronger protec-
tion of encapsulated compound, lower release rate, safety, biodegrad-
ability, and biocompatibility are the advantages of PE prepared with
biological macromolecules (Li et al., 2018). The studies regarding the
incorporation of PE stabilized active compounds into biopolymer based
films are rare and all of them are new. Mohsenabadi, Rajaei,
Tabatabaei, and Mohsenifar (2018) prepared starch-carboxymethyl
cellulose film containing rosemary EO encapsulated in chitosan na-
nogel. Liu, Wang, Qi, Huang, and Xiao (2019) encapsulated oregano EO
in a PE stabilized by complex coacervates of acid soluble soy protein
and soluble soybean polysaccharide and then fabricated soluble soy-
bean polysaccharide films containing this PE. Dammak, Lourenço, and
Sobral (2019) prepared hesperidin PE stabilized with chitosan nano-
particles and activated gelatin based film by addition of prepared PE.
The presence of hydrophobic and hydrophilic amino acids makes
whey protein isolate (WPI) as an excellent biopolymers suitable to en-
capsulate of hydrophobic bioactive compounds (Ezhilarasi, Indrani,
Jena, & Anandharamakrishnan, 2013). Protein complexation with
polysaccharides can provide higher encapsulation efficiency of core
material. Carbohydrates with shorter chains are able to act as filling
agents of protein matrix and cause to better protection of active com-
pounds by absorption of polysaccharide onto the surface of protein-
stabilized droplets (Yao et al., 2016). Inulin as a short chain
2
Food Hydrocolloids 99 (2020) 105338
polysaccharide contains β(2 → 1)-linked D-fructose units generally ter-
minated by a single glucose unit (Ronkart et al., 2006). Inulin lacks any
emulsifying properties and need to be used together with other wall
materials. The use of inulin as a secondary encapsulant has been
gaining more interests in recent years. There are few reports on the
simultaneous using of inulin together with WPI for encapsulation of
active compounds (Bakry et al., 2017; Fernandes, Borges, Botrel, &
Oliveira, 2014; Fernandes et al., 2017).
To the best of our knowledge, there are no reports on the en-
capsulation of MEO by any types of nanocarrier systems. Additionally,
any former study has not compared the characteristics of active films
containing NE and PE stabilized active compounds. The aim of this
work was to characterize and compare the properties of pectin films
activated by NE and PE encapsulated MEO. Firstly, the characteristics of
NE and PE stabilized MEO were evaluated. After that, the effect of
encapsulated MEO at different concentrations on the morphological,
water barrier properties, mechanical characteristics, antimicrobial and
antioxidant activity and release properties of pectin films was studied.
2. Materials and methods
2.1. Materials
MEO was extracted by hydrodistillation method for 4 h using a
Clevenger apparatus. Citrus pectin (galacturonic acid content, 74%;
degree of methyl esterification, 53%), Tween 80 (HLB = 15), α-di-
phenyl-β-picrylhydrazyl (DPPH), ethanol, and glycerol were procured
from Sigma-Aldrich (Germany). Inulin (food grade, DP ≥ 10, 95%
purity) and WPI (ca. 90% Kjeldahl, N × 6.38) were purchased from
Arla food ingredient (Denmark). Escherichia coli O157:H7, ATCC-11775
and Staphylococcus aureus, ATCC 19111 were procured from Iranian
Biological Resource Center (IBRC). For anti-bacterial tests Mueller-
Hinton agar were purchased from Sigma-Aldrich, USA.
2.2. Preparation of NE and PE stabilized MEO
The method of Noori et al., (2018) was used for preparation of MEO
loaded NE. At first, coarse emulsion was prepared by gradually adding
of MEO (1% wt) and Tween 80 (30% wt of MEO) to distilled water with
agitating at 3000 rpm. Then, the ultrasonic emulsification was used to
convert coarse emulsion to NE. A probe sonicator (OPTIMA, XL100K,
Germany) operating at 20 kHz and 200 W was used for nanoemulsifi-
cation. The probe of apparatus with diameter of 15 mm was dipped into
coarse emulsion in depth of 25 mm and carried out for 15 min.
The WPI/inulin stabilized PE was prepared by the method of
Hosseinnia, Alizadeh Khaledabad, and Almasi (2017). The biopolymer
suspension containing 1:1 ratio of WPI (2% wt) and inulin (2% wt) in
distilled water was prepared the day before the encapsulation and kept
at room temperature for 24 h to ensure complete saturation of the
molecules of biopolymers. After that, MEO at amount of 1 g was slowly
added to biopolymer dispersion by agitation at 5000 rpm to obtain pre-
emulsion with the core-coating ratio at 1:4. The probe sonicator with
the previously described conditions was used for encapsulation of MEO
by WPI/inulin mixtures. Ultrasonication process was performed at
room temperature for 20 min for PE preparation. In order to evaluate
the morphology of PE capsules with FE-SEM analysis, these samples
were freeze dried at −40 °C and vacuum of 0.007 atm for 48 h.
2.3. Characterization of NE and PE stabilized MEO
The particle size, zeta potential and polydispersity index (PDI) of NE
and PE stabilized MEO were measured by Dynamic Light Scattering
(DLS) technique using Zetasizer (Zetasizer Nano-ZS, Malvern, UK) at
25 °C in three replicates. Fluorescence microscopy (Bh2-RFCA,
Olympus, Japan) and field emission scanning electron microscopy (FE-
SEM; ZEISS, SIGMA, Germany) were employed to observe the shape
H. Almasi, et al.
and morphology of NE and PE stabilized MEO, respectively. For de-
termination of encapsulation efficiency (EE) of MEO in the NE and PE,
1 ml of MEO-loaded NE and PE was put into Amicon® filter (molecular
weight cutoff 100 kDa, Millipore, UK) and diluted with distilled water.
After that, the sample was centrifuged (Universal 320 centrifuge,
Hettich, Germany) at 2000 rpm for 10 min for separation of the un-
loaded MEO from capsules. Then, the concentration of unloaded MEO
was determined by UV–Vis spectroscopy (Unico, S 2100 SUV, Dayton,
Japan) and calibration curve. Absorbance was measured at 329 nm.
MEO had the maximum absorbance in this wavelength (λmax). Finally,
EE was calculated by the following equation (Noori et al., 2018):
Total MEO used in emulsion  (mg)  −  Free MEO content (mg)
EE(%) =
Total MEO used in emulsion (mg)
 × 100 (1)
2.4. Antibacterial activity of NE and PE stabilized MEO
The antibacterial activity of free MEO and encapsulated samples
against S. aureus (Gram-positive) and E. coli (Gram-negative) bacteria
was assessed by agar well diffusion technique. Suspensions containing
(1.5 × 108 CFU/mL) colonies of S. aureus and E. coli were prepared and
cultured on surface of prepared Mueller Hinton Agar plate. After that,
the wells with 7.47 mm diameter were made in the solidified media
using a sterile borer and 100 μL of the free MEO and NE or PE disper-
sions were added to each well. Subsequently, the plates were incubated
at 37 °C for 24 h. After incubation time, the diameter of the inhibition
zone around the wells was measured in triplicate by the caliper and the
means were reported.
2.5. Preparation of active films
Pectin films were prepared by method of Nisar et al. (2018). 3 g of
pectin was dispersed in 100 mL of distilled water and completely hy-
drated for 18 h at room temperature. After complete dissolution of
pectin, MEO loaded NE and PE dispersions were added to the pectin
solution in the amounts that preserve the concentrations of MEO at
constant levels (2.5, 5 and 7.5% w/w of pectin). After complete mixing
of nanocarriers by agitation for 15 min, glycerol as a plasticizer was
added at level of 20% (w/w of pectin) and mixed with magnetic stirrer.
Neat pectin film with glycerol and without MEO loaded nanocarriers
was prepared as control sample. About 25 mL of each film forming
solutions were cast on plastic petri dishes (diameter 10 cm) and dried at
25 ± 5 °C for 48 h. Finally, dried films were peeled-off and conditioned
at 55% RH prior to characterization.
2.6. Characterization of active pectin films
2.6.1. Fourier transform infrared (FT-IR) spectroscopy
The structural interactions in the film samples were investigated
with FT-IR spectroscopy (8000 S, Shimadzu, Japan) analysis. The
spectra were collected over the wavenumber range of 4000–400 cm−1.
The KBr pellet method was used for sample preparation.
2.6.2. X-ray diffraction (XRD) analyses
The XRD patterns of the film samples were recorded using the X-ray
diffractometer (Kristalloflex D500, Siemens, Germany) in the region of
the diffraction angle (2θ) from 5° to 80° at room temperature.
2.6.3. Field emission scanning electron microscopy (FE-SEM)
The morphology study of the surface of film samples was carried out
using the FE-SEM (ZEISS, SIGMA, Germany) after gold coating of
samples (DST1, Nanostructured Coating Co., Tehran, Iran). The accel-
erating voltage was from 10 to 20 KV.
3
Food Hydrocolloids 99 (2020) 105338
2.6.4. Film thickness
The thickness of film samples was determined using a digital mi-
crometer (Fowler, USA). Measurements were performed at five ran-
domly points of each film sample to calculate the average value.
2.6.5. Mechanical properties
The mechanical parameters, including Young's modulus (YM), ul-
timate tensile strength (UTS) and strain to break (STB) of the film
samples were investigated by Tensile Analyzer (Instron 5566, USA)
with a 100 N load cell. The film samples were cut into dumbbell shape
(8 × 0.5 cm2) after conditioning at RH = 55% for 24 h and were
mounted in two grips at 50 mm then the crosshead speed was set to
1.0 mm/min (Zhu, Tang, Yin, & Yang, 2018).
2.6.6. Water vapor permeability (WVP)
The WVP of the film samples was determined according to the
ASTM E96-05 (ASTM, 2005) standard method with some modifications
(Fathi, Almasi, & Pirouzifard, 2018). Film samples were sealed in glass
vials (average diameter of 2 cm and 4.5 cm height) with a diameter
slightly larger than the diameter of the vials. The RH 0% in the vials
was maintained using 3 g of anhydrous CaSO4. Then, the vials were
placed in a desiccator containing saturated K2SO4 solution with 97%
RH at 25 °C and the vials were weighed every 24 h. The changes of
weight were recorded as a function of time and the slopes were cal-
culated by a linear regression (weight vs. time). The water vapor
transmission rate (WVTR) was defined as the slope of the linear part of
the curve (g/h) divided by the transfer area (7.85 × 10−5 m2). Finally,
the WVP ( × 10−7 g m−1. h−1. Pa−1) of film samples was calculated as:
WVTR. X
WVP =
P (R1 − R2) (2)
Where P is the saturation vapor pressure of water (Pa) at the test
temperature (25 °C), R1 is the RH in the desiccator, R2 is the RH inside
the vial and X is the average thickness of film samples (m). Under these
conditions, the driving force [P (R1−R2)] is 3115.42 Pa.
2.6.7. Moisture absorption
Moisture absorption of film samples was assessed based on the
method of Ghadetaj et al. (2018). Firstly, the dried films of
(20 × 20 mm2) were conditioned at 0% RH (desiccator containing
CaSO4) for 24 h and weighed. After that, the samples were transferred
to a desiccator containing saturated Ca(NO3)2 solution to ensure a RH
of 50–55% at 20–25 °C. The samples were weighed at desired time in-
tervals until constant weight was reached. Finally, the moisture ab-
sorption of the samples was calculated using the following equation:
Wt − W0
Moisture absorption  = × 100
W0 (3)
Where W0 is the initial weight of the sample and Wt is the weight of the
sample at 55% RH after t time.
2.6.8. Color properties
The color parameters; i.e., L∗ (lightness/brightness), a∗ (redness/
greenness), and b∗ (yellowness/blueness) of film samples were in-
vestigated according to method reported by Amjadi et al. (2019).
Briefly, the film samples and RAL standard color sheets were placed in
the standard box and imaged using a digital camera (Canon Power shot
SX720 HS, Japan). The L*, a* and b* factors of film samples and RAL
standard color sheets were shown by Adobe Photoshop software. Then,
the calibration curves were obtained by drawing the actual L*, a* and
b* values of the standard sheets against the showed values by software.
Finally, the L*, a* and b* values of film samples were calculated via the
replacement of showed factors by software in the equations of cali-
bration curves. The obtained L*, a* and b* values were used for cal-
culation of the yellowness index (YI), and total color difference (ΔE) of
film samples (Ranjbaryan, Pourfathi, & Almasi, 2019):
H. Almasi, et al. Food Hydrocolloids 99 (2020) 105338

142.86  × b*
YI  =  
L* (4)

ΔE = * 2 + (Δa *)2 +  (Δb)

(ΔL) * 2
(5)
where ΔL*, Δa*, and Δb* are the difference between the color of
standard white color plate (L* = 93.49, a* = - 0.25, and b* = −0.09)
and film samples.

2.6.9. Antioxidant activity

The antioxidant activity of the films was assessed by the DPPH ra-
dical scavenging assay. Firstly, 100 mg of each film sample was dis-
solved in 2 ml of distilled water by continuous stirring for 2 min, and
1 ml of film extract solution was added to 0.2 mL of DPPH solutions
(0.04 g/L) in ethanol. The mixture was vortexed vigorously and kept in
the dark room for 30 min at room temperature. The reduction in ab-
sorbance at 517 nm was determined with UV–Vis spectrophotometer.
Finally, the antioxidant activity was measured as percentage of DPPH
free radical scavenging activity using the following equation (Zhu et al.,
2018):
Abscontrol −  Abssample
Free radical scavenging activity % =   × 100
Abscontrol (6)

2.6.10. Release properties

The release of MEO from the film samples were assessed in ethanol
95% as simulant of fatty foodstuffs according to the following proce-
dure. Briefly, the film samples were cut into 2 × 2 cm2 and totally
immersed in the vials containing 10 ml of simulant. Then, the vials were
sealed and stored in darkness at different temperature conditions (4, 25
and 40 °C) for 72 h with twice shaking per day. At each set time, 1 ml of
simulated solution was harvested for the determination of released
MEO and was replaced with fresh simulated solution. Finally, the values
of released MEO were measured using a UV–Vis spectrophotometer and
calibration curve of MEO at 329 nm (Ghadetaj et al., 2018).

2.7. Statistical analysis

Statistics on a completely randomized design were performed with

the analysis of variance (ANOVA) procedure in SPSS Statistics 23 (IBM
Corporation, Armonk, NY, USA) software. Duncan's multiple range test
(p < 0.05) was used to detect differences among mean values. All the
data were expressed as means ± standard deviation (SD).

Fig. 1. Particle size distribution of the MEO loaded NE and PE (A), fluorescence
3. Results and discussion
microscope image of NE dispersion (B) and FE-SEM image of PE stabilized
microcapsules powder (C).
3.1. Characterization of NE and PE stabilized MEO

The droplet size, PDI and zeta potential of NE and PE stabilized
MEO are shown in Fig. 1A. Size and size distribution of nanoemulsions
are important parameters for their stability, release of loaded com-
pounds, and the film properties (Haghju et al., 2016). The droplet size
of MEO-loaded NE and PE were 97.5 and 233.3 nm, respectively. The
increase in the size of MEO-loaded PE may be attributed to the surface
coverage of emulsion with macromolecular WPI and inulin. However,
the size of PE stabilized MEO is lower than those reported for other used
Pickering emulsions in the film formulations. So that, the size of thymol
loaded Pickering emulsion (Zhu et al., 2018), zein/chitosan colloid
particles stabilized Pickering emulsion (Shi et al., 2016), and rosemary
essential oil loaded Pickering emulsion (Fasihi, Fazilati, Hashemi, &
Noshirvani, 2017) were 10, 25 and 15 μm, respectively. PDI indicates
the particle size distribution which the small values of this parameter
shows a narrow size distribution (Otoni et al., 2016). The PDI values of
NE and PE stabilized MEO are 0.313 and 0.259, respectively. These
values especially the PDI value of PE (PDI < 0.3) showed a narrow
pattern of size distribution that the uniformity of the emulsion droplets
is appropriate for efficiently applying in the film formulation. More-
over, zeta potential is another important factor that defines the net
surface charge and mediates the stability of a colloidal system
(Ghadetaj et al., 2018). The zeta potential for MEO-loaded NE and PE
were −2.3 and −21.4 mV, respectively. The electrical charge of
emulsion droplets is related to the charge of surfactants adsorbed
around oil droplets (Acevedo-Fani et al., 2015). Thus, the showed
electrical charge close to zero by MEO-loaded NE might be attributed to
the used Tween 80 as a non-ionic surfactant for preparation of NE.
However, MEO-loaded PE indicated high negative zeta potential which
can be associated to incorporation of ionic biopolymers especially WPI
that has negative charge at pH values higher that its isoelectric point
(IP). The increase in the charge of emulsion enhance its stability against
creaming and/or flocculation phenomena because of the electrostatic
repulsion among the emulsion droplets (Pérez-Córdoba et al., 2018). In
this connection, Bakry et al. (2017) reported that the zeta potential of
stabilized emulsion with WPI/inulin mixture was −34 mV that this
high charge provided sufficient stability due to the electrostatic

4
H. Almasi, et al.
repulsion between droplets.
Furthermore, the fluorescence microscope image of NE dispersion
showed that the droplets of NE were spherical with diameters below
100 nm that confirmed the droplet size of MEO-loaded NE reported by
DLS (Fig. 1B). Fig. 1C presents the FE-SEM image of freeze dried PE
stabilized microcapsules powder. Such aggregated form of powder may
be attributed to the drying process. But the spherical shaped micro-
capsules with diameter about 305 nm are obvious in the figure that
reveals the successfulness of ultrasonic method in the formation of PE.
Moreover, the EE of MEO-loaded NE and PE were calculated to be
87.59 ± 2.32% and 81.58 ± 1.14%, respectively. The slightly higher
EE of NE could be attributed to the higher mobility and better surface
activity of low molecular weight surfactants. The obtained EE for MEO-
loaded PE is quite appropriate and better than those reported for other
encapsulated active compounds with Pickering emulsions for applica-
tion in the film formulations. In this regard, hesperidin (Dammak et al.,
2019), thymol (Robledo et al., 2018) and garlic EO (Pérez-Córdoba
et al., 2018) were only loaded in the PE at amounts of 78, 67 and 70%,
respectively.
3.2. Antibacterial activity of NE and PE stabilized MEO
The antibacterial activity of free MEO and emulsion samples against
Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria was in-
vestigated by well diffusion technique. Inhibition zone diameters of the
samples against bacteria are shown in Table 1 and Fig. 2. The showed
inhibition zones by free MEO were 21.20 ± 1.24 and
13.10 ± 1.09 mm against S. aureus and E. coli bacteria, respectively.
The antimicrobial activity of MEO is attributed to the high proportion
of oxygenated monoterpenes and especially to their major constituents,
such as terpin-4-ol, α-terpinol, α-pinene, and p-cymene (Ben Salha,
Herrera Díaz, Labidi, & Abderrabba, 2017). The antibacterial me-
chanism of these compounds is probably due to the increase in per-
meability of bacteria cell wall by entering between the fatty acyl chains
of the membrane lipid bilayers which disrupting lipid packing and
causing changes to membrane properties and functions (Hajlaoui et al.,
2016). However, the loading of MEO with NE and PE significantly
(p < 0.05) decreased the antibacterial activity of MEO; the anti-
bacterial activity of MEO loaded NE and PE were 32% and 24% lower
than free MEO against S. aureus, respectively. But there was no sig-
nificant difference between antimicrobial activity of PE and NE. The
main reason for encapsulation of EOs and bioactive compounds is their
low stability under different external conditions, which this low stabi-
lity limited their application as antibacterial and antioxidant com-
pounds (Liu et al., 2019). However, the controlled release of loaded
compounds by nanocarriers such as NE or PE may decrease their
functional activity (antibacterial and antioxidant activities) in the short
storage time (Ghadetaj et al., 2018). But, the release of encapsulated
active compounds enhances during storage time and this phenomenon
causes to maintain their functional activity in different milieus such as
Table 1
Antimicrobial activity of film samples.
Samples Inhibitory zone (mm)
S. aureus E. coli
MEO 21.20 ± 1.24 a
15.10 ± 1.09a
MEO-NE 14.32 ± 1.02b 12.65 ± 0.94b
MEO-PE 16.00 ± 0.97b 11.58 ± 1.15b
Data are expressed as mean ± standard deviation (n = 3) and different letters
show significant difference.
At the 5% level in Duncan's test (p < 0.05). MEO: marjoram essential oil,
MEO-NE: marjoram essential.
Oil loaded nanoemulsion, MEO-PE: marjoram essential oil loaded Pickering
emulsion.
5
Food Hydrocolloids 99 (2020) 105338
Fig. 2. The antibacterial activity of MEO and its encapsulated forms against S.
aureus and E. coli after 24 h incubation at 37 °C. MEO: marjoram essential oil,
MEO-NE: marjoram essential oil loaded nanoemulsion, MEO-PE: marjoram es-
sential oil loaded Pickering emulsion.
active films for long storage time (Li et al., 2018). Similar results were
obtained by Tastan, Ferrari, Baysal, and Donsì (2016), according to
whom the antibacterial activity of carvacrol-loaded NE was lower than
its value in the free carvacrol.
3.3. Characterization of active pectin films
3.3.1. FT-IR spectroscopy
To recognize the chemical structure of the PE stabilized MEO and
obtained films, the FT-IR analysis was used. The FTIR spectra of free
MEO, PE stabilized MEO powder and film samples are shown in Fig. 3.
The spectrum of neat pectin film displayed the specified peaks at 3440,
2923, 1638, 1382, 1113 and 931 cm−1. The broad and strong band at
3440 cm−1 could be attributed to the stretching vibration mode of O–H
bond due to intermolecular interactions through O–H bonds among
pectin monomers and the bands at 2923 cm−1 was related to C–H
stretching vibrations of methyne groups in polymer chains and methyl
group of the methyl ester (Nisar et al., 2018). Furthermore, the bands at
1638 cm−1 and 1382 cm−1 were attributed to the asymmetric and
symmetric vibrations of carboxylate present in pectin, respectively
(Oliveira et al., 2016). The peaks at 1113 and 931 cm−1 represented
C–O–C stretching vibrations of the polymer chain structure (Nisar et al.,
2018). The spectral differences between neat pectin film and other film
samples were largely related to changes of conformation and orienta-
tion of polypeptide chains by incorporation of MEO-loaded NE and PE.
By incorporation of MEO-loaded NE and PE the carboxylate peak at
1382 cm−1 was disappeared due to the change in carboxylate to car-
boxylic group. Furthermore, in the NE7.5 and PE7.5 film samples, the
peak at 3440 cm−1, related to the OH stretching vibration, was shifted
to 3434 and 3435 cm−1, respectively. These findings can be related to
the possible interaction (hydrogen bonds) between the–OH and –COOH
end groups of pectin and –OH groups of MEO-loaded NE and PE. Ad-
ditionally, the PE7.5 spectrum displayed the specified peaks at 1548
(N–H stretching of amide II), 1245 (C–N stretching of amine) and
1028 cm−1 (C–N stretching of amines) which may be related to use of
the WPI for preparation of PE.
3.3.2. X-ray diffraction (XRD) analyses
The crystalline structure of film samples and the compatibility of
pectin and MEO-loaded NE and PE were investigated by XRD analysis.
The XRD diffractograms of MEO-loaded PE powder and film samples
are shown in Fig. 4. The diffractogram of the MEO-loaded PE exhibited
different peaks between 5° and 30°, associated to polycrystalline
structure of the MEO-loaded PE. Moreover, the diffractogram of neat
pectin film showed a specific peak at 2θ of 22.4° which indicated that
the neat pectin film has semi-crystalline structure. The incorporation of
MEO-loaded PE caused to the appearance of new broad peak at 2θ of
10°. However, the incorporation of MEO-loaded PE and NE in pectin
film did not change the position of neat pectin's specific peak in the
H. Almasi, et al.
Fig. 3. FT-IR spectra of MEO, PE stabilized MEO powder (MEO-PE), neat pectin film and active films containing the highest amount of NE (NE7.5) and PE (PE7.5).
XRD pattern significantly. Generally, the profiles of diffraction spectra
of PE7.5 and NE7.5 film samples were similar to that obtained for the
neat pectin film. These results showed the compatibility between bio-
polymer and the incorporated MEO-loaded PE that and NE which lead
to preservation the crystalline structure of the films. In accordance with
these results, Nisar et al. (2018) reported that the neat pectin films
showed a broad peak at 2θ of 15.72° confirming the presence of crys-
talline structure, as well the incorporation of clove bud EO into pectin
films did not alter the peak position. Also, it has been reported that that
crystalline nature of the WPI based film was completely preserved after
addition of Grammosciadium ptrocarpum Bioss. EO loaded NE in the film
structure (Ghadetaj et al., 2018).
3.3.3. Morphology observation by FE-SEM
The FE-SEM analysis was used to investigate film morphology and
distribution of emulsion droplets in the film matrix and the FE-SEM
images of samples are shown in Fig. 5. The FE-SEM image of neat pectin
film showed a rough surface with few cracks. Incorporation of MEO-
6
Food Hydrocolloids 99 (2020) 105338
loaded NE decreased the roughness of the surface pectin film which
may be related to the presence of Tween 80 that improves the com-
pactness and density of pectin matrix. However, the incorporation of
MEO-loaded NE led to a bubble-like structure may be attributed to the
presence of NE droplets within the film matrix. The similar results were
reported with Chen et al. (2016) that chitosan based films containing
the cinnamaldehyde NE had rough surfaces with some holes. Also, it
has been reported that the holey structure of sodium alginate based
films containing EO-loaded NE may be due to the migration of oil
droplets upwards the films and further volatilization during water
evaporation (Acevedo-Fani et al., 2015). Furthermore, the FE-SEM
images of PE7.5 film sample exhibited a rougher structure, but the
density and integrity of biopolymer matrix were increased by in-
corporation of MEO-loaded PE. The resulted surface roughness by ad-
dition of MEO-loaded PE can be related to the migration of aggregates
or droplets to the top of the film during film drying, which leads to
surface irregularities (Nisar et al., 2018). There are many studies on the
increase of surface coarseness with the presence of Pickering emulsions
H. Almasi, et al.
Fig. 4. XRD patterns of PE stabilized MEO powder (MEO-PE), neat pectin film and active films containing the highest amount of NE (NE7.5) and PE (PE7.5).
that are in line with the results of the current study. In this regard,
previous studies reported that the chitosan (Shi et al., 2016) and
polylactic acid (Zhu et al., 2018) based film containing PE were dense,
but slightly heterogeneous, indicating a few microstructure-sized holes
within the film matrix.
3.3.4. Film thickness
Thickness is an effective parameter for investigation of mechanical
and water barrier properties of films. The results of thickness mea-
surement are shown in Table 2. As a result, the thickness value for
control film sample was 96.66 ± 2.21 μm, but the incorporation of 2.5
and 5% MEO-loaded NE significantly (p < 0.05) decreased the thick-
ness value of films. However, the thickness value of NE7.5 film sample
revealed no significant (p > 0.05) difference with control film sample.
According to the previous literature, the reduction of thickness value by
addition of NE might be attributed to the decrease of solids con-
centration in the film matrix because of possible losses of oil phase
during film formation (Acevedo-Fani et al., 2015). In addition, the
thickness value of film samples showed a significantly increasing trend
by incorporation of MEO-loaded PE. So that the highest thickness value
of film samples was associated with the PE7.5 film sample that was
158.43 ± 1.00 μm. This phenomenon can be described by the in-
creasing of dry matter content by addition of MEO-loaded PE and
changing structure of pectin based film matrix to dense one. Similar
results were reported by Fasihi et al. (2017) who suggested that the
increase in thickness of carboxymethyl cellulose-polyvinyl alcohol
blend films by incorporation of PE could be explained by the increase of
the solid material content by addition of these emulsions.
7
Food Hydrocolloids 99 (2020) 105338
3.3.5. Mechanical properties
Table 2 presents the measured mechanical properties of film sam-
ples. The YM, UTS and STB values for control film sample were
713 ± 6.11 MP, 2.09 ± 0.32 MP and 23.56 ± 0.87%, respectively.
The incorporation of MEO-loaded NE significantly (p < 0.05) de-
creased the YM values of film samples. Moreover, the UTS value was
reduced by incorporation of 5 and 7.5% of MEO-loaded NE, but the
addition of 2.5% of NE caused an increase in UTS value. However, the
STB values of film samples exhibited no significant (p > 0.05) differ-
ence by incorporation of MEO-loaded NE. These findings can be ex-
plained by the reduction of elasticity and increasing of plasticity by
incorporation of MEO-loaded NE. So that, the NE droplets act as a
plasticizer interfering with the hydrogen bonding of pectin based films,
restricting the rigidity and resistance while increasing flexibility and
segmental mobility of the polymer (Otoni et al., 2016). In this con-
nection, previous studies reported that the addition of NE reduced the
tensile strength of biopolymeric based films such as chitosan (Chen
et al., 2016), gelatin-chitosan (Pérez-Córdoba et al., 2018) and WPI
based films (Ghadetaj et al., 2018) due to the plasticizing effect of NE
droplets, which weaken the intermolecular interactions between poly-
meric chains and increased the extensibility of films. No significant
difference resulted in YM and UTS values by the addition of 2.5% MEO-
loaded PE, while these values increased by increasing of MEO-loaded
PE concentration to 5 and 7.5%. The STB values of film samples sig-
nificantly (p < 0.05) decreased by incorporation of 2.5 and 7.5%
MEO-loaded PE, but the STB value of PE5 film was higher than control
sample. These findings can be related to the creation of new interac-
tions between PE droplets and pectin matrix which caused to the high
mechanical resistance and ductility in the polymer network. It has been
H. Almasi, et al.
Fig. 5. FE-SEM images of neat pectin film and active films containing the highest amount of NE (NE7.5) and PE (PE7.5) at two magnifications.
reported that the mechanical properties of polylactic acid based films
were improved by incorporation of zein/chitosan-coated PE that might
be attributed to the miscible capability and interaction at the interface
of polymer matrix and PE (Zhu et al., 2018).
3.3.6. Water vapor permeability (WVP)
The WVP provides the evaluation of water barrier properties of films
and their application in food packaging (Nisar et al., 2018). The results
of WVP measurement are shown in Table 2. As a result, the addition of
8
Food Hydrocolloids 99 (2020) 105338
5 and 7.5% MEO-loaded NE significantly (p < 0.05) decreased the
WVP values of film samples, so that the WVP values of NE5 and NE7.5
film sample were 58.13 and 57.13% lower than its value in the control
sample, respectively. Additionally, the WVP values of MEO-loaded PE
incorporated film sample were significantly (p < 0.05) lower than
other film samples. The WVP values of film samples reduced more than
86.54, 84.53 and 88.05% after incorporation of 2.5, 5 and 7% MEO-
loaded PE, respectively. Thus, the highest and lowest WVP values were
to control (9.96 ± 0.76 × 10−7 g/m.h.Pa) and PE7.5
H. Almasi, et al. Food Hydrocolloids 99 (2020) 105338

Table 2
The thickness, mechanical properties and WVP values of pectin films activated by NE and PE stabilized MEO.
Film samples Thickness (μm) YM(MPa) UTS (MPa) STB(%) WVP( × 10−7 g/m.h.Pa)

Control 96.66 ± 2.21b 713 ± 6.11b 2.09 ± 0.32b 23.56 ± 0.87b 9.96 ± 0.76d
NE2.5 90.54 ± 0.87a 648 ± 9.43a 3.95 ± 0.17c 25.18 ± 1.01b 9.93 ± 0.65d
NE5 92.42 ± 1.19a 619 ± 4.14a 1.87 ± 0.45a 25.43 ± 0.87b 4.17 ± 0.09c
NE7.5 96.21 ± 0.76b 623 ± 11.32a 1.33 ± 0.05a 21.11 ± 2.32ab 4.27 ± 0.44c
PE2.5 105.43 ± 1.67c 703 ± 3.33b 2.72 ± 0.11b 19.83 ± 0.78a 1.34 ± 0.00b
PE5 115.77 ± 2.11d 736 ± 7.00c 8.67 ± 0.47d 29.50 ± 1.04c 1.54 ± 0.66b
PE7.5 158.43 ± 1.00e 719 ± 5.69b 8.18 ± 0.98d 19.47 ± 0.07a 1.19 ± 0.03a

Data are expressed as mean ± standard deviation (n = 3) and different letters show significant difference at the 5% level in Duncan's test (p < 0.05). YM: young's
modulus, UTS: ultimate tensile strength, STB: strain to break, WVP: water vapor permeability.

(1.19 ± 0.03 × 10−7 g/m.h.Pa) film samples, respectively. The WVP
value of film is dependent to the hydrophilic-lipophilic ratio of film
matrix because of water diffusion through the hydrophilic portion of
matrix (Acevedo-Fani et al., 2015). Thus, the improving of water bar-
rier properties by addition of nanoemulsions in film matrix may be
related to their hydrophobic nature. In addition, incorporation of PE
improved the water resistance of the pectin matrix by increasing cross-
linking between polymer chains and reduction mobility of chains due to
filling free spaces in the pectin matrix (Zhu et al., 2018). Moreover, it
has been reported that the reduction in WVP values by addition of PE
can be related to increasing of the tortuosity path resulted by these
emulsions (Fasihi et al., 2017). The measured WVP values are quite
appropriate and more satisfactory than those reported for other NE
incorporated films. In this regard, Ghadetaj et al. (2018) showed that
the incorporation of NE decreased the WVP value of WPI-based films
from 3.87 × 10−7 to 2.937 × 10−7 g/m.h.Pa due to the reduction of
migration rate of water vapor molecules by improving the coherence
and decreasing spaces. Acevedo-Fani et al. (2015) also reported that the
WVP value of the alginate based film containing NE was 19.49% lower
than its value in the control film sample.
3.3.7. Moisture absorption
Moisture absorption is other factor for investigation of water barrier
properties of film samples. As shown in Fig. 6, the percentage of
moisture absorption was significantly (p < 0.05) elevated after the
addition of 2.5 and 7.5% MEO-loaded NE, while this value for NE5 film
sample did not show any significant (p > 0.05) difference with neat
pectin film after 120 h. Additionally, the incorporation of PE sig-
nificantly (p < 0.05) decreased the moisture absorption percentage of
film samples and highest reduction value was obtained by addition of
2.5% PE. Therefore, the highest and lowest moisture absorption per-
centage were attributed to the NE2.5 (17.94%) and PE2.5 (10.14%)
film sample after 120 h, respectively. These results appeared to be quite
consistent with results of WVP measurement. Thus, it could be con-
cluded that the incorporation of PE improved the water barrier prop-
erties of the pectin based film by creation strong interactions between
PE particles and the hydrophilic part of the pectin chains and reduction
the mobility of these chains (Fasihi et al., 2017; Ghadetaj et al., 2018).
However, the higher amounts of PE could increase the moisture ab-
sorption of film probably due to the hydrophilic nature of inulin and
WPI.
3.3.8. Color properties
The color properties of food packaging films have a major influence
on the overall appearance and consumer acceptance (Acevedo-Fani
et al., 2015). Fig. 7a presents the ΔE and YI values of films. As shown in

Fig. 6. Moisture absorption of pectin films activated by NE and PE stabilized MEO.

9
H. Almasi, et al. Food Hydrocolloids 99 (2020) 105338

Fig. 7. Color indices (ΔE and YI) (A) and DPPH radical scavenging activity (B) of pectin films activated by NE and PE stabilized MEO. Data are expressed as
mean ± standard deviation (n = 3) and different letters in each parameter show significant difference at the 5% level in Duncan's test (p < 0.05). (For inter-
pretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7a, the YI value of film samples was significantly (p < 0.05) in-
creased by addition of 5 and 7.5% MEO-loaded PE, while the YI value of
other samples exhibited no significant (p > 0.05) difference with the
neat pectin film. Furthermore, the ΔE value increased by increasing the
concentration of emulsions and the films containing 5 and 7.5% MEO-
loaded PE exhibited the highest ΔE values. However, incorporation of
MEO-loaded NE had no drastic effect on ΔE values of pectin films. These
results indicated that the type and concentration of emulsions affected
the films' color. The NE had no significant effect on color and trans-
parency of pectin films. This could be attributed to the lower droplet
size of this type of emulsion. Nevertheless, the higher particle size of
MEO-loaded PE and also the natural white color of inulin and bright
yellow color of WPI caused a drastic decrease in transparency and in-
crease in yellowness of pectin films. This trend increased by increasing
the concentration of PE stabilized MEO in the film formulation. It has
been reported that the addition of PE increased the ΔE value in soybean
polysaccharide based films (Liu et al., 2019) and carboxymethyl cel-
lulose-polyvinyl alcohol blend films (Fasihi et al., 2017). Ghadetaj et al.
(2018) reported no significant changes in color properties of WPI film
after incorporation of NE. All of these reports are in line with the results
of the current study.
3.4. Antioxidant activity
The DPPH scavenging activity of film samples are shown in Fig. 7b.
As a result, the incorporation of MEO-loaded NE and PE significantly
(p < 0.05) enhanced the DPPH scavenging activity of film samples.
Additionally, the antioxidant activity of film samples showed increasing
trend by enhancing the percentage of MEO-loaded NE and PE. The
antioxidant activity NE incorporated film samples were meaningfully
(p < 0.05) higher than its values in the PE incorporated film samples.
Therefore, the lowest DPPH scavenging activity (7.45%) was related to

10
H. Almasi, et al. Food Hydrocolloids 99 (2020) 105338

Fig. 8. Release rate of MEO from pectin active films containing NE and PE stabilized MEO to simulant ethanol 95% in three different temperatures.

11
H. Almasi, et al.
the control sample and the highest DPPH scavenging activity (78.95%)
associated with NE7.5 film sample. The DPPH scavenging activity of
emulsion incorporated film samples was potentially attributed to the
antioxidant activity of MEO that contain phenolic acids and terpenoids
(Ben Salha et al., 2017). In another hand, the release of loaded com-
pounds in the PE is lower than their release in the NE, which this
phenomenon caused to decreased mobility of loaded compounds and
acting their functional activity in the different matrixes in the short
storage time (Dammak et al., 2019; Fasihi et al., 2017). Therefore, the
low antioxidant activity of PE incorporated film samples in comparison
with NE incorporated film samples may be attributed to the slower
release of encapsulated MEO in the film matrix. In another study, the
antioxidant activity of the WPI based films containing EO loaded NE
revealed no significant difference with the film samples containing free
EO; which this finding attributed to the inhibitory effect of encapsula-
tion on release of EO to antioxidant measuring medium (Ghadetaj et al.,
2018).
3.4.1. Release properties
The release profiles of MEO from film samples in the three different
incubation temperatures (4, 25 and 40 °C) are shown in Fig. 8. The
release profiles of MEO in the all film samples revealed increasing trend
during incubation time at all incubation temperatures. The increasing
of MEO-loaded NE and PE percentage in the film samples significantly
(p < 0.05) increased the release ratio of MEO in the film samples.
According to the previous studies, the increasing of the amount of re-
leased EO by increasing of NE content in the film may be attributed to
the changes in the microstructure and properties of the film matrix by
high EO content (Chen et al., 2016). Moreover, the release ratio of MEO
were enhanced by increasing the incubation temperature. The finding
can be described by enhancing the mobility of pectin chains and mi-
gration of MEO from film matrix to the release medium which this
result was in line with the previous literature (Ghadetaj et al., 2018).
Furthermore, the release ratio of MEO in the NE incorporated film
samples were significantly (p < 0.05) higher than its ratio in the PE
incorporated film samples. These results appeared to be quite consistent
with antioxidant activity analyses results where encapsulation of MEO
by PE decreased the release rate of MEO in the different incubation
conditions. So that, the PE stabilized by absorbed WPI-inulin complex
at the interface of oil-water phase which caused more protection of
loaded MEO against external conditions (Ghadetaj et al., 2018). In this
regard, Zhu et al. (2018) reported that the release rate of thymol from
the polylactic acid based film samples were reduced by encapsulation of
thymol with PE, as well the increasing the thymol concentration in the
film samples increased its release rate.
4. Conclusion
The pectin films activated by MEO-loaded NE and PE were prepared
and characterized with FT-IR, FE-SEM and XRD analyses. The film
samples containing PE revealed appropriate mechanical and water
barrier properties due to their dense and less permeable structure.
Moreover, the encapsulation of MEO with PE provided slower release
profile from the film samples at different incubation temperature as
compared to the encapsulation with NE. Generally, the fabricated ac-
tive film provided unique features by the addition of MEO-loaded PE as
compared to the MEO-loaded NE. In conclusion, this study paves the
way for application of the pectin films activated by MEO-loaded PE as a
new active food packaging system, representing a great promise for
improving quality and shelf life of food products.
Conflicts of interest
The authors declare that there is no conflict of interest in prepara-
tion and submission of this manuscript.
12
Food Hydrocolloids 99 (2020) 105338
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