International Journal of Biological Macromolecules 245 (2023) 125562

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International Journal of Biological Macromolecules

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

Development and characterization of edible films based on flaxseed gum

incorporated with Piper betle extract
Arshied Manzoor a, Saghir Ahmad a, Basharat Yousuf b, c, *
a
Department of Post-Harvest Engineering and Technology, Faculty of Agricultural Sciences, A.M.U., Aligarh, U.P, India
b
Department of Food Science & Technology, School of Applied Sciences & Technology, University of Kashmir, Srinagar 190006, J&K, India
c
Department of Food Technology, School of Engineering & Technology, Islamic University of Science & Technology, Awantipora 192122, J&K, India

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

Keywords: There has been a shift from use of petroleum-based plastics, causing serious environmental pollution, towards
Active packaging innovative and biodegradable edible packaging. The present study documents the development of composite
Antibacterial edible films edible films based on the flaxseed gum (FSG) modified by the incorporation of betel leaf extract (BLE). The films
Biodegradable films
were assessed for physicochemical, mechanical, morphological, thermal, antimicrobial and structural charac­
Biological activity
Water vapor permeability
teristics. Scanning electron microscopy images indicated that the roughness decreased with an increase in BLE
concentration. The water vapor permeability of the FSG-BLE films ranged from 4.68 to 1.59 × 10− 9 g s− 1 m− 2
Pa− 1, lower than that of the control sample (6.77 × 10− 9 g s− 1 m− 2 Pa− 1). The BLE4 (containing 10 % BLE)
films had the highest tensile strength of 32.46 MPa compared to the control sample (21.23 MPa). Similarly, EAB
and seal strength of the films incorporated with BLE were ameliorated. X-ray diffraction pattern and FTIR
illustrated the shift of amorphous to crystalline behavior and a significant interaction among the BLE and FSG
functional groups. Furthermore, the thermal stability of the treated films was not affected significantly however,
they showed improved antimicrobial activity with the highest diameter of inhibition zone in the BLE4 sample.
This study concluded that the FSG-BLE composite films (BLE4 in particular) can be considered as novel pack­
aging material for food conservation coupled with a potential to enhance the shelf life of perishable food
products.

1. Introduction alternatives to low-quality and non-biodegradable products and pack­

The survival of living beings is under severe threat because of the
accumulation of non-biodegradable materials in the environment which
is creating havoc by generating toxicity, hence causing limitless oblit­
erations to the living world. This serious issue is further provoked by the
prevalent use of non-biodegradable conventional packaging materials
(films/coatings) manufactured and used in the packaging industries
[1,2]. Moreover, consumer safety is at risk by using harmful synthetic
packaging materials. Giving credibility to the aforementioned concerns,
scientific research has shifted the focus on the exploration, development
and usage of edible films/coatings from different natural materials with
biodegradable and eco-friendly features. Yousuf, Qadri, & Srivastava [3]
have reported that polysaccharides, being a larger group (among lipids,
polysaccharides and proteins), have found major exploration in the field
of edible film fabrication for food packaging in the past few years. Edible
polymers are high-quality, biocompatible and biodegradable
ages used in different food industries [4].
Another group of plant-based polysaccharides called natural gums
are proclaimed as readily available with eco-friendly nature and
generally recognized as safe (GRAS) status. Hence, natural gums are
being explored immensely instead of synthetic materials for food ap­
plications focused on film/coating formulations as the former ones exist
abundantly at low cost and are devoid of toxicity [5]. Seed gums are
bestowed with exceptional film-forming properties such as physical,
mechanical and barrier properties. In this regard, flaxseed (Linum usi­
tatissimum L.) gum (FSG), extracted by an aqueous extraction process
from flaxseed (of which 8–10 % total seed weight constitutes FSG) could
be used for the development of films/coatings with excellent functional
properties in general, and rheological properties in particular [6]. FSG is
mostly found in the outermost flaxseed coat and the nutritional (50–80
% carbohydrates, 4–20 % of proteins and ash) and health benefits of FSG
make it a potential candidate for exploration in food industries. FSG has

* Corresponding author at: Department of Food Technology, Islamic University of Science & Technology, 192122, J&K, India.
E-mail address: yousufbasharat@gmail.com (B. Yousuf).

https://doi.org/10.1016/j.ijbiomac.2023.125562
Received 15 January 2023; Received in revised form 10 June 2023; Accepted 23 June 2023
Available online 26 June 2023
0141-8130/© 2023 Elsevier B.V. All rights reserved.
A. Manzoor et al.
been majorly explored for functional and rheological properties along
with the extraction followed by its characterization [7–9]. Nevertheless,
FSG laden with excellent active ingredients (antioxidants and antimi­
crobials) has been rarely explored for food packaging applications
through the development of films for food packaging.
Fresh foods (meat, fruits and salads) are best preserved by the
incorporation of plant-based extracts into them or by packaging them in
films with excellent oxygen and water vapor barrier properties that help
in preventing food dehydration [10]. Moreover, chemical compounds
and polymers present in edible films could interact with each other and
influence the mechanical properties (TS and EAB) of edible films [4].
Packaging material also protects against food contamination and dis­
eases caused by food-borne pathogens. This has brought great relief to
the packaging industries by channelizing the plant-based bioactive
compounds (antioxidants, antimicrobial, and flavoring agents) into
biodegradable packaging for the aforementioned concerns [1]. In this
concern, several studies have reported the usage of natural compounds
from plants (peel, leaf or extract thereof) and fruits in edible film/
coating formulations with improved antimicrobial and antioxidant po­
tential [11–13]. Similarly, flaxseed gum films fabricated with the
addition of carvacrol showed potential antibacterial activities however,
the authors suggested limited use of the developed film in food in­
dustries [14]. Cellulose nanocrystals were loaded onto the flaxseed gum
films with glutaraldehyde as a crosslinking agent which led to the
reduction in film solubility and hydrophobicity of the film matrix [15].
FSG could also be incorporated for the enhancement of physico-
chemical attributes of edible films developed from gluten [16]. More­
over, FSG (0.3 and 0.6 %) was explored for the development of an edible
coating with the addition of lemongrass essential oil (0, 200, 500 and
800 ppm) [17].
Piper betle L., commonly known as betel leaf (family-Piperaceae,
color-deep green) is reported to deliver antiseptic, antibacterial, anti­
oxidant, antidiabetic, anti-inflammatory, anticancer and immunomod­
ulatory functions attributed to the presence of phytochemicals, such as
phytol, eugenol, hexadecanoic acid, alkaloids, flavonoids, steroids, sa­
ponins, and tannins [18,19]. Betel leaf extract (BLE) has been unveiled
for its antioxidant and antimicrobial properties by several recent studies
that could meet the increasing consumer demands for healthier food
[19–21]. Keeping in view the advantages and convenience of BLE
particularly towards the efficaciousness for human well-being, the
development of flaxseed gum-based edible films with BLE incorporation
can be an amazing and promising approach to active packaging for
different food products to uphold quality. To the best of our knowledge,
there is no study regarding the antibacterial properties of films based on
flaxseed gum loaded with betel leaf extract. Besides, the conventional
petroleum-based plastics have consistently affected the environment
adversely. Hence, to tackle the concerns related to the environment (due
to plastics) along with that associated with agro-waste management the
objective of this study was to fabricate and investigate the quality
characteristics of novel FSG composite films incorporated with BLE for
their prospective applications in food/non-food industries. The results
from the present study suggest the exploration of BLE for antioxidant
and antimicrobial purposes and its incorporation into the edible film
development as an alternative to synthetic films for packaging of
different food products.
2. Material and methods
2.1. Material
Flaxseeds used in this study were purchased from Spencer super­
market, Aligarh, India. The seeds were then manually cleaned and
stored in an airtight container. Betel leaves were obtained from the local
supermarket, Aligarh. Ethanol (99 % pure, CAS number = 64–17-5) was
purchased from Merck suppliers, New Delhi, India. Glycerol (analytical
grade) was acquired from Sigma Aldrich Mumbai, India. Foli-Ciocalteu
2
International Journal of Biological Macromolecules 245 (2023) 125562
reagent was purchased from Loba Chemie Pvt. Ltd. Mumbai, India and 2,
2-diphenyl-1- picrylhydrazyl (DPPH) was purchased from Fluka Goldie.
Pseudomonas aerogenosa - PA01 and Staphylococcus aureus - ATCC 25923
were obtained from the Department of Agricultural Microbiology, Ali­
garh Muslim University, India. All other chemicals/reagents used in this
study were of analytical grade and were obtained from CDH Chemicals,
New Delhi, India.
2.2. Preparation of flaxseed gum powder
Extraction of flaxseed gum powder (FSG) from the flaxseeds was
undertaken by following the method illustrated by Wang et al. [22]
along with some modifications. Briefly, the flaxseeds after washing with
running water (2 min) for dirt removal were stirred continuously (300
RPM) in distilled water in the ratio (1:10 W/V) at ambient temperature
for 12 h. Then, a sieve and a cotton cloth were used to separate the seeds
and filter them, respectively to ascertain the resulting viscous solution is
free of any fine husk particles. A hot air oven (York Scientific Industries,
Ghaziabad, India) was arranged to dry the solution for 12 h (105 ◦ C)
which was converted into FSG powder by a laboratory blender and
finally stored in an air-tight glass container to avoid moisture ingress.
2.3. Preparation of betel leaf extract
Fresh leaves of Piper betle L. (desi bangla) with no apparent damage
were washed three times with distilled water. After washing, the clean
betel leaves were subjected to drying at room temperature (25 ± 1 ◦ C)
for 4 days in the laboratory at the Department of Post-Harvest Engi­
neering and Technology, Aligarh Muslim University, India. The extrac­
tion was done by following the method described by Nouri & Nafchi [1]
with slight modifications. Briefly, powdered dried betel leaves (100 g,
prepared previously) after mixing with a 1 L solution of ethanol (90 %)
were extracted for 24 h with continuous stirring (1200 rpm) at ambient
temperature and filtered through Whatman No. 1 filter paper. The
extraction process of the residue hence obtained was repeated 2–3 times
until the residue was colorless. A rotary vacuum film evaporator (Kshitij
R&D Lab Instruments, CAT No. 2199A, Haryana, India) at 40 ◦ C was
employed for separating the solvent (ethanol), thereby concentrating
the residue which was finally lyophilized to convert the extract into
powder. Vacuum-packed bags were used for the storage of powder at
− 18 ◦ C until used.
2.4. Preparation of films
For the film preparation, the method described by Abedinia et al.
[23] was followed. FSG concentration was selected based on the pre­
liminary experiments that suggested 1 % FSG as the base material for
films. FSG concentrations higher than 1 % (1.5 and 2 %) produced film-
forming solution which had excessive thickness, posing difficulties in
film forming process such as casting and drying. Whereas the concen­
tration below 1 % (0.5 % and 0.75 %) posed difficulty in the process of
separation of the edible films from the petri plate surface. FSG (1 %) was
dissolved in distilled water for preparing the film-casting solution and
kept in a hot water bath set at 80 ◦ C for 30 min (Water bath shaker, Cat
no. BGS-116, Biogen Scientific-India). Further, glycerol (30 % of the
weight of film-forming material; Thermo Fisher Scientific India Pvt. Ltd.
Mumbai, India; CAS No.: 56–81-5; MW: 92.09 gmol− 1) was added as a
plasticizer to the solution. The mixture after the addition of glycerol was
stirred (1000 rpm) on a magnetic stirrer for the next 20 min at 60 ◦ C
during which the desired BLE levels were added in different batches. The
mixture was cooled and air bubbles were removed by subjecting the
solution to sonication (20 kHz) for 20 min using an ultrasonic bath
(Digital ultrasonic cleaner, Labman Scientific Instruments, S.No.
L32195). Finally, 25 g of the prepared casting solution was poured and
spread onto the petri plates (90 mm diameter) preventing any bubble
formation followed by drying in a hot air oven (30 ◦ C for 24 h). The
A. Manzoor et al.
films, after cooling at room temperature (20 min) were stripped off the
surface of petri plates and stored for a minimum period of 24 h (at 25 ◦ C,
50 % RH) in a desiccator having bromide solution (saturated) before
analysis (Fig. 1(a)). In total, five types of films were prepared namely
BLE0 (film with 0 % BLE), BLE1 (film with 2.5 % BLE), BLE2 (film with
5 % BLE), BLE3 (film with 7.5 % BLE) and BLE4 (film with 10 % BLE) as
shown in Fig. 1(b).
2.5. Characterization of films
2.5.1. Physicochemical characteristics
2.5.1.1. Moisture content. A well-known gravimetric method was
employed to determine the moisture content (MC) of films as done by
Zhai et al. [24]. Briefly, moisture content was recorded as the loss in film
piece (3 cm × 3 cm) weight recorded before and after drying in a hot air
oven (110 ◦ C, 24 h) and drying was done until the weight of the film
sample was constant. Results were obtained in triplicates from which an
Fig. 1. (a) Preparation (b) Visual appearance of flaxseed gum-betel leaf extract composite edible films added with different betel leaf extract (BLE) concentrations.
BLE0, BLE1, BLE2, BLE3, BLE4 correspond to films with 0, 2.5, 5, 7.5, 10 % BLE respectively.
3
International Journal of Biological Macromolecules 245 (2023) 125562
average was taken. Eq. (1) was employed for calculation of the film
moisture content where the weight of the film piece before and after
drying was denoted by W1 and W2, respectively.
(W1 − W2)
MC(%) = × 100 (1)
W1
2.5.1.2. Film solubility. Film solubility was estimated by the procedure
followed by Kaya et al. [25] with certain adjustments. Briefly, the film
sample was dried in a laboratory scale oven (60 ◦ C for 24 h) followed by
immersion in distilled water (20 mL) at room temperature and left un­
disturbed for 48 h. This was followed by draining the water through
Whatman filter paper 2 and again drying the film sample at 60 ◦ C for 24
h. Eq. (2) was used for the determination of film solubility;
(X1 − X2)
Film solubility (%) = × 100 (2)
X1
where X1 = initial weight and X2 = final weight of the film sample.
A. Manzoor et al.
2.5.1.3. Film thickness. A digital micrometer (Precise, Sudershan
Measuring & Engg (P) Ltd., New Delhi India) with an accuracy of 0.001
mm was used for the estimation of average film thickness taken from five
different places as done by Abedinia et al. [26], which were expressed in
millimeters (mm).
2.5.1.4. Water vapor permeability (WVP). This study employed the
gravimetric method of the American Society for Testing & Materials
with slight modifications for the determination of water vapor perme­
ability (WVP) of the films. Briefly, the mouth of the centrifuge tube
(containing dried silica gel) was sealed with film samples (4 × 4 cm)
which were then stocked up in a desiccator at 100 % RH and room
temperature and after every 1 h interval, the weight change in the
weight of the cup was recorded for 8 h. Finally, the film WVP was
calculated following the Eq. (3) as suggested by Sarıcaoglu & Turhan
[27]:
wL
WVP = (3)
t × ΔPA
where A and L denote the area of the mouth of the centrifuge tube and
the average film thickness respectively. ΔP (kPa) denotes the pressure
difference of water vapor across the film (25 ◦ C).
2.5.2. Mechanical properties
2.5.2.1. Elongation at break (EAB) and tensile strength (TS). The pro­
cedure followed by Wu et al. [28] along with certain adjustments was
employed to determine these film properties for which the films were
conditioned for 2 days (50 ± 5 % RH and 25 ± 2 ◦ C) before testing by a
Texture analyzer (TA.XT. plus, Stable Micro Systems., Vienna Court,
UK). A load cell of 5 kN with a 30 mm min− 1 crosshead speed and with 1,
0.50 and 2 mm s− 1 of pre-test speed, test speed, and post-test speed
respectively. Moreover, the equipment was equipped with a 10 mm s− 1
and 10 g of distance and break sensitivity respectively for the analysis of
films (dimension = 2 cm × 5 cm).
2.5.2.2. Seal strength. Seal strength was estimated using a texture
analyzer (TA.XT. plus, Stable Micro Systems., Vienna Court, UK),
operational with a static load cell (30 kg and 60 mm min− 1 speed) that
held each film (sealed) leg vertically to the pull direction. An average
gap of 2.5 cm was maintained between the grips. Finally, the seal
strength (Nm− 1) is calculated as the ratio of force (maximum) causing
seal fracture to the width of the film sample.
2.5.3. Optical properties
2.5.3.1. Light transmittance and attenuation coefficient (k). The method
suggested by Ramos et al. [29] was followed for the evaluation of the
film transmittance with some modifications. UV–vis spectrophotometer
(Double beam UV-viz Spectrophotometer-2206, Systronics India
Limited, Gujrat, India) was employed to determine the film barrier
properties against ultraviolet and visible light at some selected wave­
lengths. Briefly, small strips were cut out from the dried film samples
and placed in a cuvette perpendicular to the direction of the incident
light. In this process, the transmittance was recorded at 600 nm against
an empty cuvette (as control). The Eq. (4), as used by Wang et al. [30]
was employed for the calculation of relative film light transmittance
where A600 and X represent the absorbance of the film at a wavelength of
600 nm and the film thickness (mm) respectively.
A600
Transparency = × 100 (4)
X
The attenuation coefficient (k) was determined by following the Eq.
(5) as done by Cavallaro et al. [31] and the values were calculated using
the absorbance (A) data.
4
International Journal of Biological Macromolecules 245 (2023) 125562
k = A/2.3* D (5)
where, A and D is the absorbance and thickness of the FSG-BLE com­
posite films respectively.
2.5.3.2. Color parameters. Film color parameters (L*, a*, and b* values)
were determined using a Hunter Lab Colorimeter (Mini Scan XE Plus,
Hunter Associates Laboratory, Inc., Reston, USA) having a 5 mm aper­
ture size viewed at 10o angle geometry that was calibrated previously by
black and white plates. The instrument is equipped with a pulsed xenon
arc lamp filtered to illuminant D65 lighting and at least three mea­
surements were taken for each sample. Briefly, L*, a*, and b* values
were determined as the reading recorded from the film sample that was
placed on the light source and covered superficially by a white plate. Eq.
(6) was employed to estimate the color difference of the films and Eq. (7)
was utilized for the film chroma determination.
[ ]1/2
ΔE = (ΔL* )2 + (Δa* )2 + (Δb* )2 (6)
[ ]1/2
Chroma = (a* )2 + (b* )2 (7)
where, ΔL*, Δb* and Δa* are the differences in L*, a* and b* values of
the standard plate and the film sample.
ΔL* = L*std – L*sample, Δa* = b*std – b*sample and Δb* = a*std – a*sample
For the standard white plate, L*std = 93.45, a*std = − 0.64 and b*std =
1.73.
L*std – L*sample = a*std – a*sample = b*std – b*sample.
Eq. (8) was employed to determine the whiteness index of the
composite films.
[ ]1/2
WI = 100 − (100 − L* )2 + (a* )2 + (b* )2 (8)
2.5.4. Scanning Electron Microscopy (SEM)
Scanning Electron Microscope (JEOL, JSM6300 SEM, JEOL, Tokyo,
Japan) was employed for the determination of the surface morphology
and cross-section structure of the films. Before analysis, the films were
conditioned by keeping the composite films in air-tight desiccators
packed with silica gel (~0 % RH). Liquid nitrogen was then employed to
break the composite films evenly into small fragments for the cross-
section analysis. After fracturing the films, the film fragments were
gently mounted on the aluminum stubs with a double-sided adhesive
tape to get a thin gold layer uniformly sputtered for the conductivity
amelioration which was finally analyzed using a 15 kV accelerating
voltage.
2.5.5. X-ray diffraction
Film samples with an area of 10 mm2 were conditioned/dehydrated
efficiently in a desiccator loaded with silica gel (~0 % RH). After con­
ditioning, the film crystallinity was estimated by an XRD device (Bruker
D8 Advance powder diffractometer, Japan) usually operated at room
temperature with current (40 mA) and voltage (45 kV) by radiating Cu
radiations having a wavelength of 1.54 Å on to the sample. The films
were observed under an X-ray diffractometer over an angular (2θ) range
of 5–80◦ to obtain an XRD pattern.
2.5.6. Fourier transform infrared spectroscopy (FTIR)
Analysis of functional groups and their interaction in films was ob­
tained by Spectrum Two (Perkin Elmer, spectrometer) FR-IR Fourier-
Transform using the KBr pellet method where the pre-conditioned film
samples were mounted on a plate and the transmittance data of the film
samples was observed between a 4500–380 cm− 1 transmittance region
at a 4 cm− 1 resolution.
2.5.7. Thermal properties
The thermogravimetric analysis (TGA) of the flaxseed gum-based
A. Manzoor et al. International Journal of Biological Macromolecules 245 (2023) 125562

composite films was measured by employing Shimadzu TA-60 WS
thermal analyzer to track weight change as a function of time or tem­
perature caused by thermal degradation of compounds by heating from
room temperature to 800 ◦ C in an inert nitrogen atmosphere (to avoid
thermo-oxidative reactions) of 30 and 20 mL/min nitrogen flows for the
sample and the balance respectively with a ramp of 10 ◦ C/min. The
different degradation phases were obtained from the plots of weight loss
values against temperature as done by Casariego et al., [32].
2.5.8. Total phenolic content (TPC)
Estimation of phenol content in the film samples was done by
following the method of Moradi et al. [33] with certain modifications. A
film sample (25 mg) was dissolved in distilled water (3 mL) to prepare
the film extract. After this, a mixture was prepared by mixing the film
extract (0.5 mL) and 2.5 mL of 10 % folin-Ciocalteau reagent which was
further added with 2 mL of 7.5 % sodium carbonate solution. The whole
mixture was then put under incubation (in dark) for 2 h at 50 ◦ C fol­
lowed by analysis with a spectrophotometer (Double beam UV-viz
Spectrophotometer-2206, Systronics India Limited, Gujrat, India) at
765 nm and compared to the gallic acid calibration curve. TPC was re­
ported as mg gallic acid equivalents (GAE)/g of films.
2.5.9. DPPH radical scavenging activity
The 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging
method as followed by Yousuf, Wu, & Gao [5] was undertaken for the
3. Results and discussion
3.1. Physicochemical characteristics
3.1.1. Moisture content (MC)
Table 1 depicts the MC of different flaxseed gum-based composite
films modified with BLE incorporation at different concentrations.
Polymer and water molecules are reported to interact with each other
through hydrogen bonding in two ways namely polymer-water system
(MC <10 %) and water–water interactions (MC >10 %) [1]. In our
study, moisture content decreased progressively with increased incor­
poration of BLE in flaxseed gum-based films (Table 1). The highest
(14.47 ± 0.70 %) and the lowest (9.21 ± 0.65 %) moisture content was
observed in BLE0 and BLE4 films developed by incorporating 0 % and
10 % BLE concentration respectively. This may be credited to the hy­
drophobic nature of the extract/oil present and the amount of hydro­
phobic inclusions within the film increases with the increase in BLE
Table 1
Physicochemical properties (moisture content, solubility, thickness, and water
vapor permeability); Mechanical properties (seal strength, tensile strength (TS)
and elongation at break (EAB); Color parameters (light transmittance, Attenu­
ation coefficient at 600 nm, L*, a*, b* and chroma) of flaxseed gum-based films
added with different concentrations of betel leaf extract (BLE)).
Sample Moisture Solubility Thickness WVP (×10− 9
g s− 1

determination of the antioxidant activity of films with some modifica­ content (%) (%) (mm) m− 2 Pa− 1)
tions. 100 mL of distilled water was used for dissolving 1 g of the film BLE0 14.47 ± 0.70a 22.16 ± 0.032 ± 6.77 ± 0.67a
sample to prepare the film extract. From this extract, 3 mL was mixed 0.49a 0.003a
with 1 mL of 0.1 mM ethanolic solution of DPPH followed by 2 min BLE1 12.65 ± 0.60b 25.83 ± 0.042 ± 4.68 ± 0.40b
0.54b 0.006b
vortex shaking and incubating for 30 min in dark. A spectrophotometer
BLE2 11.23 ± 0.33c 29.20 ± 0.058 ± 3.75 ± 0.45bc
(Double beam UV-viz Spectrophotometer-2206, Systronics India 0.25c 0.002c
Limited, Gujrat, India) was finally used for measuring the absorbance of BLE3 10.39 ± 0.53c 33.18 ± 0.052 ± 2.83 ± 0.65c
the resultant extract at 517 nm and calculated using the Eq. (9) where 0.19d 0.005cd
‘As’ and ‘Ab’ represents the absorbance of the sample extract and the BLE4 9.21 ± 0.65d 36.32 ± 0.063 ± 1.59 ± 0.80d
0.24e 0.003d
DPPH solution (ethanolic) respectively.
(Ab − As)
DPPH scavenging activity (%) = × 100 (9)
Ab Mechanical properties

Sample Seal strength (N/m) Tensile strength Elongation at break (%)

2.5.10. Antimicrobial properties (MPa)
For the determination of antimicrobial properties (against Pseudo­
BLE0 351.86 ± 0.56a 21.23 ± 0.93a 15.19 ± 1.05a
monas aerogenosa - PA01 and Staphylococcus aureus - ATCC 25923), the BLE1 382.18 ± 0.26b 25.20 ± 1.11b 18.04 ± 1.86b
films were cut into discs of 1.2 cm in diameter using a cork borer. The cut BLE2 400.88 ± 0.31c 28.17 ± 1.05c 21.52 ± 1.46c
discs were inserted into the nutrient agar-laden petri dishes which were BLE3 443.90 ± 0.15d 29.87 ± 0.54c 25.20 ± 0.99d
inoculated and already contained cell culture (0.1 mL). The culture BLE4 515.55 ± 0.52e 32.46 ± 1.52d 21.14 ± 0.96c

(already grown in nutrient agar for 24 h) was incorporated into the

peptone water (0.1 %) for the preparation of microbial inoculums and
the cell culture was set to 1.0 McFarland unit corresponding to 13 × 107 Color parameters

CFU/mL. Afterward, microbial growth was allowed by incubating the Sample Light Attenuation L* a* b* Chroma
prepared petri dishes for 36 h at 37 ◦ C and the growth was later on transmittance coefficient
measured by the inhibitory zone estimation using a caliper which gives BLE0 1.51 ± 0.03a 3.83 ± 0.14a 23.36 0.22 6.16 6.14 ±
the antimicrobial activity of the composite films expressed in millime­ ± ± ± 0.12bc
0.81a 0.14a 0.98a
ters (mm).
BLE1 1.06 ± 0.02b 4.74 ± 0.26b 14.92 2.43 4.40 5.08 ±
± ± ± 0.07a
2.6. Statistical analysis 0.73b 0.83b 0.81b
BLE2 1.03 ± 0.01b 5.82 ± 0.21c 12.31 4.80 2.93 5.81 ±
The data obtained from the average of three readings were analyzed ± ± ± 0.16b
0.35bc 0.50c 0.12c
statistically using SPSS 19.0 software (Chicago, IL, USA). Significant
BLE3 0.97 ± 0.02c 7.14 ± 0.36d 13.73 6.02 2.13 6.45 ±
differences (p < 0.05) among data were obtained by using one-way ± ± ± 0.57c
ANOVA technique and Duncan's tests. All the data shown in tables and 1.05bc 0.66d 0.09c
figures are mean ± standard deviation and were obtained by performing BLE4 0.90 ± 0.02d 9.24 ± 0.12e 8.15 7.88 0.95 7.28 ±
the tests in triplicate. ± ± ± 0.78d
0.26c 0.54e 0.11d

Results are expressed as mean ± standard deviation, n = 3. Different superscript

letters in a same column represent significant (p < 0.05) differences among the
different films. BLE0, BLE1, BLE2, BLE3, BLE4 correspond to films with 0, 2.5, 5,
7.5, 10 % BLE respectively.

5
A. Manzoor et al.
concentration which in turn results in the reduction of interactions be­
tween water, protein and polysaccharide within the film [34]. Our re­
sults are supported by the findings from the study on the whey protein
based-films containing candelilla wax and butter fat [35] and the
study on whey protein isolate films containing soya oil [36]. The in­
teractions mentioned above could lower the availability of hydroxyl and
amino groups thereby restricting the interactions such as hydrogen
bonding among water and polysaccharides. Similar results of decreasing
moisture content up on incorporation of tea polyphenols were reported
by Wang et al. [37]. They explained the fact with the explanation that
tea polyphenols limited the chitosan-water interaction. The MC of BLE4
was lower than BLE0 which might be related to the less compact
structure of treated film samples produced by short and flexible chains of
flaxseed gum which ultimately leads to a poor water retention capacity.
Hence, the loose network of the film matrix caused by the decrease in
inter- and intramolecular interactions between polysaccharide mole­
cules results in the easy removal of water molecules by evaporation
during the process of film drying [38].
3.1.2. Film solubility
Film solubility, considered an important parameter, describes the
integrity of films in aqueous phase and also defines film biodegradability
in which lower film solubility means a higher resistance to water and
vice versa. The solubility profile of the FSG-BLE composite films is
presented in Table 1. Higher solubility of films incorporated with the
BLE was found as compared to the pure FSG film without BLE addition.
A significant progressive increase was witnessed as the concentration
increased in the composite films and the highest film solubility of 36.32
± 0.24 was found in films developed from FSG with 10 % BLE. This is
because of the presence of hydroxyl groups on BLE and glycerol which
lead to an increased film solubility as stated by Laohakunjit & Noom­
horm [39]. Our results are in line with the studies reported earlier
[1,40]. Packaging films with lower solubility are favored for food stor­
age purposes however, higher solubility proves to be beneficial when the
food products coated with edible films are cooked.
3.1.3. Film thickness
Film thickness is an essential property influencing the physical
properties of edible composite films. The thickness of all the films
developed in this study is presented in Table 1 and as expected the pure
sample presented the least thickness which increased with an increase in
BLE incorporation and ranged between 0.032 mm and 0.063 mm.
However, the films did not show huge variation in thickness and the
highest thickness was recorded in the BLE4 film sample. The results
revealed that with an increase in BLE concentration, the film thickness
increased which is supported by the fact that an increase in film thick­
ness is directly proportional to the increase in hydrophobic molecules
such as oil or extract in composite films. In addition, the water vapor
permeability results (Table 1) of films developed from FSG added with
different BLE levels was observed to be lesser as compared to the control
film, which further justifies our results of increase in film thickness
values as suggested by Mir et al. [41].
3.1.4. Water vapor permeability (WVP)
Water vapor permeability (WVP) depicts the moisture transfer be­
tween the packed food and the surroundings thereby defining its role in
combating adverse effects caused by unfavorable environmental con­
ditions. Hence, WVP is considered one of the most important film
characteristics in food packaging applications that compel the re­
searchers to modify packaging films to reduce the WVP through the
films. The oil/extract-containing composite films are known for their
good water vapor barrier characteristics bestowed by the presence of
oil/extract. The data regarding the WVP of the flaxseed gum-based
(FSG) films are gathered in Table 1 and as expected, pure FSG films
displayed the highest WVP however, the incorporation of BLE resulted
in the progressive reduction (P < 0.05) of WVP in FSG-BLE composite
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International Journal of Biological Macromolecules 245 (2023) 125562
films. The WVP in the BLE4 sample decreased to a value of 1.59 × 10− 9
g s− 1 m− 2 Pa− 1 as compared to pure flaxseed gum-based films with
WVP recorded as 6.77 × 10− 9 g s− 1 m− 2 Pa− 1. This implies a 76.51 %
WVP decrease in FSG-BLE composite films as the BLE concentration was
increased from 0 to 10 %. The reduction in WVP may be credited to the
hydrophobic behavior of the films attributed to BLE addition and the
components along with their spatial alignment in the film matrix [5]. In
addition, the transfer of water vapors takes place through the hydro­
philic portion of the packaging film which is a function of the ratio of
hydrophilic to hydrophobic film ingredients and in this study, the BLE
incorporation increased the hydrophobic fraction thereby improving the
water vapor permeability. Moreover, it may be due to the dense and
compact structure formed through the intermolecular interaction be­
tween BLE and the polysaccharides within the film [42]. The results are
in line with the study conducted by Wu et al. [28] who incorporated
green tea extract in silver carp (Hypophthalmichthys molitrix) skin gelatin
film.
3.2. Mechanical properties
3.2.1. Tensile strength (TS) and elongation at break (EAB)
Pertaining to use in different applications, the development of
composite films is focused to ameliorate the mechanical properties along
with the permeability which in turn depend on the characteristics of the
film and the interactions among the components within the film matrix
[43]. The composite films were studied for tensile strength and elon­
gation at break. The TS and EAB constitute the mechanical properties of
the films and reflect the mechanical resistance and the flexibility of films
respectively. TS is the maximum force per unit area that a film can
withstand before breaking and the EAB is related to the film's flexibility
if subjected to mechanical stress and tension. In packaging applications,
mechanical properties are considered vital parameters of edible films
and composite films with good tensile strength coupled with adequate
flexibility and elongation qualify for efficient food packages. Table 1
depicts the mechanical properties of flaxseed gum-based films incor­
porated with betel leaf extract. As evident from the table, BLE4 samples
possess higher TS (32.46 ± 1.52 MPa) and EAB (21.14 ± 0.96 %) values
as compared to the FSG films without BLE (TS = 21.23 ± 0.93 MPa, EAB
= 15.19 ± 1.05 %) which might be due to the formation of compact
structure based on hydrogen bonding between phenolic hydroxyl groups
and the polysaccharides present in BLE and the flaxseed gum respec­
tively. The appearance of more intense peaks in case of the films con­
taining BLE, as evident from the results of FTIR, confer the formation of
different types of interactions that lead to further increase in tensile
strength [41]. Moreover, the BLE polyphenolic compounds may pose a
hindrance in the interaction among the FSG matrix chains. This infers
the enhancement in mechanical resistance and flexibility in FSG films
with the incorporation of BLE and our results are in agreement with the
study reported recently by Chong & Brooks [44]. Liu et al. [42] also
reported similar results for κ-Carrageenan-based films incorporated with
the mulberry extract.
3.2.2. Seal strength
Seal strength is correlated to the film's ability in keeping the foodstuff
and its constituents intact during handling and storage. Hence, the
efficient seal strength of packaging film is highly appreciated as it resists
harsh operations. Compared to the control there was a progressive in­
crease in the seal strength of composite films with an increase in BLE
concentration as presented in Table 1. The increase in BLE from 0 to 10
% increased the seal strength of composite films by 31.75 %. The reason
for the increase in seal strength is credited to the increase in hydrogen
bonds within the film matrix. Similar results were also reported by one
more study on sago starch-based composite films added with an extract
from betel leaves [1].
A. Manzoor et al.
3.3. Optical properties
3.3.1. Light transmittance
Light transparency of the composite films is an important parameter
which is a function of the internal structure of the film. Hence, the light
transmittance determines the effect of extract addition on the pure
edible films quantitatively. The light transmittance of flaxseed gum-
based composite films developed by the incorporation of BLE is pre­
sented in Table 1 which confirms that the transmittance of the composite
films depends on the internal film matrix microstructure coupled with
the distribution of the components/ingredients of the extract added to
the film. Transmittance is decreased by the occurrence of large amounts
of heterogeneous structures contained by the film and vice versa. This is
because light dispersion is more prevalent in heterogeneous structures.
In this study, the maximum light transmittance of 1.51 ± 0.03 was
witnessed in pure film (without BLE) and the minimum value (0.90 ±
0.02) was recorded in the composite films added with 10 % of BLE. The
reduction in transparency was seen due to the presence of lipids within
the gum matrix in a dispersed manner that led to light scattering within
the BLE-added films. Moreover, the composite films with higher BLE
presented further lower transmittance values credited to the absorbance
caused by the extract present. This depicts that the BLE-added films were
more opaque in visual appearance with higher heterogeneous integrity
as compared to the control counterpart. During the light exposure of the
composite film, the polyphenols in the extract carry out selective ab­
sorption of light at low wavelengths hence decreasing the transmittance
values of the films. Similar results were also presented by Fabra et al.
[45] who witnessed an increase in film opaqueness of alginate-based
films by the incorporation of green tea extract+grape seed extract.
The current study also determined attenuation coefficient at the
specific investigated wavelength of 600 nm (Table 1). It is evident that
the addition of the BLE resulted in a significant enhancement in the
attenuation coefficient of the films with a conclusion that the composite
films exhibited a lower transparency as compared to the control one and
the results are consistent with the light transmittance data. The effect on
the attenuation coefficient is stronger and prominent by increasing the
BLE and the results obtained are in line with the results reported by the
Cavallaro, et al. [31] in their recent study on nanocomposites based on
halloysite nanotubes and sulphated galactan from red seaweed
Fig. 2. Color change and whiteness index of flaxseed gum based composite films added with different betel leaf extract (BLE) concentrations. BLE0, BLE1, BLE2,
BLE3, BLE4 correspond to films with 0, 2.5, 5, 7.5, 10 % BLE respectively.
7
International Journal of Biological Macromolecules 245 (2023) 125562
Gloiopeltis. Similar results were also depicted in studies on pectin/HNT
and hydroxylpropyl cellulose/HNT nanocomposites [46,47].
3.3.2. Color parameters
Color is an important characteristic of composite films as most
biodegradable films are used in the food and pharmaceutical industries.
Color parameters of different flaxseed gum-based composite films are
gathered in Table 1. The pure FSG films appeared white and transparent
in appearance when visualized apparently, which turned dark on BLE
addition that further intensified progressively with an increase in BLE
concentration. It is evident that the L* value decreased with an increase
in BLE from 0 to 10 %. L* value of the pure FSG film (without BLE) was
23.36 ± 0.81 and that of the composite film (with 10 % BLE addition)
was recorded as 8.15 ± 0.26. The decrease in L* value signifies the
decrease in film lightness that is attributed to the dark green color of BLE
[1]. The composite films showed an increase in a* value however, the b*
value was found to show a progressive and significant decrease in the
composite films with an increase in BLE concentration. A decrease in the
b* value defines the dark yellow appearance in films which was also
reported by some recent studies [1,5]. Fig. 2 presents the other colori­
metric parameters [color change (ΔE) and whiteness index, (WI)] of the
films. An increase in ΔE value with an increase in BLE concentration can
be seen however, lower ΔE values were observed in films with lower BLE
concentration (2.5 and 5 %). This explains that the addition of BLE to the
films brought about significant changes in the color of the films which is
again attributed to the dark green color of the extract. Regarding the
whiteness index (WI) a decreasing trend was shown by the composite
films on increasing the BLE concentration. BLE4 film samples with the
highest BLE (10 %) concentration showed the lowest WI value against
the pure flaxseed gum-based film (BLE0) that had the highest WI value.
The results parallel to this study were also reported by Yousuf, Wu & Gao
[5] in their study on karaya gum-based film incorporated with Schi­
sandra chinensis oil.
3.4. Scanning Electron Microscopy (SEM)
The morphology of the BLE-FSG films determined by scanning
electron microscopy (SEM) is depicted in Fig. 3(a) and (b), which reveals
the surface and cross-sectional morphologies. The micrographs depict
A. Manzoor et al.
Fig. 3. SEM, (a) surface (viewed at magnification 500×) (b) cross section (viewed at magnification 1000×) images of flaxseed gum based composite films added with
different betel leaf extract (BLE) concentrations. BLE0, BLE1, BLE2, BLE3, BLE4 correspond to films with 0, 2.5, 5, 7.5, 10 % BLE respectively.
how the film components are arranged in the matrix thereby controlling
many other characteristics of biodegradable substances that make it an
important feature [48]. It was observed that the control sample had a
rough and non-homogenous surface. However, the films added with the
BLE were seen to have a little grainy structure shown by the presence of
a few agglomerates formed by the BLE powder that was unable to
dissolve completely in the polymer solution. This grainy structure went
on increasing with the increase in BLE concentration in the films.
The cross-section images of the BLE-FSG films (control and films
added with the extract) are depicted in Fig. 3(b) which shows a general
rough appearance in all the films. Further, it showed that the control
film showed some pores which went on decreasing by increasing the BLE
concentration. Compared to the control film which developed cracks,
the films added with BLE showed negligible cracks which decreased
with an increase in BLE level. Similar results were observed by Yousuf,
Wu & Gao [5] in their study on Karaya gum-based films incorporated
8
International Journal of Biological Macromolecules 245 (2023) 125562
with Schisandra chinensis oil and its oleogel.
3.5. X-ray diffraction
Fig. 4 depicts the X-ray diffractogram pattern of the pure and com­
posite films developed from flaxseed gum with the absence and presence
of betel leaf extract which is reported to be associated with the me­
chanical behavior. It is clear from the data that there was one specific
peak at 2θ of 20◦ , confirming that a semi-crystalline structure was
associated with all the film samples. Moreover, flaxseed gum film dis­
played an amorphous behavior which is in line with the results reported
recently by Nguyen et al. [49]. The diffractogram was seen devoid of
sharp peaks in all the film samples which designate the crystalline
structure. However, the addition of betel leaf resulted in the increase
and broadening of the peak intensity at 20.5◦ which means a gradual
shift from amorphous to crystalline behavior. All the film samples
A. Manzoor et al.
Fig. 3. (continued).
presented an initial peak corresponding to 2θ = 20◦ however, the BLE
samples additionally showed some small peaks at 2θ =26.4◦ , especially
in the BLE3 sample with the addition of 7.5 % extract. With an increase
in betel leaf extract concentration, the degree of crystallization
increased insignificantly as compared to the control film sample. This
explains the possible ordered arrangement of polyphenolic chains
caused by the interactions (physical and chemical) between the func­
tional components and the flaxseed gum-based film matrix. Our results
are in line with the results reported by Nguyen et al. [49] who also re­
ported comparable broadness and strength of peaks of the chitosan-
based films. Moreover, other researchers also reported a similar array
of results by incorporating different phenol-rich extracts (grapefruit seed
extract and Nigella sativa seedcake extract) into chitosan-based films
[50].
9
International Journal of Biological Macromolecules 245 (2023) 125562
3.6. Fourier transform infrared spectroscopy (FTIR)
The molecular interactions among the matrix components are
investigated by Fourier transform infrared spectroscopy (FTIR) and the
results of pure and composite films in the presence and absence of betel
leaf extract are displayed in Fig. 5. Concerned to the control films, the
spectrum presented characteristic peaks of gum and lipid components.
In the region of 2895–3335 cm− 1, the broad spectrum is allocated to -OH
stretching of water and the characteristic shifts are observed due to in­
teractions between water and the matrix coupled with the changes in
water content thereby disturbing the organization of the network within
the film matrix [51]. On incorporation of betel leaf extract, a shift of the
C–– O stretching of triglycerides from lower wavenumbers to a higher
wavenumber (1125 cm− 1) was observed. Our results are in agreement
with those reported for sodium alginate-carboxymethylcellulose films
A. Manzoor et al. International Journal of Biological Macromolecules 245 (2023) 125562

Fig. 4. X-ray diffraction patterns of flaxseed gum based composite films added with different betel leaf extract (BLE) concentrations. BLE0, BLE1, BLE2, BLE3, BLE4
correspond to films with 0, 2.5, 5, 7.5, 10 % BLE respectively.

Fig. 5. FT-IR spectra of flaxseed gum based composite films added with different betel leaf extract (BLE) concentrations. BLE0, BLE1, BLE2, BLE3, BLE4 correspond
to films with 0, 2.5, 5, 7.5, 10 % BLE respectively.

incorporated with Ziziphora clinopodioides essential oil, apple peel
extract and zinc oxide nanoparticles [52]. Moreover, the incorporation
of BLE resulted in the splitting of the band associated with the bending
vibration of the methylene group. However, the control did not show
remarkable peaks at the respective bands at 2875 and 2895 cm− 1. The
intensity of the peak at 1400 cm− 1 assigned to the amide III vibration
and 1650 cm− 1 attributed to the amide II, significantly declined with the
increase in BLE. These changes were likely due to the possible physical
and chemical interactions between OH and CO groups of BLE with
functional groups in flaxseed gum (–OH, –NH2). In addition, these

10
A. Manzoor et al. International Journal of Biological Macromolecules 245 (2023) 125562

interactions can also be correlated to the functional properties (antiox­ Table 2

idant and antimicrobial) of the composite films. Similar results were also Degradative temperature and mass loss of flaxseed gum-based films added with
reported by Fabra et al. [45] and Nguyen et al. [49] by incorporating the different concentrations of betel leaf extract (BLE).
green tea+grape seed phenolic extracts into alginate-based films and Mass loss (%)
betel leaf extract into chitosan-based films respectively. Temperature range (◦ C) BLE0 BLE1 BLE2 BLE3 BLE4

35–170 13.03 12.18 11.17 10.24 9.8

3.7. Thermal properties
The Thermogravimetric technique, an important technique for
examining the thermal feasibility of polymer composites in fabricating
packaging materials, was employed to study the thermal properties of
FSG-BLE composite edible films (Fig. 6). Table 2 depicts the thermal
properties of composite films with or without betel leaf extract recorded
within the temperature range of 30–800 ◦ C. Four stages of decomposi­
tion were presented by all the composite films with only the control
sample showing early degradation and a three-stage degradation. In the
control samples (BLE0), the first stage of decomposition corresponding
to a temperature range (35–170 ◦ C) is associated with the evaporation of
free and bound water and witnessed a 13.03 % loss in weight. The
second stage of degradation (180–240 ◦ C) designates the weight loss
caused by the decomposition of glycerol constituting the film makeup
[25,30]. The third stage of degradation (250–345 ◦ C) corresponds to the
degradation and depolymerization of polysaccharide units present in
flaxseed gum [25].
The degradative temperature range in the composite films was
recorded in the range of 250–345 ◦ C with the addition of betel leaf
extract (BLE) which is correlated to the decomposition of efficiently
embedded stable phenolic compounds within the polysaccharide matrix
[53]. TGA analysis of the composite films containing BLE witnessed a
progressive reduction in weight loss possibly credited to the reduced
amount of moisture absorbed within the polymeric matrix due to the
strong BLE-polysaccharide chemical interaction. Compared to the con­
trol, the main stage of decomposition (250–345 ◦ C) showed a lower
weight loss in the BLE composite films. This confirms that the addition of
BLE ameliorates the thermal stability of the composite films attributed to
the physical and chemical interactions between the functional com­
pound of BLE and the film matrix. Moreover, the composite films
showed comparatively lower values of decomposition temperature than
the control film as the phenolic compounds in the extract may be heat
180–240 18.41 16.00 14.29 13.49 13.27
250–345 30.68 27.28 25.92 23.73 21.26
350–475 56.91 – – – –
BLE0, BLE1, BLE2, BLE3, BLE4 correspond to films with 0, 2.5, 5, 7.5, 10 % BLE
respectively.
sensitive and hence easily decomposed. Our results are in line with the
results explained by Wang et al. [30] in a study on chitosan films
modified with black soybean seed coat extract. The control and the
composite films (with 0, 2.5, 5, 7.5 and 10 % BLE) remained with an ash
content of 30.68, 27.28, 25.92, 23.73 and 21.26 %, respectively at the
end of the thermal degradation supporting that the addition of the
extract did not affect the thermal stabilities significantly which is in line
with some previous reports of blend films [25,49]. Hence, it is impera­
tive to conclude that the inclusion of BLE into the FSG film matrix
increased the strength along with the thermal stability of the composite
films making them ideal for application to packaging of foods developed
through heat processing.
3.8. Total phenolic content
The antioxidant activity of betel leaf extract is majorly derived from
the phenolic and polyphenolic compounds which act as effective free
radical scavengers besides being potential antioxidants. Hence, a close
correlation exists between the content of polyphenolic compounds and
the antioxidant activity [10]. Fig. 7 sums up the total phenolic content of
the flaxseed gum-based composite films which show the lowest phenol
value for the pure flaxseed gum-based film (30.21 ± 0.55 mg GAE/100
g) and the highest value (111.98 ± 0.76 mg GAE/100 g) for the BLE4
film sample with the highest concentration of BLE incorporated (10 %).
This means that the addition of BLE increased the total phenolic content
significantly compared to the control sample. The reason for the TPC

Fig. 6. Thermogravimetric analysis (TGA) of flaxseed gum based composite films added with different betel leaf extract (BLE) concentrations. BLE0, BLE1, BLE2,
BLE3, BLE4 correspond to films with 0, 2.5, 5, 7.5, 10 % BLE respectively.

11
A. Manzoor et al.
Fig. 7. Total Phenol content and antioxidant (DPPH) activity, of flaxseed gum
based composite films added with different betel leaf extract (BLE) concentra­
tions. BLE0, BLE1, BLE2, BLE3, BLE4 correspond to films with 0, 2.5, 5, 7.5, 10
% BLE respectively.
increase in the composite films is attributed to the addition of BLE as
betel leaf extract contains a pool of phenolic and other bioactive com­
pounds. Das et al. [54] reported a total phenolic content of 50 mg GAE/
10 mg extract for betel leaf extract. Akter et al. [55] also reported a
comparable amount (124.42 ± 0.14 mg of GAE/ g of extract) of total
phenolic content in betel leaf extract. Hence, betel leaf extract incor­
poration significantly improved the phenolic content of the composite
films. Similar results were reported by Yousuf, Wu & Gao [5] who added
Schisandra chinensis oil/extract to the film based on karaya gum. Fabra
et al. [45] also reported a boost in phenolic content in alginate-based
active films modified with green tea extract.
3.9. DPPH radical scavenging activity
The antioxidant activity of FSG-BLE composite films is depicted in
Fig. 7. The pure sample (without BLE) showed the lowest antioxidant
activity as depicted by the percent scavenging activity of DPPH (21.76
± 0.25 %). However, the films containing BLE were found to be having
higher DPPH scavenging activity than films without BLE addition and
the highest DPPH scavenging potential (83.05 ± 0.38 %) was reported
in the BLE4 sample with 10 % BLE. The BLE-incorporated film samples
showed significant differences (P < 0.05) with increasing the BLE
concentration as observed by Tagrida et al. [56] who reported an un­
expected upsurge in antioxidant activity of fish/gelatin blend films
modified with betel leaf extract. This increase in antioxidant activity as
determined by DPPH radical scavenging activity is again credited to the
betel leaf extract addition that possesses numerous phytochemicals such
as phytol, eugenol, and hexadecanoic acid with strong antioxidant ac­
tivity [57]. Further, the availability of phenolic compounds also con­
tributes to the upsurge in antioxidant activity which is evident from the
results of TPC, showing significant improvement when the BLE con­
centration was increased from 0 to 10 %. Hence, the antioxidant activity
has a definite correlation with the total phenolic content as reported by
Aryal et al. [58] in their study on different wild vegetables. Similar re­
sults of enhancement in antioxidant activity of alginate-based active
films by the addition of green tea extract and a grape seed extract were
reported by Fabra et al. [45].
3.10. Antimicrobial characteristics of developed films
In active packaging, antimicrobial activity is considered one of the
essential factors because food spoilage is caused by pathogenic micro­
organisms. Several studies have already reported the antimicrobial ac­
tivity of betel leaf extract advocating for its potential to be incorporated
into composite films for use in active packaging applications [59,60].
12
International Journal of Biological Macromolecules 245 (2023) 125562
Two species of microorganisms, namely Pseudomonas aeruginosa – PA01
(Gram-negative) and Staphylococcus aureus – ATCC 25923 (Gram-posi­
tive) were taken for the determination of the antimicrobial activity of
the developed composite films depicted by the inhibitory zone measured
in millimeter (mm) using a vernier caliper. The antimicrobial activity of
the composite films added with different levels of BLE is presented in
Table 3. It can see that the control sample did not produce any zone of
inhibition against any microorganism indicating no antimicrobial ac­
tivity as the films were developed with no BLE addition. The highest
zone of inhibition (24.22 ± 0.42 mm) was detected in film sample BLE4
containing 10 % of the extract against Staphylococcus aureus however,
the same film sample displayed a lower inhibition zone (17.58 ± 0.86
mm) against Pseudomonas aeruginosa. Generally, the inhibitory zone
increased with the increase in BLE concentration in treated samples
irrespective of the type of microorganism hence, deducing that BLE
incorporation increased the antimicrobial potential of the composite
films. The BLE-added composite films were more effective against the
Gram-positive as compared to the Gram-negative bacteria as depicted by
the zone of inhibition due to the less susceptibility of Gram-negative
bacteria to microbial action, credited to the difference in morphology
between the two [1]. The antimicrobial activity of the composite films is
attributed to the strong antimicrobial potential of BLE due to the pres­
ence of bioactive compounds such as eugenol propenylphenols, allyl­
pyrocatechol diacetate, chavibetol, chavibetol acetate, chavicol, and
hydroxychavicol [61]. Similar results were also reported for sago starch
composite films modified with betel leaf extract [1].
4. Conclusion
The study reported that an increase in betel leaf extract concentra­
tion (0–10 %) lead to the development of FSG-BLE composite films with
superior physicochemical, mechanical, and functional characteristics.
Moreover, water vapor permeability, the deciding factor of the film for
packaging applications was reduced by the BLE incorporation however,
the solubility and the seal strength of the film increased with an increase
in the BLE level. The composite film containing 10 % BLE was observed
to be darker in color and presented the highest antimicrobial confron­
tation against Staphylococcus spp. SEM and XRD analysis reported an
enhancement in film smoothness and crystallinity as compared to the
control film sample whereas thermogravimetric analysis reported an
improvement in thermal stability. The study revealed the addition of
BLE (10 % in particular) increased the film solubility and enhanced the
film strength compared to the film without BLE. Moreover, amelioration
in the phenol content and the antioxidant activity of the composite films
was also witnessed as the BLE concentration increased, supporting its
utilization in food packaging applications for enhancing the shelf life of
spoilage-prone food products. Contrary to the control sample, a reduced
mass loss (%) was observed in the treated films by increasing the BLE
concentration as depicted by the thermal properties. To widen the ho­
rizon of the present study, the developed films can be explored by
Table 3
Antimicrobial activity of flaxseed gum-based films added with different con­
centrations of betel leaf extract (BLE).
Sample Test microorganism (antimicrobial activity measured as inhibitory zone in
mm)
Pseudomonas aeruginosa Staphylococcus aureus
a
BLE0 0 ± 0.00 0 ± 0.00a
BLE1 5.23 ± 0.02b 7.24 ± 0.41b
BLE2 8.37 ± 0.36c 11.13 ± 0.38c
BLE3 12.27 ± 0.39d 16.24 ± 0.36d
BLE4 17.58 ± 0.86e 24.22 ± 0.42e
Results are expressed as mean ± standard deviation, n = 3. Different lowercase
superscript letters in the same column indicates statistical significance for the
test microorganism. BLE0, BLE1, BLE2, BLE3, BLE4 correspond to films with 0,
2.5, 5, 7.5, 10 % BLE respectively.
A. Manzoor et al.
investigating their effect on food products such as meat/meat products
and fresh-cut fruits and vegetables.
CRediT authorship contribution statement
Arshied Manzoor: Investigation, Formal analysis, Data curation,
Writing - original draft, Methodology, Software. Saghir Ahmad: Su­
pervision, Project administration, editing. Basharat Yousuf: Concep­
tualization, Validation, Writing -review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The first author would like to extend his gratitude to the Indian
Council of Medical Research (ICMR), New Delhi for providing financial
assistance in the form of a Senior Research Fellowship (SRF). Moreover,
the authors are also grateful to the Department of Post-Harvest Engi­
neering and Technology, Department of Chemistry, University Poly­
technic and University Sophisticated Instrumentation Facility (USIF),
Aligarh Muslim University for making the sample analysis possible.
References
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