International Journal of Biological Macromolecules 169 (2021) 352–361

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International Journal of Biological Macromolecules

j ou rn al h om e p age : ht tp :// w w w . e l s e v i e r . c o m / l o c at e / i j biom ac

Preparation of nanobiocomposite film based on lemon waste

containing cellulose nanofiber and savory essential oil: A new
biodegradable active packaging system
Mitra Soofi a, Ainaz Alizadeh a,⁎, Hamed Hamishehkar b, Hadi Almasi c, Leila Roufegarinejad a
a
Department of Food Science and Technology, Tabriz Branch, Islamic Azad University, Tabriz, Iran
b
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
c
Department of Food Science and Technology, Faculty of Agriculture, Urmia University, Urmia, Iran

a r t i c l e info
abstract
Article history:
Received 27 October 2020 Lemon waste after industrial juice extraction encompasses of valuable bio-components that stimulated the
Received in revised form 14 December de- velopment of novel and biodegradable films. Lemon waste powder (LWP) based nanobiocomposite films
2020 Accepted 15 December 2020 were prepared by incorporating different concentrations of cellulose nanofiber (CNF) (3 and 6% w/w) and
Available online 24 December 2020 savory es- sential oil (SEO) (1.5 and 3% w/w) in order to modify physical, mechanical and antimicrobial
properties of the films. The fabricated film samples were characterized in terms of FTIR, XRD, FE-SEM and
Keywords: DSC analyses as well as mechanical, water vapor permeability and antimicrobial properties. FTIR and FE-
Cellulose nanofiber SEM results indicated a good compatibility between LWP matrix and incorporated CNF and SEO. Physical
Lemon waste
and thermal analysis showed a significant effect of incorporating SEO and CNF on enhancing glass transition
Nanobiocomposite film
temperature, tensile strength and water barrier properties of the film samples. SEM analysis revealed non-
Physical properties
Savory essential oil
uniform dispersion of CNF at higher concentration, while SEO incorporation improved the structure of the
films. In addition, the LWP based films sig- nificantly showed antimicrobial properties against five food borne
pathogens and this effect improved consider- ably by elevating the SEO loading concentration. In conclusion,
LWP based nanobiocomposite films containing 3% CNF and 3% SEO could be introduced as a good candidate
for development of active food packaging.
© 2020 Published by Elsevier B.V.

1. Introduction wastes would be considered the best source for biopolymer production
due to their low cost and abundance. Citrus waste is globally produced
Nowadays, environmental pollution is one of the severe threats in vast quantities with the challenges related to it's disposal [7,8].
fac- ing mankind all over the world. In this regard, food packaging Among citrus fruits, lemon production estimated about 22 million tons
materials include petroleum-based plastics are enormously utilized annually in the world [9] and the residue generated by industrial pro-
owing to their availability in large quantities, cost effective, superb cessing of lemons such as lemon juice production accounts for the 50–
mechanical properties and exceptional barrier to water [1–3]. 60% of the original lemon mass [6,7]. Although, lemon waste dis- posal
However, the non- biodegradability of these materials and is tough due to its negligible economic value and high cost of
consumers demand for healthy and high quality food products, transportation, but high content of total solid, water, pH 3–4 of lemon
have led to the development of biode- gradable packaging such as waste could cause serious environmental drawbacks when handling in-
bioactive films and coatings as an alterna- tive to petroleum-based appropriately [8,10]. Moreover, lemon waste contains sufficient quanti-
plastics [4,5]. On the contrary, bio-polymers used for production of ties of pectin (30–40% from peel on dry matter basis), cellulose,
these films are biodegradable and mainly deriv- atives of cultivated hemicellulose, soluble sugars, starch, lignin, protein, fats and flavonoids
crops and fruits, while the land dedicated to biopoly- mer that indicates its technological characteristics as well as nutritional
production is still negligible [4,6]. Therefore, the use of bio-based properties, which are suggesting the importance of recovery and reuse
and biodegradable food wastes as available source for biopolymer of lemon waste [6,10,11]. Although, these compounds have great poten-
with no land use is a new approach to deal with high cost and low tial to form biodegradable films, commercial utilization of edible films
avail- ability of biopolymers [6]. Wastes from food plants including fabricated from fruits and vegetables puree has been somewhat limited
citrus partially due to the presence of low molecular weight compounds that
prevent the formation of continuous film network and insufficient me-
chanical and barrier properties [4,12]. Nanocomposites and cellulose
⁎ Corresponding author at: Department of Food Sci. & Tech., Tabriz Branch, Islamic Azad
University, Tabriz, Iran.
E-mail address: a.alizadeh@iaut.ac.ir (A. Alizadeh).

https://doi.org/10.1016/j.ijbiomac.2020.12.114
0141-8130/© 2020 Published by Elsevier B.V.
M. Soofi, A. Alizadeh, H. Hamishehkar et International Journal of Biological Macromolecules 169 (2021) 352–
al. 361

nano-fibers as a filler and promising nano reinforcement agents had the dried waste was milled to a very fine powder and used in the
be- come more considered to enhance biopolymer functionality prepara- tion of the LWP based film samples [6].
restrictions such as increase in mechanical strength, transparency,
thermal resis- tance, improved film appearance, reduced vapor and
gas permeability in addition to the control release of active
compounds when well- dispersed in biopolymer matrix [13–15].
According to the previous studies CNF was used widely as
reinforcement agent in whey protein isolate/polydextrose [15],
chitosan [16], mango puree [12], sodium car- boxymethylcellulose
[14,17], sodium alginate [18] and starch [19] based nanocomposite
films and exhibited favorable mechanical, water barrier and thermal
properties. Furthermore, the incorporation of bioactive agents such
as essential oils in the nanocomposite films provide out- standing
functionality for active packaging [20].
Nowadays, packaging films have gained a specific attention as car-
riers of antioxidant, antimicrobial and active compounds to reduce mi-
crobial growth, improve the quality and extending the shelf life of
foods by continuous migration or direct contact of packaging with
food surface [5]. Savory (Satureja khuzistanica L.) essential oil
belonging to the Lamiaceae family has a numerous therapeutic effects.
Savory es- sential oil contains 94% carvacrol as the main compound
and is known as seasoning agent with remarkable antioxidant
characteristics that have been widely demonstrated [2,20]. The
antimicrobial activity of this essential oil is allocated to its phenolic
compounds and its antioxi- dant properties which is greater than or
equal to the synthetic antioxi- dants [2].
To the best of our knowledge, no studies have been conducted to
prepare bioactive film based on lemon waste in combination with
other reinforcing or active agents. However, there are only a few
studies published about the integration of savory essential oil as an
antimicro- bial agent in active films [2,20]. Therefore, the purpose
of this study was to investigate the feasibility of nanobiocomposite
film production based on lemon waste containing cellulose nano
fiber and savory essential oil.

2. Materials and methods

2.1. Materials

Lemon waste (obtained from lemon juice production) was

procured from Asia shoor Industry Co. (Tabriz, Iran). Savory
essential oil (SEO) was purchased from Tabibdaru Industry Co.
(Kashan, Iran). Calcium sul- fate, magnesium nitrate, potassium
sulfate, citric acid (monohydrate,
>99.5) and glycerol were purchased from Merck Chemicals Co. (Darm-
stadt, Germany). CNF (average diameter and length was 35 nm and
5 μm, respectively, 99% purity) was prepared from Nano Novin
Polymer Co. (Sari, Iran). Listeria monocytogenes (PTCC 1298),
Salmonella enterica (PTCC CiP104115), Pseudomonas aeruginosa
(PTCC 1310), Staphylococ- cus aureus (PTCC 1764), and Escherichia
coli (PTCC 1163) were procured from Persian Type Culture Collection
(PTCC) (Tehran, Iran). For microbi- ological tests Mueller-Hinton agar
was purchased from Sigma-Aldrich Co. (St. Louis, USA).

2.2. Preparation of lemon waste powder (LWP)

Lemon waste was processed according to the method of Batori et

al. (2017) with a slight change. In this regard, the lemon waste was
washed with water to remove dissolved sugar, as sugar
involvement led to for- mation of cracks and heterogeneous surface
in the film samples. For this purpose, firstly waste was soaked in
water overnight and then two fur- ther washing steps were
applied. The ratio of water to lemon waste in all washing steps
was 1.5 to 1 and each washing step involved stirring the lemon
waste immersed in a water bath at 115 rpm for 20 min at 35 °C.
After draining, the lemon waste was collected and dried in the
oven (UF 55, Memmert, Germany) for 16 h at 40 °C. Consequently,
35
M. Soofi, A. Alizadeh, H. Hamishehkar et International Journal of Biological Macromolecules 169 (2021) 352–
al. Preparation of nanobiocomposite LWP based films
2.3. 361
LWP/CNF6%/SEO1.5% 3 6 1.5
LWP/CNF6%/SEO3% 3 6 3

The fabricated LWP-based nanobiocomposite film samples LWP: lemon waste powder, CNF: cellulose nanofiber, SEO: savory essential oil.
were prepared by the method of Batori et al. (2017) with some
modifications and were coded as Table 1. The filmogenic solutions
were prepared by incorporating 3% (w/v) of LWP and CNF (3 and
6% (w/w) based on LWP weight) in 100 mL acidic aqueous
solution containing 7% (w/w) glycerol (based on solution weight)
as a plasticizer and 1% (w/v) citric acid. In order to uniform and
homogeneous dispersion of nanoparticles and reduced aggregation,
CNF was sonicated (UP200H, Hielscher, Teltow, Germany) with a
nominal frequency of 40 kHz for 10 min before used in first
solution. Next, the prepared suspension was stirred by using
magnetic stirrer at 115 rpm for 30 min at 70 °C and then filtered to
separate large particles. After that, the temperature of the film
solu- tions lowered and maintained at the room temperature. In
order to ob- tain emulsions, SEO was incorporated into previously
filtered solution as an antimicrobial agent to reach final
concentration of 1.5 and 3% (w/w) (based on LWP weight) and
then homogenized (IKA T25- Digital Ultra Turrax, Staufen,
Germany) for 3 min in 13,500 rpm. The SEO incorporation in the
film samples was carried out without the need to emulsifying
agents. The pectin itself has emulsifying property which is well
documented in literature [11]. Consequently, the mixture was
treated by ultrasound for 20 min at 40 kHz to remove dissolved air
bubbles. Furthermore, LWP-based films without any CNF and
essential oil inclusion were also prepared for later comparison.
Finally, film solu- tions were casted onto polystyrene plates and
dried at 35 °C for 36 h. Prepared film samples were also
conditioned in a desiccator containing saturated magnesium
nitrate solution (53% relative humidity (RH)) at 25 °C for 48 h for
subsequent analysis [6].

2.4. Characterization of films

2.4.1. Fourier transforms infrared (FT-IR) spectroscopy

The infrared (IR) spectroscopy was used to investigate the
structure interactions in the nanobiocomposite film samples that
determined with FT-IR spectroscopy (Tensor27, Bruker, Germany).
The KBr-pellet method was used for sample preparation and all
FTIR spectra were col-
lected over the spectral range of 4000–500 cm−1 with a resolution
of 4 cm−1.

2.4.2.Field emission scanning electron microscopy (FE-SEM)

FE-SEM analysis was used to examine morphological properties of
LWP based film samples by studying the surface and cross section im-
ages of films, carried out by scanning electron microscope (SIGMA
VP, ZEISS Co., Germany) after gold coating of samples (DST1,
Nanostruc- tured Coating Co., Tehran, Iran).

2.4.3. Differential scanning calorimetry (DSC)

Differential scanning calorimetry (DSC) analysis was done to
inves- tigate thermal properties of LWP based films containing CNF
and SEO by using DSC system (DSC400 SANAF, Iran). The glass
transition tempera- ture (Tg) and melting temperature (Tm) of
samples were recorded by

Table 1
The fabricated LWP-based bioactive film samples.

Samples LWP (% w/v) CNF (% w/v) SEO (% w/v)

LWP/CNF0%/SEO0% 3 0 0
LWP/CNF0%/SEO1.5% 3 0 1.5
LWP/CNF0%/SEO3% 3 0 3
LWP/CNF3%/SEO0% 3 3 0
LWP/CNF3%/SEO1.5% 3 3 1.5
LWP/CNF3%/SEO3% 3 3 3
LWP/CNF6%/SEO0% 3 6 0

35
placing 20± 5 mg of each film sample in DSC sample pan and
scanned by increasing the temperature over the range of −100 to
250 °C with a heating rate of 10 °C·min−1. An empty pan was used as
a reference.

2.4.4. X-ray diffraction (XRD) analyses

The XRD analysis of the LWP based film samples were assisted
using the X-ray diffractometer (Kristalloflex D500, Siemens, Germany)
oper- ating at 40 kV and 40 mA, within the diffraction angular (2θ)
range of 5°–40° at room temperature that produced Cu Kα radiation
with wave- length of 0.154 nm for XRD analysis.

2.4.5.Mechanical properties
The mechanical properties of film samples including ultimate
tensile strength (UTS) and elongation at break (EB) were assessed
by Tensile Analyzer (DBBP-20, Bongshin, Korea) according to the
method proposed by Zabihollahi et al. [14]. The film samples were cut
into dumbbell shape with dimension of 8 × 0.5 cm as well as
crosshead speed and initial grip spacing were set to 10 mm·min−1 and
50 mm, respectively [14].

Fig. 1. Fourier transforms infrared (FT-IR) spectra of LWP/CNF0%/SEO0%,

2.4.6. Water vapor permeability (WVP)
LWP/CNF0%/ SEO3%, LWP/CNF6%/SEO0 and LWP/CNF6%/SEO3%
The WVP of the LWP based film samples was measured gravimetri- nanobiocomposite film samples. LWP: lemon waste powder, CNF: cellulose nanofiber,
cally according to the ASTM E96-05 [21] standard method. In this re- SEO: savory essential oil.
gard, glass vials used for this experiment were filled with dried
anhydrous CaSO4 to reach the vials inside RH to 0% and sealed with
film samples and then each vials placed in a desiccator containing
and proteins, thus spectra revealed the presence of different functional
K2SO4 solution to maintain the RH of 97% at 25 °C. The vials were
groups of pectin include carboxyl, carboxylates, methylene, alcohol, es-
weighed periodically every 24 h using a digital balance (AND, Model
ters and ketones along with other groups in reactions [22]. Spectrum of
HR 200, Tokyo, Japan) by accuracy of ±0.0001 g. Consequently, the
LWP/CNF0%/SEO0% film sample exhibited different specified peaks.
slope (changes of weight versus time) was calculated by a linear regres-
The broad absorption band around 3302 cm−1 corresponded to the
sion [21]. The water vapor transmission rate (WVTR) and water vapor
N\\H groups and free and intermolecular stretching vibration bonds
permeability (WVP) of film samples were calculated as:
of -OH groups related to the pectin monomers as well as asymmetric
and sym-
WVTR ¼ Curve slope metric stretching vibrations associated with the H2O [5,22]. The
Film area ð1Þ
peak around 2928 cm−1 referred to the C\\H stretching vibrations
of the methylene and methyl groups in polymer chains and methyl
Thickness × ester of galacturonic acid in pectin structure, respectively [5,22].
WVP ¼ WVTR Pressure ð2Þ
difference Moreover the band situated at the wavenumber of 1714 cm−1
represented symmetric stretching vibrations of ester carbonyl
(COOCH3) groups [23]. Further- more the peak around 1714 cm−1
2.4.7. Antibacterial activity wavenumber could be ascribed by the existence of ester functional
group, related to the natural lemon es-
The antimicrobial effect of LWP based films was carried out by using The FT-IR spectra of LWP/CNF0%/SEO0%, LWP/CNF0%/SEO3%, LWP/
agar disc diffusion method against five food-borne pathogenic bacteria CNF6%/SEO0 % and LWP/CNF6%/SEO3% film samples are presented in
(S. enterica, P. aeruginosa, L. monocytogenes, S. aureus, and E. coli). Fig. 1. The main component of LWP is pectin with combination of sugars
The films were cut to form of 7 mm diameter discs and placed on the
surface of Mueller Hinton agar plates, which prepared by cultured
suspensions containing 1.5 × 108 CFU/mL bacteria on surface of
prepared Mueller Hinton Agar plate. Subsequently, plates were
incubated at 37 °C for 24 h and the diameter of the inhibition zone
around the film samples were measured by the caliper (AOS Digimatic
Caliper, Mitutoyo, U.S.A.) in triplicate [15].

2.5. Statistical analysis

Data analysis was carried out based on one-way analysis of variance

(ANOVA) using IBM SPSS Statistics 26 (IBM Corporation, Armonk,
NY, USA). Additionally, the Duncan's mean comparison tests were per-
formed at 5% significant level. All the analyses were performed in
three replicates and data presented as mean ± SD.

3. Results and discussion

3.1. Fourier transforms infrared (FT-IR) spectroscopy

sential oil existence in LWP and also could be related to
polyphenol compounds that are naturally conjugated to
polysaccharides and ap- peared as functional groups such as esters
[5,24,25]. In addition, the peak around 1323 cm−1 attributed to
the existence of amide III vibra- tions bonds caused by C\\N and
N\\H groups [15]. Some peaks are ap- peared around 1212 and
1019 cm−1 in all samples containing pectin, such as fruit puree,
that ascribed to the characteristics of saccharides and attributed to
the carboxylic acids C\\O and C\\C groups of the pec-
tin ring structure, respectively [3,23,25–27]. Additionally, a small
peak around 888 cm−1 assigned to C\\H stretching vibrations
[27,28] and the peak around 779 cm−1 is associated with glycerol
absorption bands [15,29]. Spectrum of the film containing 3% SEO
represented no new absorption peaks; however, the amplitude and
intensity of their
specific peaks had slight differences compared to control film. In this
re- gard, decrease in the range and intensity of the peaks wavenumber
in- dicates a reduction in the number of free groups and also
associated with the formation of few interactions that are in line with
previous studies [5,30]. The important changes caused by inclusion of
SEO in LWP/CNF0%/SEO3% film sample were as follow: 1) the
absorption peak around 3302 cm−1 got more flattened and shifted
to the 3278 cm−1 wavenumber that indicated binding interactions
between SEO and pectin, which decreased free hydroxyl groups [5];
2) peaks around 2928, 1212, 888 and 779 cm −1 shifted slightly to the
lower wavenumber; and 3) peak intensities in the wavenumbers were
de- creased compared to control sample that indicated the slight
chemical interactions of phenolic compounds of SEO with the
functional groups
of LWP [5,28]. However spectrum of LWP/CNF6%/SEO0% film content of free hy- droxyl groups in CNF and its competition with
sample represented appearance of new peak around 1597 cm −1 that polymer matrix to cause interactions. In this regard, inclusion of CNF at
allocated to the asymmetric stretching vibrations of the carboxylate high concentration (6%) can weaken the interface bonds between
ion (–COO) [23,26]. Also, a slight shift of peak around 3302 cm −1 to CNF and pectin and
lower wavenumbers by the addition of CNF was corresponded to the
forma- tion of hydrogen bonds between CNF and pectin of LWP based
film. In
addition, incorporating 6% CNF shifted the peaks around 2928 and
1714 slightly to lower wavenumber and peaks around 779, 1212 and
1323 cm−1 to higher wavenumbers as well as decrease the bonds inten-
sities which were reported with several authors [18,23,31]. It was ob-
served that in spectrum of LWP/CNF6%/SEO3% film sample, the peak
around 3302 cm−1 disappeared by incorporating the combination of
SEO and CNF due to the formation of hydrogen bonds, as a result of lu-
brication effect of SEO that can facilitate polymer chains rearrangement
and formation of new hydrogen bonds [30]. Moreover, the peaks
around 2928, 1714, 1019 and 779 cm −1 shifted to the lower wavenumber
and the peaks around 1597, 1323 and 1212 cm −1 shifted to the higher
wavenumber as well as fall in all peaks intensities that could be associ-
ated with the interactions occurred between LWP, CNF and SEO.

3.2. Field emission scanning electron microscopy (FE-SEM)

Microstructure of the film shows the arrangement of its components

and provides better understanding noticeable differences in biopolymer
film matrix. FE-SEM images of LWP/CNF0%/SEO0% film sample
indi- cated a rough and very porous surface (Fig. 2a and b). This
phenomenon is common in films produced from more than one
component, such as films made from fruit puree due to the existence of
soluble and insolu- ble polymers in film forming solution during drying
condition [6,22]. The results were in line with findings of Viana et al.
about heteroge- neous and rough surfaces of films made from guava
and mango puree, due to the complex nature of the fruit purees [23].
However, the surface and cross section images of LWP/CNF0%/SEO3%
film sample (Fig. 2c and
d) presented the improvement of film compactness and indicated a
dense sheet like structure that suggested uniformly incorporation of
SEO in the film matrix and desirable compatibility between film com-
pounds. The SEM images also exhibited that SEO emulsion system was
stable and droplets were homogenously entrapped in continuous net-
work of film matrix which led to no collapse of emulsion and phase sep-
aration on the surface of the film during the drying. Moreover, this
could also be due to the liquid form of the SEO and its existence in the
void spaces of polymer chains that caused the surface of the film
smoother as a result of formation some interactions and reduction of
surface roughness [32]. Results were in accordance with previous
findings about improvement the uniformity of surface micrograph with
incorpo- rating citrus essential oil in fish skin gelatin film and Zataria
multiflora essential oil nanoemulsion in basil seed gum-based edible
film [33,34]. Furthermore, according to the cross-sectional
micrographs, micro- cavities were observed in the films containing 3%
SEO. In fact, these cav- ities were spaces filled with essential oil
droplets that homogenously disturbed in film matrix and evaporate
from the surface of the film as the film dried [35–37]. However, the
SEO droplets were localized inside the LWP film matrix and no
dispersion was noticeable on the surface of film. The similar results were
reported with Nisar et al. about integrated clove bud essential oil in
citrus pectin films, Shojaee-Aliabadi et al. about incorporating Satureja
hortensis L. essential oil in k-carrageenan film and Ojagh et al. about
incorporating cinnamon essential oil in chitosan film [2,5,38]. As can be
found in Fig. 2e and f, incorporation of CNF into the
LWP/CNF6%/SEO0% film sample led to limit the formation of deep
cracks compared to the control film. According to the surface
micrograph, cracks and porous structure were also shown by
incorporating 6% CNF in LWP based film sample, which could be due
to agglomeration of CNF. This phenomenon could be as a result of high
create hydrogen bonds between cellulose nanofibers that associated to the control film. Obtained results could be due to the
consequently led to break the film matrix near the CNF existence of SEO between the biopolymer chains that suggested good
aggregation area [39,40]. Al- though the agglomeration of CNF compatibility between SEO and CNF with LWP when used in
was evident in some parts of the film, but these masses are also combination.
distributed uniformly in all areas which indicated the compatibility
between CNF and polymer and suggested the production of a
composite with the compact structure at lower levels of CNF. In
this regard, Bagheri et al. concluded that surface image of the
gluten-carboxymethyl cellulose film contains 10% CNF showed
spherical components due to high CNF loading; however cross-
sectional images showed compact and cohesive structure [41]
which were similar with results reported with previous studies
[17,42–44]. Interestingly, according to the Fig. 2g and h, surface
micro- graph of LWP/CNF6%/SEO3% indicated reduction in crack
caused by ag- glomeration of CNF and improved film structure
compared to the film containing 6% CNF. These findings could be
associated with the appro- priate interactions between LWP, CNF
and SEO and a good compatibility could be obviously deduced from
the SEM micrographs.

3.3. X-ray diffraction (XRD) analyses

X-ray analysis was used to study crystalline structure of films. Fig. 3

shows the diffractograms of LWP/CNF0%/SEO0%,
LWP/CNF0%/SEO3%, LWP/CNF6%/SEO0% and LWP/CNF6%/SEO3%
film samples. The films based on LWP constitute of different
compounds like sugars and pro- teins, however pectin demonstrated
as the main compound. It has been reported that pure pectin
crystallinity was associated to specific peaks appeared at 2θ of 12.72°,
16.30°, 18.45°, 25.32° and 40.14° in X- ray analysis [5]. In this regard,
the diffractograms of LWP/CNF0%/ SEO0% film sample exhibited
two specific peaks near at 2θ of 11.52° and 12.72° and a large
amorphous area, which indicated the semi- crystalline and relatively
amorphous structure of the film obtained from lemon waste and also
presented the existence of some small crys- tal structures due to
hydrogen bonds between film compounds [19]. In accordance with
these results, Nisar et al. [5] reported the abroad peak and semi-
crystalline structure of pectin film that extracted from citrus. However,
the illustrated diffraction spectrum of LWP/CNF0%/SEO3%
suggested one specific peak near at 2θ of 13.8°, which indicated a
slight interaction of SEO with the components of the film and showed
differ- ences in the crystalline structure. Although, intermolecular
interactions between the components of the composite limited the
movement of the polymer chains, but the mobility of the chains
increased in presence of SEO [30]. In this regard, the same
observation has been reported with other authors [30,45] As can
found, pure CNF diffractogram showed three defined peaks near at 2θ
of 14.54°, 16.92° and 22.6° [16,42,46]. However, pattern of
LWP/CNF6%/SEO0% revealed two specific peaks near at 2θ of 13°
and 21.5° which was related to characteristic of cellu- lose
diffractogram in the CNF structure. Accordingly, inclusion of CNF,
caused disappearance of peak at 2θ of 11.52° and slight shifted the
peak at 2θ of 12.72° to higher degree (13°) as well as appearance of
new crystalline regions in the amorphous area at 2θ of 21.5°
[15,31,39,43]. The findings indicated the compatibility between the
CNF and LWP due to the existence of some nanofibers in void spaces
of the polymer and formation of some interactions. However, the ap-
pearance of new peak near at 2θ of 21.5°, which was close to pure
CNFs specific peak could suggest the agglomeration of some rigid
CNF between the polymer chains and limited the chains interactions
that could be associated with improperly dispersion of CNF in the film
structure and consequently appearance of pure CNF specific peak in
lower intensities in diffractogram [30,40]. Diffractogram pattern
pertaining to LWP/CNF 6%/SEO 3% film showed only one specific
peak at 2θ of 22.5°, which corresponded to the crystalline regions
created by the addition of CNF. The illustrated diffractogram of
composite pre- sented disappearing of basic crystalline peaks
Fig. 2. Field emission scanning electron microscopy (FE-SEM) images of surface and cross section of LWP/CNF0%/SEO0% (a and b), LWP/CNF0%/SEO3% (c and d), LWP/CNF6%/SEO0%
(e and
f) and LWP/CNF6%/SEO3% (g and h) nanobiocomposite film samples. LWP: lemon waste powder, CNF: cellulose nanofiber, SEO: savory essential oil.
 glass transition temperature and Tm: melting temperature.

Fig. 3. X-ray diffraction (XRD) patterns of LWP/CNF0%/SEO0%, LWP/CNF0%/SEO3%,

LWP/ CNF6%/SEO0% and LWP/CNF6%/SEO3% nanobiocomposite film samples. LWP:
lemon waste powder, CNF: cellulose nanofiber, SEO: savory essential oil.

3.4. Differential scanning calorimetry (DSC)

Thermal analysis of the films and composites as summarized in

Table 2 is important to investigate processing temperature limit of
food packaging. Thermograph of LWP/CNF0%/SEO0% film sample
showed a glass transition temperature (Tg) of −90 °C and melting tem-
perature (Tm) of 197.4 °C. The lower Tg could be related to the hydro-
philic nature and high soluble solids content (especially mono and
disaccharides) of the LWP based films that had plasticizing effect by en-
hancing polymer chains mobility [47]. Although, the sub-zero Tg of the
produced films indicated the relatively poor chemical stability, which
was due to the high molecular mobility and consequently the high reac-
tivity of these compounds but low Tg value presented the very desirable
flexibility of the film at refrigeration temperatures [5]. According to the
thermographs, incorporating 3% SEO individually could enhance the Tg
value of the film up to −89.5 °C, which was associated to homogeneous
distribution of SEO in LWP based film and a slight improvement in the
barrier properties of LWP/CNF0%/SEO3% film sample. This
phenomenon could also be owing to high molecular weight and
crosslinking forma- tion between polyphenol compounds of SEO and
LWP based film sam- ple [5,48]. Additionally, the reasons of increased
Tg value by incorporating CNF in to LWP/CNF6%/SEO0% film
sample could be

Table 2
Thermal properties of LWP-based bioactive film samples.

Samples Tg (°C) Tm (°C)

LWP/CNF0%/SEO0% −90 197.4
LWP/CNF0%/SEO3% −89.5 169.6–197.4
LWP/CNF6%/SEO0% −88.3 194.4
LWP/CNF6%/SEO3% −86.9 194.8

LWP: lemon waste powder, CNF: cellulose nanofiber, SEO: savory essential oil, Tg:
summarized as follow: 1) the reaction of cellulose with water that in- larly, the same trend was reported about incorporating oleic acid in
cluded redistribution of water molecules in the film matrix and reduc- the films based on CMC [56] and essential oils in the films based on chi-
ing the plasticizing effect of water, 2) interactions between matrix tosan [57,58]. Furthermore, by increasing SEO loading concentration,
compounds and CNF, thereby reduced matrix mobility, and 3) the EB value of the film increased (p < 0.05); this result can be related
increased crystallinity due to the addition of CNF and subsequently to the plasticizing effect of SEO bioactive compounds on polymer
limited film matrix mobility [5,12,49]. Additionally, Pelissari et al.
reported high cor- relation of glycerol with cellulose that caused
glycerol to move from high polymer zones to CNF containing parts of
the film and reduced the glycerol plasticizing effect that led to increase
Tg value of CNF incor- porated films [47]. According to the results,
thermograph of LWP/ CNF0%/SEO3% film sample presented two
Tm indicating the Tm values of the SEO (169.6 °C) and the film
biopolymer base (197 °C). However, there was an overlap between the
two Tm peaks, which could be due to the low cross-linking formation
between SEO and pectin content of the lemon waste [22].
Furthermore, incorporating 6% CNF significantly de- creased Tm
value of the film from 197.4 °C to 194.8 °C, which was re- lated to the
melting of crystalline regions. This could also associate with non-
uniformly distribution of CNF in film matrix and decrease in the
nucleation capacity of CNF as a result of agglomeration that led to
degradation of composite structure [18]. However, Tg of
LWP/CNF6%/ SEO3% film samples increased from −90 °C in
control film up to
−86.9 °C, which indicated the improvement of the barrier
properties
of the film. Similar findings regarding the effect of CNF on
enhancement of Tg value of films has been reported [12,49–51].
Increased Tg value as well as decreased Tm indicated effectiveness
of CNF on the reduced mo- bility of the chains in amorphous
regions. However, the strength of the bonds between CNF and SEO
with biopolymers in amorphous regions was not high enough to
cause a significant enhancement in intermolec- ular density.
Therefore, with increasing amorphous regions and decom- position
of compacted crystalline regions due to agglomeration of CNF, the
Tg and Tm values of the film changed in contrary. As a result,
incor- porating high concentration of CNF (6%) had reduced the
compactness of the film structure and changed the orientation of
the chains in the biopolymer matrix. Results were in line with
previous studies [17,18,39]. Furthermore, reduction of Tm by
incorporating CNF could be the result of morphological alterations
and might be associated with the reduction of pectin crystal sizes
due to the formation of cellu- lose network in the matrix;
therefore, the growth of pectin crystals was limited. Similar results
reported by Samir et al. about the decrease of polyoxyethylene
films Tm by incorporating CNC [52].

3.5. Mechanical properties

The effect of incorporating SEO and CNF on mechanical properties

of LWP based film is summarized in Table 3. According to the results
of tensile test, LWP/CNF0%/SEO0% film sample had UTS and EB
values of
7.64 ± 0.08 MPa and 24.24 ± 1.01%, respectively. In this regard,
studies on the production of fruit puree films had shown less tensile
strength compared to the films made from pure biopolymers, which
was owing to the dilution of the matrix by the fruit components and
the plasticizing effects of low molecular weight sugars and fruit acids
[53,54]. As can be noticed, incorporating SEO significantly (p < 0.05)
increased the UTS and EB values of the film samples simultaneously,
compared to the con- trol film and the effect of individual
incorporating 3% SEO on increasing the UTS of LWP/CNF0%/SEO3%
film samples (9.59 ± 0.25 MPa) was more than 1.5% SEO (8.46 ±
0.15 MPa). In consistent with the results of SEM image, these findings
could be attributed to the high reactivity of SEO and cross-links
formation between hydroxyl groups of the LWP film constituents such
as carbohydrates with phenolic compounds of SEO that led to
reduction of void spaces between polysaccharide chains and
improvement of uniform and continuous structure [35,55]. Simi-
M. Soofi, A. Alizadeh, H. Hamishehkar et International Journal of Biological Macromolecules 169 (2021) 352–
al. 361

Table 3
Mechanical and WVP properties of LWP-based bioactive film samples.

Samples EB (%) UTS (MPa) WVP (×10−7 gPa−1 h−1 m−1)

LWP/CNF0%/SEO0% 24.24 ± 1.01c 7.64 ± 0.08i 2.46 ± 0.14a
LWP/CNF0%/SEO1.5% 25.86 ± 0.90b 8.46 ± 0.15 h 2.10 ± 0.13b
LWP/CNF0%/SEO3% 27.67 ± 0.46a 9.59 ± 0.25 g 1.79 ± 0.06c
LWP/CNF3%/SEO0% 13.59 ± 0.82i 13.63 ± 0.37c 0.90 ± 0.10 g
LWP/CNF3%/SEO1.5% 14.98 ± 0.95 h 14.27 ± 0.16b 0.57 ± 0.11 h
LWP/CNF3%/SEO3% 16.64 ± 0.38 g 15.22 ± 0.29a 0.21 ± 0.04i
LWP/CNF6%/SEO0% 17.89 ± 0.56f 11.10 ± 0.67f 1.61 ± 0.02d
LWP/CNF6%/SEO1.5% 19.68 ± 0.72e 11.81 ± 0.23e 1.40 ± 0.08e
LWP/CNF6%/SEO3% 21.59 ± 0.36d 12.90 ± 0.16d 1.12 ± 0.09f

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); LWP: lemon waste powder,
CNF: cellulose nanofiber, SEO: savory essential oil, EB: elongation at break, UTS: ultimate tensile strength, WVP: water vapor permeability.

which can enhance extensibility of the films [5,20,30]. In general, the concentrations of SEO and CNF. According to the results, the highest and
SEO reactive compounds were able to form cross-links in LWP film ma- the lowest WVP values were related to the LWP/CNF0%/SEO0% (2.46 ±
trix and its lipophilic compounds played plasticizing effect in some re- 0.14 × 10−7 gPa−1 h−1 m−1) and LWP/CNF3%/SEO3% (0.20 ±
gions. This is the reason for simultaneously increased UTS and EB of
LWP film after incorporation of SEO. According to the obtained results,
although incorporating 3% CNF revealed significantly (p < 0.05) in-
crease in UTS value of the LWP/CNF3%/SEO0% film sample from
7.64 ± 0.08 MPa for the control film up to 13.63 ± 0.37 MPa,
noticeable reduction of UTS values to 11.10 ± 0.67 MPa was shown
by elevating CNF loading concentration up to 6%. In general,
increasing the UTS value of the films by incorporating of 3% CNF
can summarized as follow:
1) similar chemical structure of cellulose and pectin led to a strong
in- teraction between the functional groups of the pectin molecule
(includ- ing carboxyl and hydroxyl groups) and cellulose through
the intermolecular hydrogen reaction and ionic interaction
[15,44,47,59],
2) reduction of moisture content in CNF containing film samples
that led to decrease the plasticizing effect of water, 3) existence of
CNF in void spaces between polymer chains, which led to reduction
in chain mobility and associated with the development of crystalline
regions and the high mechanical strength of CNF in comparison
with the bio- polymer matrix, and 4) rigidity and strength of CNF
that is known as a better reinforcement agent in film matrix
[4,15,41,60]. However, EB values of LWP based films decreased (p
< 0.05) with incorporating CNF that could be associated with
increasing the crystallinity and reduc- ing the mobility of the
polymer chains [16,17]. Our findings were com- parable with the
findings of Chaichi et al. who noticed the increased UTS and
decreased EB by adding CNF (5%) into pectin based films [4].
More- over, the same results were observed in previous studies
[13,15–17,61]. The UTS value of the film samples containing 6%
CNF declined notice- ably compared to films containing 3% CNF.
This could be indicated the non-uniform dispersion and
agglomeration of CNF when incorporating at high concentration and
the reduction of interactions between the film matrix and CNF
which were in consistent with FE-SEM results. Therefore,
aggregation of CNF at high loading concentration had led to the
formation of non-uniform regions within the matrix and weakened
stress transfer from the polymer matrix that caused failure of films
at low strains. The similar trend have been reported for the
addition of CNC in polyhydroxybutyrate nanocomposites [39],
pectin [4], agar- based nanocomposite [43] and inclusion of high
concentration of CNF in alginate film [18]. However, the negative
effect of CNF aggregation on UTS and EB values of film samples
could be compensated by inclu- sion of SEO due to the plasticizing
effect of oil which was in line with FE-SEM results.

3.6. Water vapor permeability (WVP)

Table 3 shows WVP of lemon waste based films containing different

35
M. Soofi, A. Alizadeh, H. Hamishehkar et International Journal of Biological Macromolecules 169 (2021) 352–
al. 361

Table 3
0.04 × 10−7 gPa−1 h−1 m−1), respectively. LWP/CNF0%/SEO0% film
sample presented the highest WVP value as a result of carbohydrate
na- ture of the film that had high hydrophilic groups in the polymer
chains and consequently low resistance to water vapor [23,29,62].
However, WVP value of SEO incorporated films significantly (p <
0.05) decreased by enhancing the concentration of SEO. This may be
due to the fact that water vapor is transferred from the hydrophilic
parts of the film, so in- clusion of SEO as a hydrophobic agent can
interfere with the structure of the polysaccharide film network and
decrease WVP value [5]. Further- more, SEO incorporation was able
to reduce the solubility of the film and therefore had improved the
inhibitory properties to water vapor [2,5]. As can be found, WVP of
LWP/CNF0%/SEO3% film sample de- creased significantly(p < 0.05)
and reached to 1.79 ± 0.06 × 10−7 gPa−1- h−1 m−1 which could be also
associated with increasing diffusion path of water vapor due to the
presence of oil globules through the film structure [63]. Additionally
incorporating SEO in LWP based film re- sulted to the formation of
some intra molecular hydrogen and covalent bonds between phenolic
compounds of SEO and the active groups of pectin in the film matrix
as well as decrease free hydroxyl groups (OH) of the film to bond
with water and suppressed the transfer of water vapor through the
film [2,64]. Moreover, the results were in con- sistent with SEM
images and FTIR spectra that presented smooth and compact
structure as well as decrease in free hydroxyl groups, respec- tively by
incorporating SEO in film samples. The findings are also in line with
the results of Tongnuanchan et al. about decrease of WVP value by
incorporating citrus essential oils into fish skin gelatin film and
Sánchez-González et al. about the effect of inclusion bergamot, lemon
and tea tree essential oils in reduction WVP value of films based on
hydroxylpropyl methylcellulose and chitosan films [34,65]. A similar
trend was also seen in different films based on alginate [66], chi-
tosan [38], whey protein concentrate [67] and carrageenan [2] with in-
corporating different types of essential oils. According to the obtained
results, by incorporating 3% CNF in the LWP based films,
significantly
(p < 0.05) fall in WVP trend of LWP/CNF3%/SEO0% film sample
(0.85 ± 0.10 × 10−7 gPa−1 h−1 m−1) had been realized compared to
the control film. In this regard Viana et al. reported the noticeable
effect of bacterial cellulose nanofiber on WVP reduction of pectin
based film samples [23], which were in consist with effect of CNF
inclusion into the agar based film and bacterial cellulose nanofiber
into the buckwheat protein based film [31,42]. Decrease of WVP by
inclusion of 3% CNF could be ascribed as follow: 1) formation of
hydrogen bonds between the matrix and the hydroxyl groups of
CNF, which led to the decrease of free hydroxyl groups, 2) water
insoluble properties of CNF that caused formation of long and
tortuosity (zigzag) pathway and acts as an obstacle to extent
diffusion path of water vapor thorough the film thickness, 3) filling
the voids between the biopolymer chains, which re- duced the
mobility of the chains and consequently decrease the pene- tration
rate of water molecules, and 4) high crystalline properties of CNF
and low permeability and hydrophilicity of this compound com-
pared to the biopolymer matrix [4,28,41]. However, the results
revealed

35
M. Soofi, A. Alizadeh, H. Hamishehkar et International Journal of Biological Macromolecules 169 (2021) 352–
al. 361

Table 4
Antimicrobial activity of LWP based films containing CNF and SEO.

Film samples Inhibition zone (mm) of films

P. aeroginosa S. enterica E. coli L. monocytogenes S. aureus

LWP/CNF0%/SEO0% 6.33 ± 0.57c 5.33 ± 0.57c 6.33 ± 1.15c 11.33 ± 2.08c 13.33 ±
1.52c
LWP/CNF0%/SEO1.5% 10.33 ± 1.15b 7.66 ± 0.57b 12.33 ± 0.57b 18.00 ± 1.00b 19.33 ± 2.08b
LWP/CNF0%/SEO3% 13.33 ± 0.57a 11.00 ± 1.00a 17.33 ± 2.08a 23.00 ± 1.00a 25.00 ± 1.73a
LWP/CNF3%/SEO0% 6.00 ± 1.00c 5.00 ± 1.00c 6.00 ± 1.00c 11.66 ± 0.57c 13.00 ±
1.00c
LWP/CNF3%/SEO1.5% 9.66 ± 1.52b 8.00 ± 1.73b 11.66 ± 2.30b 17.66 ± 2.30b 20.00 ± 2.00b
LWP/CNF3%/SEO3% 13.00 ± 1.73a 11.33 ± 0.57a 17.66 ± 0.57a 23.00 ± 1.73a 25.33 ± 2.08a
LWP/CNF6%/SEO0% 6.66 ± 1.52c 5.33 ± 0.57c 5.66 ± 1.52c 10.66 ± 1.52c 12.33 ± 0.57c
LWP/CNF6%/SEO1.5% 9.66 ± 0.57b 7.33 ± 0.57b 12.33 ± 1.15b 17.66 ± 0.57b 20.00 ±
1.73b
LWP/CNF6%/SEO3% 13.33 ± 1.15a 10.33 ± 0.57a 16.66 ± 1.52a 23.66 ± 1.52a 26.00 ±
1.00a
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); LWP: lemon waste powder,
CNF: cellulose nanofiber, SEO: savory essential oil.

the increase of WVP value by incorporating higher concentration of their antibacterial properties through various mechanisms as follow: a)
CNF in LWP/CNF6%/SEO0% film sample (1.61 ± 0.02 × 10−7 gPa−1 h−1 interact with bacterial cell membrane, b) attack the phos- pholipid
m−1) compared to LWP/CNF3%/SEO0% film. This could be due to the bilayer of the cell membrane, c) coagulate the cytoplasm,
CNF ag- glomeration in some parts of the film at higher concentration d) degrade proteins and lipids, e) disrupt the enzyme system,
(6%) of CNF that led to formation voids and cracks in the composite
structure as could be seen in SEM images. Similar observations were
reported for pectin and agar based films incorporated with high
amounts of cel- lulose nanocrystals [4,44]. Mahardika et al. also
reported the aggrega- tion of CNF in starch based nanobiocomposites
that led to increase the WVP value by elevating CNF loading [19].

3.7. Antibacterial activity

Effect of incorporating CNF and SEO on to the antimicrobial

properties of LWP based nanobiocomposite films against Gram-
positive (S. aureus and L. monocytogenes) and Gram-negative
bacteria (P. aeruginosa, E. coli and S. enterica) are shown in Table 4.
According to the obtained results, LWP/CEO0%/SEO0% film sample
had a signifi- cant (p < 0.05) inhibitory effect on all tested bacteria.
These results ver- ified the antibacterial potential of the LWP based film
due to the low pH of the film solution and the natural active
ingredients in the lemon waste, such as natural lemon essential oil
(citral), quercetin, flavonoids and phenolic acids [68]. Similarly, such
results were obtained by Viuda Martos et al. who presented a high
antimicrobial effect of lemon essential oil against food spoilage bacteria
[69]. However, the results suggested that no significant (p > 0.05)
inhibition zone was observed by incorporating of CNF compared to the
control film against all tested bacteria, which were in line with previous
studies [14,15]. Although, incorporating of SEO had a significant (p <
0.05) inhibitory effect against evaluated bacteria, but the diameter of
inhibition zones increased significantly (p < 0.05) by ele- vated the SEO
loading concentration from 1.5 to 3% in all samples. Obvi- ously, the
findings could be due to major inhibitory components of SEO such as
carvacrol, g-terpinene, p-Cymene and synergistic effect between carvacrol
and p-Cymene that had a wide spectrum of antimicrobial prop- erty
[2,20]. Similar result was reported with Dashipour et al. about the high
antimicrobial effect of carvacrol on S. aureus, B. cereus, E. coli,
P. aeruginosa and S. typhimurium [35]. According to the results,
Gram- positive bacteria (L. monocytogenes and B. cereus) presented the
highest susceptibility in the films containing SEO and diameters of
inhibition zone were significantly (p < 0.05) increased followed
the order of
S. enterica < P. aeruginosa < E. coli < L. monocytogenes < S.
aureus. This phenomenon could be due to the high resistance of
Gram-negative bacte- ria that was associated to the
lipopolysaccharide wall surrounded the peptidoglycan cell wall and
consequently prevent active components of SEO from reaching the
cytoplasmic membrane [2,20]. Essential oils extracted from plant
sources are rich in biologically active compounds and presented

35
M. Soofi, A. Alizadeh, H. Hamishehkar et International Journal of Biological Macromolecules 169 (2021) 352–
al. compromised of bacterial
f) genetic material and g) produced 361

hydroperoxidase from oxidation of unsaturated fatty acids [35,70]. As

can found, SEO had noticeable (p < 0.05) inhibitory effect to all
examined bacteria, however the highest and the lowest inhibition effect of
incorpo- rating SEO into LWP/CNF0%/SEO3% based film was related
to S. aureus (25.00 ± 1.73 mm) and S. enterica (11.00 ± 1.00 mm)
respectively. In this regard, Skocibusic et al. observed the potential of
antibacterial activity of SEO against 13 bacteria and 9 fungi and
concluded that Gram-positive bacteria were more sensitive than Gram
negative ones [71]. Atef et al. also suggested that Gram-positive bacteria
were more sensitive to antimicro- bial compounds of SEO compared to
Gram-negative bacteria, which could be due to their cell wall
lipopolysaccharides that prevent the penetration of the active
compounds of SEO through the cytoplasmic membrane [20].
Furthermore, Shojaee Aliabadi et al. reported that S. aureus was the most
sensitive bacteria to antimicrobial compounds of SEO [2].

4. Conclusion

In this study, for the first time, LWP based nanobiocomposite

were successfully fabricated. SEO as antimicrobial agent and CNF
as nanoreinforcement agent were incorporated into LWP based
films. The control film showed the highest WVP and elongation at
break and the lowest tensile strength which was in accordance with
SEM images. The proper distribution of CNF and SEO as well as
interactions between them and LWP polymers are related to
microstructure, mechanical and WVP characteristics of
nanobiocomposite film samples. The results re- vealed that LWP
based film could act as carrier for additives and the for- mation of
interactions between film matrix, CNF and SEO, indicated their
good compatibility. The SEM images confirmed the aggregation of
CNF at high concentration (6%) and revealed the effect of SEO in
im- prove the surface structure of the film. The XRD results
revealed that CNF in the film matrix retained its crystalline form.
In addition, the thermal properties, and crystallinity of CNF and
SEO incorporated LWP-based films were acceptable. Neat LWP
film showed drastically an- timicrobial effect which increment
significantly at higher concentration of SEO. It was found that SEO
incorporated films were more effective against Gram positive
bacteria especially S. aureus, while CNF loaded films had shown
no significant antimicrobial effect against tested bacte- ria. As can
be concluded, development of environmentally friendly
nanobiocomposite based on LWP, could open a new horizon for
the challenges on disposal of lemon waste and has a potential to
the re- placement of conventional synthetic plastics. Nevertheless,
further in- vestigations are needed to evaluate the films
performance on different food systems.

CRediT authorship contribution statement

Mitra Soofi: Investigation, Formal analysis, Writing – original

draft. Ainaz Alizadeh: Supervision, Writing – original draft. Hamed
Hamishehkar: Supervision, Data curation, Writing – original draft.

36
Hadi Almasi: Supervision, Investigation, Formal analysis. Leila [17] A.A. Oun, J.W. Rhim, Preparation and characterization of sodium carboxymethyl cel-
lulose/cotton linter cellulose nanofibril composite films, Carbohydr. Polym. 127 (2015)
Roufegarinejad: Supervision, Formal analysis. 101–109, https://doi.org/10.1016/j.carbpol.2015.03.073.

Declaration of competing interest

The authors wish to confirm that there are no known conflicts of

in- terest associated with this publication and there has been no
significant financial support for this work that could have influenced
its outcome.

Acknowledgment

The authors gratefully thanks the supports of the Islamic Azad

University, Tabriz Branch, Tabriz, Iran. This research did not
receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors.

References

[1] C.G. Otoni, B.D. Lodi, M.V. Lorevice, R.C. Leitão, M.D. Ferreira, M.R.d. Moura,
L.H.C. Mattoso, Optimized and scaled-up production of cellulose-reinforced
biodegradable composite films made up of carrot processing waste, Ind. Crop. Prod.
121 (2018) 66–72, https://doi.org/10.1016/j.indcrop.2018.05.003.
[2] S. Shojaee-Aliabadi, H. Hosseini, M.A. Mohammadifar, A. Mohammadi, M.
Ghasemlou, S.M. Ojagh, S.M. Hosseini, R. Khaksar, Characterization of antioxidant-
antimicrobial κ-carrageenan films containing Satureja hortensis essential oil, Int. J.
Biol. Macromol. 52 (2013) 116–124, https://doi.org/10.1016/j.ijbiomac.2012.08.
026.
[3] K.K. Dash, N.A. Ali, D. Das, D. Mohanta, Thorough evaluation of sweet potato starch
and lemon-waste pectin based-edible films with nano-titania inclusions for food
packaging applications, Int. J. Biol. Macromol. 139 (2019) 449–458, https://doi.org/
10.1016/j.phrs.2020.104743.
[4] M. Chaichi, M. Hashemi, F. Badii, A. Mohammadi, Preparation and characterization
of a novel bionanocomposite edible film based on pectin and crystalline
nanocellulose, Carbohydr. Polym. 157 (2017) 167–175, https://doi.org/10.1016/j.
carbpol.2016.09.062.
[5] T. Nisar, Z.C. Wang, X. Yang, Y. Tian, M. Iqbal, Y. Guo, Characterization of citrus
pectin films integrated with clove bud essential oil: physical, thermal, barrier,
antioxidant and antibacterial properties, Int. J. Biol. Macromol. 106 (2018) 670–680,
https://doi. org/10.1016/j.ijbiomac.2017.08.068.
[6] V. Bátori, M. Jabbari, D. Åkesson, P.R. Lennartsson, M.J. Taherzadeh, A. Zamani, Pro-
duction of pectin-cellulose biofilms: a new approach for citrus waste recycling,
Int. J. Polym. Sci. (2017) 1–9, https://doi.org/10.1155/2017/9732329.
[7] A.M. Balu, V. Budarin, P.S. Shuttleworth, L.A. Pfaltzgraff, K. Waldron, R. Luque,
J.H. Clark, Valorisation of orange peel residues: waste to biochemicals and
nanoporous materials, ChemSusChem 5 (2012) 1694–1697,
https://doi.org/10.1002/cssc. 201200381.
[8] H. Kowalska, K. Czajkowska, J. Cichowska, A. Lenart, What’s new in biopotential of
fruit and vegetable by-products applied in the food processing industry, Trends
Food Sci. Technol. 67 (2017) 150–159, https://doi.org/10.1016/j.tifs.2017.06.016.
[9] FAO, Food and Agriculture Organization of United Nation Cropping Database,
http:// www.fao.org/faostat/en/#data/QC 2018 accessed July 10, 2020.
[10] P.P. Borah, P. Das, L.S. Badwaik, Ultrasound treated potato peel and sweet lime
pom- ace based biopolymer film development, Ultrason. Sonochem. 36 (2017)
11–19, https://doi.org/10.1016/j.ultsonch.2016.11.010.
[11] U.S. Schmidt, K. Schmidt, T. Kurz, H.U. Endreß, H.P. Schuchmann, Pectins of
different origin and their performance in forming and stabilizing oil-in-water-
emulsions, Food Hydrocoll. 46 (2015) 59–66,
https://doi.org/10.1016/j.foodhyd.2014.12.012.
[12] H.M.C. Azeredo, L.H.C. Mattoso, D. Wood, T.G. Williams, R.J. Avena-Bustillos, T.H.
McHugh, Nanocomposite edible films from mango puree reinforced with cellulose
nanofibers, J. Food Sci. 74 (2009) 31–35, https://doi.org/10.1111/j.1750-3841.
2009.01186.x.
[13] H.M.C. Azeredo, L.H.C. Mattoso, R.J. Avena-Bustillos, G.C. Filho, M.L. Munford, D.
Wood, T.H. McHugh, Nanocellulose reinforced chitosan composite films as affected
by nanofiller loading and plasticizer content, J. Food Sci. 75 (2010) N1–N7.
[14] N. Zabihollahi, A. Alizadeh, H. Almasi, S. Hanifian, H. Hamishekar, Development
and characterization of carboxymethyl cellulose based probiotic nanocomposite film
containing cellulose nanofiber and inulin for chicken fillet shelf life extension, Int.
J. Biol. Macromol. 160 (2020) 409–417, https://doi.org/10.1016/j.scitotenv.2019.
136126.
[15] N. Karimi, A. Alizadeh, H. Almasi, S. Hanifian, Preparation and characterization
of whey protein isolate/polydextrose-based nanocomposite film incorporated
with cellulose nanofiber and L. plantarum: a new probiotic active packaging
system, LWT - Food Sci. Technol. 121
(2020)https://doi.org/10.1016/j.lwt.2019.108978.
[16] S.C.M. Fernandes, L. Oliveira, C.S.R. Freire, A.J.D. Silvestre, C.P. Neto, A. Gandini,
J. Desbriéres, Novel transparent nanocomposite films based on chitosan and
bacterial cellulose, Green Chem. 11 (2009) 2023–2029,
https://doi.org/10.1039/b919112g.
[18] B. Deepa, E. Abraham, L.A. Pothan, N. Cordeiro, M. Faria, S. Thomas, cellulose/cellulose
Biodegradable nanocomposite films based on sodium alginate and cellulose
nanofibrils, Materials (Basel) 9 (2016) 1–11, https://doi.org/10.3390/ma9010050.
[19] M. Mahardika, H. Abral, A. Kasim, S. Arief, F. Hafizulhaq, M. Asrofi, F. Ha,
Properties of cellulose nanofiber/bengkoang starch bionanocomposites: effect of
fiber loading, LWT - Food Sci. Technol. 116 (2019) 108554.
[20] M. Atef, M. Rezaei, R. Behrooz, Characterization of physical, mechanical, and
antibac- terial properties of agar-cellulose bionanocomposite films incorporated
with savory essential oil, Food Hydrocoll. 45 (2015) 150 –157,
https://doi.org/10.1016/j.foodhyd. 2014.09.037.
[21] ASTM-E96, Standard Test Methods for Water Vapor Transmission of Materials,
ASTM Int, West Conshohocken, PA,
2005https://doi.org/10.1520/E0096_E0096M- 05.
[22] S. Tulamandi, V. Rangarajan, S.S.H. Rizvi, R.S. Singhal, S.K. Chattopadhyay, N.C.
Saha, A biodegradable and edible packaging film based on papaya puree, gelatin,
and defatted soy protein, Food Packag. Shelf Life 10 (2016) 60 –71,
https://doi.org/10. 1016/j.fpsl.2016.10.007.
[23] R.M. Viana, N.M.S.M. Sá, M.O. Barros, M.F. de Borges, H.M.C. Azeredo,
Nanofibrillated bacterial cellulose and pectin edible films added with fruit purees,
Carbohydr. Polym. 196 (2018) 27–32, https://doi.org/10.1016/j.carbpol.2018.05.017.
[24] P. Suppakul, R. Boonlert, W. Buaphet, P. Sonkaew, V. Luckanatinvong, Efficacy of
su- perior antioxidant Indian gooseberry extract-incorporated edible Indian
gooseberry puree/methylcellulose composite films on enhancing the shelf life of
roasted cashew nut, Food Control 69 (2016) 51–60,
https://doi.org/10.1016/j.foodcont. 2016.04.033.
[25] R.M.S. Andrade, M.S.L. Ferreira, É.C.B.A. Gonçalves, Development and characteriza-
tion of edible films based on fruit and vegetable residues, J. Food Sci. 81 (2016) E412 –
E418, https://doi.org/10.1111/1750-3841.13192.
[26] S. Šešlija, A. Nešić, J. Ružić, M. Kalagasidis Krušić, S. Veličković, R. Avolio, G.
Santagata, M. Malinconico, Edible blend films of pectin and poly(ethylene
glycol): preparation and physico-chemical evaluation, Food Hydrocoll. 77
(2018) 494–501, https://doi.org/10.1016/j.foodhyd.2017.10.027.
[27] M. Szymanska-Chargot, A. Zdunek, Use of FT-IR spectra and PCA to the bulk
charac- terization of cell wall residues of fruits and vegetables along a fraction
process, Food Biophys 8 (2013) 29–42, https://doi.org/10.1007/s11483-012-
9279-7.
[28] A. Orsuwan, S. Shankar, L.F. Wang, R. Sothornvit, J.W. Rhim, Preparation of
antimi- crobial agar/banana powder blend films reinforced with silver
nanoparticles, Food Hydrocoll. 60 (2016) 476–485,
https://doi.org/10.1016/j.foodhyd.2016.04.017.
[29] A. Sadeghi-Varkani, Z. Emam-Djomeh, G. Askari, Physicochemical and
microstruc- tural properties of a novel edible film synthesized from Balangu seed
mucilage, Int. J. Biol. Macromol. 108 (2018) 1110–1119,
https://doi.org/10.1016/j.ijbiomac. 2017.11.029.
[30] R. Zhang, W. Wang, H. Zhang, Y. Dai, H. Dong, H. Hou, Effects of hydrophobic
agents on the physicochemical properties of edible agar/maltodextrin films, Food
Hydrocoll. 88 (2019) 283–290, https://doi.org/10.1016/j.foodhyd.2018.10.008.
[31] X. Wang, N. Ullah, X. Sun, Y. Guo, L. Chen, Z. Li, X. Feng, Development and
character- ization of bacterial cellulose reinforced biocomposite films based on
protein from buckwheat distiller’s dried grains, Int. J. Biol. Macromol. 96 (2017)
353–360, https://doi.org/10.1016/j.ijbiomac.2016.11.106.
[32] L. Atarés, J. Bonilla, A. Chiralt, Characterization of sodium caseinate-based edible
films incorporated with cinnamon or ginger essential oils, J. Food Eng. 100 (2010)
678–687, https://doi.org/10.1016/j.jfoodeng.2010.05.018.
[33] H. Hashemi Gahruie, E. Ziaee, M.H. Eskandari, S.M.H. Hosseini, Characterization
of basil seed gum-based edible films incorporated with Zataria multiflora
essential oil nanoemulsion, Carbohydr. Polym. 166 (2017) 93–103,
https://doi.org/10.1016/ j.carbpol.2017.02.103.
[34] P. Tongnuanchan, S. Benjakul, T. Prodpran, Properties and antioxidant activity of
fish skin gelatin film incorporated with citrus essential oils, Food Chem. 134 (2012)
1571–1579, https://doi.org/10.1016/j.foodchem.2012.03.094.
[35] A. Dashipour, V. Razavilar, H. Hosseini, S. Shojaee-Aliabadi, J.B. German, K. Ghanati,
M. Khakpour, R. Khaksar, Antioxidant and antimicrobial carboxymethyl cellulose
films containing Zataria multiflora essential oil, Int. J. Biol. Macromol. 72 (2015)
606–613, https://doi.org/10.1016/j.ijbiomac.2014.09.006.
[36] P.J.P. Espitia, R.A. Batista, H.M.C. Azeredo, C.G. Otoni, Probiotics and their
potential applications in active edible films and coatings, Food Res. Int. 90 (2016)
42–52, https://doi.org/10.1016/j.foodres.2016.10.026.
[37] M. Jouki, F. Tabatabaei Yazdi, S.A. Mortazavi, A. Koocheki, Physical, barrier and anti-
oxidant properties of a novel plasticized edible film from quince seed mucilage, Int.
J. Biol. Macromol. 62 (2013) 500–507, https://doi.org/10.1016/j.ijbiomac.2013.09.
031.
[38] S.M. Ojagh, M. Rezaei, S.H. Razavi, S.M.H. Hosseini, Development and evaluation
of a novel biodegradable film made from chitosan and cinnamon essential oil with
low affinity toward water, Food Chem. 122 (2010) 161–166,
https://doi.org/10.1016/j. foodchem.2010.02.033.
[39] B. Zhang, C. Huang, H. Zhao, J. Wang, C. Yin, L. Zhang, Y. Zhao, Effects of
cellulose nanocrystals and cellulose nanofibers on the structure and properties
of polyhydroxybutyrate nanocomposites, Polymers (Basel) 11
(2019)https://doi.org/ 10.3390/polym11122063.
[40] X. Niu, Y. Liu, Y. Song, J. Han, H. Pan, Rosin modified cellulose nanofiber as a
reinforc- ing and co-antimicrobial agents in polylactic acid/chitosan composite film
for food packaging, Carbohydr. Polym. 183 (2018) 102–109,
https://doi.org/10.1016/j. carbpol.2017.11.079.
[41] V. Bagheri, B. Ghanbarzadeh, A. Ayaseh, A. Ostadrahimi, A. Ehsani, M.
Alizadeh- Sani, P.A. Adun, The optimization of physico-mechanical
properties of bionanocomposite films based on gluten/carboxymethyl
 nanofiber using response surface methodology, Polym. Test. 78 (2019) https://
doi.org/10.1016/j.polymertesting.2019.105989. [58] S.M. Ojagh, M. Rezaei, S.H. Razavi, S.M.H. Hosseini, Effect of chitosan coatings
[42] X. Wang, C. Guo, W. Hao, N. Ullah, L. Chen, Z. Li, X. Feng, Development and char- enriched with cinnamon oil on the quality of refrigerated rainbow trout, Food
acterization of agar-based edible films reinforced with nano-bacterial cellulose, Chem. 120 (2010) 193–198, https://doi.org/10.1016/j.foodchem.2009.10.006.
Int. J. Biol. Macromol. 118 (2018) 722–730, https://doi.org/10.1016/j.ijbiomac. [59] R.A. Carvalho, T.A. Santos, V.M. de Azevedo, P.H.C. Felix, M.V. Dias, S.V. Borges,
2018.06.089. Bio- nanocomposites for food packaging applications: effect of cellulose nanofibers
[43] M. Atef, M. Rezaei, R. Behrooz, Preparation and characterization agar-based nano- on morphological, mechanical, optical and barrier properties, Polym. Int. 67 (2018)
composite film reinforced by nanocrystalline cellulose, Int. J. Biol. Macromol. 70 386–392, https://doi.org/10.1002/pi.5518.
(2014) 537–544, https://doi.org/10.1016/j.ijbiomac.2014.07.013. [60] H.M.C.C. Azeredo, L.H.C. Mattoso, R.J. Avena-Bustillos, G.C. Filho, M.L. Munford, D.
[44] J.P. Reddy, J.W. Rhim, Characterization of bionanocomposite films prepared with Wood, T.H. McHugh, R.J. Avena-Bustillos, G.C. Filho, M.L. Munford, D. Wood, T.H.
agar and paper-mulberry pulp nanocellulose, Carbohydr. Polym. 110 (2014) 480– McHugh, Nanocellulose reinforced chitosan composite films as affected by
488, https://doi.org/10.1016/j.carbpol.2014.04.056. nanofiller loading and plasticizer content, J. Food Sci. 75 (2010) N1–N7, https://
[45] C. Valenzuela, L. Abugoch, C. Tapia, Quinoa protein-chitosan-sunflower oil edible doi.org/10.1111/j.1750-3841.2009.01386.x.
film: mechanical, barrier and structural properties, LWT - Food Sci. Technol. 50 [61] N. Vahedikia, F. Garavand, B. Tajeddin, I. Cacciotti, S.M. Jafari, T. Omidi, Z. Zahedi,
(2013) 531–537, https://doi.org/10.1016/j.lwt.2012.08.010. Bio- degradable zein film composites reinforced with chitosan nanoparticles and
[46] X. Sun, Q. Wu, X. Zhang, S. Ren, T. Lei, W. Li, G. Xu, Q. Zhang, Nanocellulose films cinna- mon essential oil: physical, mechanical, structural and antimicrobial
with combined cellulose nanofibers and nanocrystals: tailored thermal, optical attributes, Colloids Surf. B Biointerfaces 177 (2019) 25–32,
and mechanical properties, Cellulose 25 (2018) 1103–1115, https://doi.org/10. https://doi.org/10.1016/j.colsurfb. 2019.01.045.
1007/s10570-017-1627-9. [62] P. Zhang, Y. Zhao, Q. Shi, Characterization of a novel edible film based on gum ghatti:
[47] F.M. Pelissari, M.M. Andrade-Mahecha, P.J. do A. Sobral, F.C. Menegalli, Nanocom- effect of plasticizer type and concentration, Carbohydr. Polym. 153 (2016) 345–355,
posites based on banana starch reinforced with cellulose nanofibers isolated from https://doi.org/10.1016/j.carbpol.2016.07.082.
banana peels, J. Colloid Interface Sci. 505 (2017) 154–167, https://doi.org/10.1016/ [63] M. Anker, J. Berntsen, A.M. Hermansson, M. Stading, Improved water vapor barrier
j.jcis.2017.05.106. of whey protein films by addition of an acetylated monoglyceride, Innov. Food Sci.
[48] N. Tammineni, G. Ünlü, B. Rasco, J. Powers, S. Sablani, C. Nindo, Trout-skin
Emerg. Technol. 3 (2002) 81–92, https://doi.org/10.1016/S1466-8564(01)00051-0.
gelatin- based edible films containing phenolic antioxidants: effect on physical
[64] B. Saberi, Q.V. Vuong, S. Chockchaisawasdee, J.B. Golding, C.J. Scarlett, C.E.
properties and oxidative stability of cod-liver oil model food, J. Food Sci. 77 (2012)
Stathopoulos, Physical, barrier, and antioxidant properties of pea starch-guar
1–6, https://doi.org/10.1111/j.1750-3841.2012.02964.x.
gum biocomposite edible films by incorporation of natural plant extracts, Food
[49] A. Alemdar, M. Sain, Biocomposites from wheat straw nanofibers: morphology,
Bioprocess Technol. 10 (2017) 2240–2250, https://doi.org/10.1007/s11947-017-
thermal and mechanical properties, Compos. Sci. Technol. 68 (2008) 557–565, 1995-z.
https://doi.org/10.1016/j.compscitech.2007.05.044.
[65] L. Sánchez-González, A. Chiralt, C. González-Martínez, M. Cháfer, Effect of
[50] L. Petersson, K. Oksman, Biopolymer based nanocomposites: comparing layered sil-
essential oils on properties of film forming emulsions and films based on
icates and microcrystalline cellulose as nanoreinforcement, Compos. Sci. Technol.
hydroxypropylmethylcellulose and chitosan, J. Food Eng. 105 (2011) 246–
66 (2006) 2187–2196, https://doi.org/10.1016/j.compscitech.2005.12.010.
253, https://doi.org/10.1016/j.jfoodeng.2011.02.028.
[51] J.P. Borges, M.H. Godinho, A.F. Martins, A.C. Trindade, M.N. Belgacem, Cellulose-
[66] S. Benavides, R. Villalobos-Carvajal, J.E. Reyes, Physical, mechanical and
based composite films, Mech. Compos. Mater. 37 (2001) 257–264.
[52] M.A.S. Azizi Samir, F. Alloin, J.Y. Sanchez, A. Dufresne, Cellulose nanocrystals rein- antibacterial properties of alginate film: effect of the crosslinking degree and oregano
forced poly(oxyethylene), Polymer (Guildf) 45 (2004) 4149–4157, https://doi.org/ essential oil concentration, J. Food Eng. 110 (2012) 232–239,
10.1016/j.polymer.2004.03.094. https://doi.org/10.1016/j.jfoodeng. 2011.05.023.
[53] H.M.C. Azeredo, R. Morrugares-Carmona, N. Wellner, K. Cross, B. Bajka, K.W. [67] S. Bahram, M. Rezaei, M. Soltani, A. Kamali, S.M. Ojagh, M. Abdollahi, Whey protein
Waldron, Development of pectin films with pomegranate juice and citric acid, concentrate edible film activated with cinnamon essential oil, J. Food Process.
Food Chem. 198 (2016) 101–106, https://doi.org/10.1016/j.foodchem.2015.10.117. Preserv. 38 (2014) 1251–1258, https://doi.org/10.1111/jfpp.12086.
[54] C.G. Otoni, M.R.d. Moura, F.A. Aouada, G.P. Camilloto, R.S. Cruz, M.V. Lorevice, N. [68] S. Jafari, S. Esfahani, M.R. Fazeli, H. Jamalifar, M. Samadi, N. Samadi, A. Najarian Toosi,
de M.R. Shams Ardekani, M. Khanavi, Antimicrobial activity of lime essential oil against
F.F. Soares, L.H.C. Mattoso, Antimicrobial and physical-mechanical properties of food-borne pathogens isolated from cream-filled cakes and pastries, Int. J. Biol.
pec- tin/papaya puree/cinnamaldehyde nanoemulsion edible composite films, Food Chem. 5 (2011) 258–265, https://doi.org/10.3923/ijbc.2011.258.265.
Hydrocoll. 41 (2014) 188–194, https://doi.org/10.1016/j.foodhyd.2014.04.013. [69] M. Viuda-Martos, Y. Ruiz-Navajas, J. Fernandez-Lopez, J. Perez-Álvarez, Antibacterial
[55] D. Dehnad, H. Mirzaei, Z. Emam-Djomeh, S.M. Jafari, S. Dadashi, Thermal and activity of lemon (Citrus lemon L.), mandarin (Citrus reticulata L.), grapefruit
antimi- (Citrus paradisi L.) and orange (Citrus sinensis L.) essential oils, J. Food Saf. 28
crobial properties of chitosan-nanocellulose films for extending shelf life of
(2008) 567–576.
ground meat, Carbohydr. Polym. 109 (2014) 148–154,
[70] L. Atarés, A. Chiralt, Essential oils as additives in biodegradable films and
https://doi.org/10.1016/j.carbpol.
coatings for active food packaging, Trends Food Sci. Technol. 48 (2016) 51–62,
2014.03.063.
https://doi.org/ 10.1016/j.tifs.2015.12.001.
[56] B. Ghanbarzadeh, H. Almasi, Physical properties of edible emulsi fied films based on
[71] M. Skočibušić, N. Bezić, V. Dunkić, Phytochemical composition and antimicrobial
carboxymethyl cellulose and oleic acid, Int. J. Biol. Macromol. 48 (2011) 44 –49,
https://doi.org/10.1016/j.ijbiomac.2010.09.014. ac- tivities of the essential oils from Satureja subspicata vis. growing in Croatia, Food
[57] M. Moradi, H. Tajik, S.M. Razavi Rohani, A.R. Oromiehie, H. Malekinejad, J. Aliakbarlu, Chem. 96 (2006) 20–28, https://doi.org/10.1016/j.foodchem.2005.01.051.
M. Hadian, Characterization of antioxidant chitosan film incorporated with
Zataria multiflora Boiss essential oil and grape seed extract, LWT - Food Sci.
Technol. 46 (2012) 477–484, https://doi.org/10.1016/j.lwt.2011.11.020.