Progress in Organic Coatings 183 (2023) 107772

Contents lists available at ScienceDirect

Progress in Organic Coatings

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

Fabrication of antimicrobial edible films from chitosan incorporated with

guava leaf extract
Thiet Anh Dinh a, b, Yen Nhi Le a, b, Nhat Quyen Pham a, b, Phu Ton-That a, b, Thinh Van-Xuan a, b,
Thanh Gia-Thien Ho c, Tri Nguyen c, d, *, Ha Huynh Ky Phuong a, b, **
a
Vietnam National University Ho Chi Minh City, Linh Trung Ward, Thu Duc Dist., Ho Chi Minh City, Viet Nam
b
Faculty of Chemical Engineering, Ho Chi Minh City University of Technology (HCMUT), 268 Ly Thuong Kiet Str., Dist. 10, Ho Chi Minh City, Viet Nam
c
Institute of Chemical Technology, Vietnam Academy of Science and Technology, No.1A, TL29 Str., Thanh Loc Ward, Dist. 12, Ho Chi Minh City, Viet Nam
d
Ho Chi Minh City Open University, 97 Vo Van Tan Str., District 3, Ho Chi Minh City, Viet Nam

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

Keywords: With the aim of ramping up the production of edible films that are both environmentally friendly and anti­
Fabrication bacterial for packaging applications, we researched biofilms and evaluated the extract incorporation on the
Antimicrobial antioxidant, antibacterial, and physical properties based on a blend of chitosan (CS), guava leaf extract (GLE),
Edible film
and glycerol for the first time. This study used the casting technique to produce chitosan-based films, and guava
Chitosan
Guava leaf
leaf extract was prepared at different concentrations (0–3 %). Cross-section morphologies, film’s thickness, the
Extract functional group interaction between chitosan, glycerol, and guava leaf extract, thermal behaviours of chitosan
films comprised of DSC (differential scanning calorimetry) and TGA (thermogravimetric analysis), mechanical
and physiochemical properties including tensile strength and elongation at break, optical, moisture content,
water permeability, coating as well as biodegradation were employed in this research. The results showed that
the antioxidant properties of the films improved when increasing the concentration of GLE. Likewise, increasing
the concentration of GLE also lead to improvement in the biodegradation of chitosan-based films. Among these
films, the scavenging activity of C2G35E2.0 (84.59 ± 0.73 %) was two times as much as the control chitosan film
(45.33 ± 1.14 %). Moreover, C2G35E2.0 had the highest tensile strength (26.97 ± 0.46 MPa), elongation at
break (42.18 ± 1.35 %), and the smoothest surface in SEM images. Due to these combined properties, the
produced blend films containing guava leaf extracts are suitable candidates for edible and eco-friendly packaging
applications, as they may keep fruits fresher in an eco-friendly, sustainable manner.

1. Introduction films are derived from crude oil and natural gas and may contain haz­
ardous additives [4]. They are durable and can take up to 1000 years to
Food preservation, particularly food packaging, can improve food decompose completely. This can cause severe environmental pollution.
utilization and prevent bacterial spoilage. In a more detailed way, cus­ According to the statistics from OCED in 2021, just 9 % of plastic
tomers pay attention to high-quality fruits and vegetables since those garbage generated worldwide will be recycled, 19 % will be destroyed,
fresh foods, including many healthy nutrients such as vitamins, min­ and around 50 % will be buried in landfills. The remaining 22 % is
erals, and amino acids, are beneficial to the human body [1,2]. How­ plastic waste that has either been improperly handled or leaked to the
ever, one of the significant issues for fruit and vegetable storage safety is outside. Researchers are focusing on investigating and developing new
the fast decay rate and short shelf-life time. Improper food storage generations of bioactive and friendly films. Switching from conventional
during preservation can cause physical changes such as surface films to biodegradable biofilms will significantly enhance environ­
shrinkage, loss of luster, and fruit rot due to high water transpiration, mental protection because they can degrade [5].
nutrient loss, and microbial spoilage [3]. Conventional food packing Chitosan (CS), the second-largest waste product in the world, is

* Correspondence to T. Nguyen, Institute of Chemical Technology, Vietnam Academy of Science and Technology, No.1A, TL29 Str., Thanh Loc Ward, Dist. 12, Ho
Chi Minh City, Viet Nam.
** Correspondence to: H.H.K. Phuong, Vietnam National University Ho Chi Minh City, Linh Trung Ward, Thu Duc Dist., Ho Chi Minh City, Viet Nam.
E-mail addresses: ntri@ict.vast.vn (T. Nguyen), hkpha@hcmut.edu.vn (H.H.K. Phuong).

https://doi.org/10.1016/j.porgcoat.2023.107772
Received 17 March 2023; Received in revised form 6 June 2023; Accepted 22 June 2023
Available online 26 June 2023
0300-9440/© 2023 Elsevier B.V. All rights reserved.
T.A. Dinh et al. Progress in Organic Coatings 183 (2023) 107772

obtained from a deacetylated chitin derivative by chemical or packaging films could result in a new functionality for the guava in­
biochemical treatment of the outer skeleton of crustacean [5]. Chitosan dustry converting the waste guava leaves into a highly useful product.
has many uses because of its remarkable properties, including antibac­ Therefore, this research was to evaluate the antioxidant and antibacte­
terial, antifungal, biodegradable, highly biocompatible, and safe for rial property of chitosan films loaded with guava leaf extract having
humans [5]. According to the United States Food and Drug Adminis­ both antioxidant and antimicrobial activities. In addition, the micro­
tration (FDA), chitosan is a safe dietary supplement additive and can structural, mechanical and physical properties such as optical, moisture
lower cholesterol [6]. Nevertheless, most polysaccharide-based films are content, water vapor, swelling degree, biodegradability of the films were
brittle and prone to cracking during use and storage. Thus, glycerol was investigated. Moreover, the external morphological change of banana
added as a plasticizer to the film to improve its mechanical properties, fruit and tomato vegetable protected by chitosan-based film was also
such as elasticity, and reduce the brittleness and fragility of pure chi­ assessed during storage. The results obtained are discussed and pre­
tosan films [7]. Besides that, to enhance chitosan films’ antibacterial, sented, in conjunction with relevant explanations.
antifungal, and antioxidant properties, biological substances such as
fruit peels extract, leaves, seeds, or nanomaterials can be successfully 2. Experimental
associated with the material [8]. Additionally, composite films based on
plant extracts from the previous study, such as banana peel extract [9], 2.1. Materials
byrsonima crassifolia extract [10], and mango peel extract [11], have
been effectively employed to increase the quality and shelf life of several Chitosan (medium molecular weight and 85 % de-acetylation de­
fruits. Polyphenols are physiologically active chemicals with antioxidant gree), Acetic acid (CH3COOH), Glycerol (C3H8O3), Silica Gel, Sodium
and antibacterial properties in many naturally occurring plants. Poly­ Chloride (NaCl), Sodium carbonate anhydrous (Na2CO3) were supplied
phenols can protect the body against a variety of diseases. Polyphenols’ from Xilong ScientificCo, China. Folin-Ciocalteu’s phenol were pur­
antioxidant mechanism primarily depends on their abilities to scavenge chased from MERCK, Germany, and Methanol (CH3OH) were provided
reactive oxygen species and chelate metal ions [12]. from Chemsol, Vietnam. The plant material (Psidium guajava L.) was
Guava (Psidium guajava Linn) is a popular fruit plant in many tropical collected from District 12, Ho Chi Minh City, south of Vietnam.
and subtropical areas. Guava leaf has been utilized as an herbal tea for
many years in Vietnam, China, Japan, India, and other countries due to 2.2. Preparation and characterization of Guava leaves extract
its extensive bioactivities and pharmacological benefits. Guava leaves
contain a variety of antioxidant and antibacterial compounds, including Guava leaf extraction (GLE) was prepared in accordance with
tannins, phlorotannins, saponins, terpenoids, alkaloids, and polyphenols Jongkwon Seo et al. [19] with some slight modifications. Briefly, a
[12]. The beneficial ingredients in guava leaves, such as quercetin, sample of 20 g guava leaves powder in 300 mL of distilled water was
lycopene, and vitamin C, assist in the fight against cancer. Guava leaf boiled for 4 h. The extracted solution was then collected after the sample
extract is used in food preservation to extend fruit’s shelf life. Guava leaf had been vacuum filtered. Then, the obtained extract was stored at 4 ◦ C
extract exhibits highly potent antibacterial activity against strains of for further experiments.
Staphylococcus, Shigella, E. coli, Clostridium, etc. [13]. These benefits The Folin-Ciocalteau method was used to determine the total
make Guava leaf extracts ideal for edible film production. phenolic content (TPC) of GLE [20]. During the reduction process, the
In the food industry, several bacteria are harmful to human health. hydroxyl groups are easily oxidized, resulting in a blue color with a
Escherichia coli (E. coli) is a Gram-negative bacterium in human and maximum absorbance of 765 nm. In this study, 2 mL of GLE solution was
animal gastrointestinal tracts. They may be found in the intestines of mixed with 5 mL Folin Ciocalteau 10 % and reacted for 5 min in the
people and animals such as cows, pigs, and goats. It is also present in raw dark. After the reaction, 4 mL of saturated Na2CO3 solution was added
meat, unpasteurized milk and juices, and contaminated water [14]. and reacted for 60 min. The absorbance of the reaction solution was
Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative aerobic bac­ recorded at 760 nm. The total phenolic content of GLE was determined
terium that causes disease in animals and humans. P. aeruginosa is based on the calibration curve of gallic acid.
widely spread in the environment and is frequently carried by humans. Rutin’s calibration curve was used to calculate the total flavonoid
P. aeruginosa has been found in water, dairy products, meat, and plant- content (TFC), which is represented as milligrams of rutin equivalent per
based foods [15]. Staphylococcus aureus (S. aureus) is a Gram-positive gram of extract (mg RE/g extract). The total flavonoid content was
bacterium that belongs to the Bacillus and is a standard part of the measured in accordance with Nidal Jaradat [21]. Briefly, 100 mg of
body’s microbiota, commonly found in the upper respiratory system and rutin was dissolved in 10 mL of distilled water and then diluted to make
on the skin. The existence of S. aureus toxin in food is frequently caused 100 mL. Then, the stock solution was subsequently diluted to create a
by cross-contamination of prepared food with raw food [16]. Entero­ range of concentrations (5, 10, 20, 40, and 100 μg/mL). A total of 5 mL
coccus faecalis (E. faecalis) is a Gram-positive, facultative anaerobe. It of distilled water, 3 mL of methanol, 0.2 mL of 10 % AlCl3, 0.2 mL of
can cause a wide range of infections, including endocarditis, urinary potassium acetate 1 M, and 0.5 mL of each diluted solution were com­
tracts, prostatitis, intra-abdominal infections, cellulitis, surgical site in­ bined. Finally, the mixtures were incubated at room temperature for 30
fections, and infectious disease blood infection [12]. Moreover, adding min. The GLE sample was prepared using the same procedure, which
guava leaf extract (GLE) can inhibit Methicillin-resistant Staphylococcus included distilled water with methanol, 10 % AlCl3, and potassium ac­
aureus (MRSA), a group of Gram-positive bacteria genetically distinct etate as a blank. The absorbance of all the samples was measured at a
from other strains of Staphylococcus aureus. MRSA may cause various wavelength of 415 nm.
potentially fatal infections, including septic shock, endocarditis, abscess
formation, and severe pneumonia [17]. MRSA infection transmission 2.3. Fabrication of films
mechanisms may be similar to S. aureus, generally found in raw food,
prepared food products (such as meat and fish), and among individuals The films were developed following the solution casting method.
[18]. For instance, meals provided in public hospitals and retail food First, chitosan 2 % (w/v) was dissolved in 100 mL of 2 % (in volume)
environments should be given extra consideration since they are meant acetic acid aqueous solution, which was thoroughly stirred for 5 h. The
for consumption by patients, a group at high risk of contracting chitosan solution was then given glycerol 35 % (v/w) as a plasticizer and
numerous S. aureus-caused disorders. Leaf extracts as an ideal material stirring was continued for 30 min. After that, different ratios of guava
for edible film production. As a result, developing food packaging to leaf extract (GLE) were added to the mixed solution and homogenized
prevent the hazards of the presence of these germs is critical. for 4 min. Then, the mixture was placed into the ultrasonic bath to
A scientific study with the objective of GLE incorporated active food remove the air bubbles. After being poured into 12 cm diameter Petri

2
T.A. Dinh et al. Progress in Organic Coatings 183 (2023) 107772

plates, the film-forming solutions were dried for 24 h at 60 ◦ C. Before the

Δm x
further examination, the generated film samples were peeled at room WVP = × (7)
ΔtA ΔP
temperature and stored in plastic bags within desiccators [22]. The
composite films was labelled with 0 %, 1 %, 1.5 %, 2 %, 2.5 % and 3 % where ΔtA
Δm
is the weight of the moisture gain per unit of time (g/s), A is an
GLE were named C2G35E0, C2G35E1.0, C2G35E1.5, C2G35E2.0,
area of the exposed film surface (m2 ), x is the film thickness (mm), and
C2G35E2.5, C2G35E3.0 films, respectively [8,23].
ΔP is the difference of partial pressure.
The swelling degree experiment was conducted according to Bierhalz
2.4. Characterization of films et al. [27] with some modifications. The film samples were cut with 5
cm × 5 cm dimensions, and their initial weight was measured. Then the
2.4.1. Physiochemical properties film samples were immersed in distilled water at room temperature for
A colorimeter equipped with a D65 illuminant and a 10o observer 60 min. At different time intervals, the swelled films were put between
was used to detect the film’s color characteristics (L*, a*, and b* values), two Whatman filter papers to remove the remaining water from the film
which were determined after the film had been attached to a standard surface and reweigh. The swelling degree (SD) was calculated by Eq. (8):
white plate. The following equation was used to determine the hue angle mf − mi
SD (%) = × 100 (8)
(H) and total color difference (ΔE) of film [24]: mi
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
ΔE = (Ls − L* )2 + (as − a* )2 + (bs − b* )2 (1) where mf is the mass of the wet (swollen) film (g), and mi is the mass of
the dried film.
( *)
b
H = tan− 1
when a* > 0 and b* > 0 (2) 2.4.2. Antioxidant activity
a*
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) method was used to
( *) determine the antioxidant activity of CS with guava leaf extract by
b
+ 180 when a* < 0 (3) scavenging the free radical of DPPH, which followed the technique of
1 ◦
H = tan−
a*
Siripatrawan and Harte with some modifications [23]. CS was mixed
( *) with guava leaf extracts (from 0 %–3 % concentration of the extractions)
b
+ 360 when a* > 0 andb* < 0 (4) and then the mixture was combined with 3 mL of the solution including
1 ◦
H = tan−
a* (
mol
methanol and DPPH 10 3 L.) Then, the solution was shaken in a glass

where Ls = 85.8, as = 0.32 and bs = 0.33 are the color parameters of bottle for at least 1 min before incubating in the dark at room temper­
standard white plate. ature for a half hour [28]. The absorbance of the final solution was
A field emission scanning electron microscope (Hitachi S4800 in­ measured at 517 nm by utilizing a microplate spectrophotometer DPPH
strument) was used to observe the surface microstructure and cross- scavenging activity was calculated by the Eq. (9).
section of the films at 10 kV accelerating voltage. Fourier transform
infrared (FTIR) spectrum of the film was measured by a spectrometer (a ADPPH − Ab
DPPH scavenging activity (%) = × 100 (9)
Tensor 27-Bruker spectrometer in the range of 400–4000 cm− 1). The ADPPH
TGA and DSC analysis was carried out using a Setaram Labsys evo TG-
with ADPPH : the absorbance value at 517 nm of the DPPH methanol so­
DSC 1600C. The investigation was conducted under an N2 atmosphere
lution, Ab : the absorbance value at 517 nm of the DPPH solution.
in room temperature (25 ◦ C) to 600 ◦ C. The mechanical strength of films
was evaluated by tensile test using the Texture Analyser (TA. XTplus,
2.4.3. Antibacterial activity
Stable Micro Systems, Surrey, UK). Samples were cut into 50 mm × 10
The agar disk diffusion technique was used to determine a zone of
mm rectangles and vertically held by two mechanical miniature grips,
inhibition against MRSA, E. coli, S. aureus, P. aeruginosa, E. faecalis. After
leaving 30-mm-length for tensile loading. The crosshead speed of the
autoclaving and drying, the agar medium was injected with liquid
experiment was fixed at 0.005 mm/s at room temperature. The sample
overnight with culture at a cell density of 5105 CFU/mL. After the agar
thickness was predetermined using an electronic micrometre. The film
had been set, the wells in the agar were punched and filled with 10 μL of
sample’s moisture content (MC) is determined according to Priyadarshi
the film solution. After incubating the plates for 24 h at 37 ◦ C, the
et al. [25]. Firstly, the film (2 cm × 2 cm) was dried at 105 ◦ C for 24 h.
inhibitory zone width was determined. All of the tests were done in
The results were calculated as a function of the mass loss of the film
triplicate, and the average result was used [29].
before and after drying. The MC value was determined by Eq. (5):
W0 − W1 2.4.4. Biodegradability
MC (%) = × 100 (5)
W0 The biodegradation test of the film samples was carried out using a
modified version of the procedure outlined in the literature [30]. The
where Wo (g) was the initial weight of the film sample, W1 (g) was the soil was acquired from the garden at the Institute of Chemical Tech­
weight of the film after drying. nology (Ho Chi Minh City, Vietnam). The film samples were cut into
The film sample’s Water Vapour Permeability (WVP) was modified rectangular pieces (2 cm × 2 cm) and then weighed to get the initial
by ASTM standard method E96-95 [26]. The film sample tightly sealed mass (M0 ). After that, they were buried at 1–2 cm depth in plastic trays
the circular plate content with silica gel (0 % RH). After recording the containing the same dirt mentioned above. Water was sprayed twice a
initial weight, the plates were placed in a desiccator maintained at 25 ◦ C day to sustain the moisture of the soil. After 7 days, the film samples
and 75 % RH with a saturated sodium chloride solution. Then these were taken out, washed, and dried in the oven at 105 ◦ C for 24 h before
plates were weighed accurately at hour intervals over 6 h. The change of being weighed to get the dry mass (M1 ). The weight loss of the film
these plates’ weight was accurately 0.0001 g. Eqs. (6) and (7) were used samples is calculated by Eq. (10).
to evaluate and calculate the water vapor transmission rate and water
vapor permeability below: WL (%) =
(M0 − M1 )
× 100 (10)
M0
Δm
WVTR = (6)
ΔtA

3
T.A. Dinh et al. Progress in Organic Coatings 183 (2023) 107772

2.5. Coating application 3.2. Textural properties of the film

At room temperature, bananas, and tomatoes were coated in pure When it comes to film appearance, color characteristics are crucial
chitosan solution and chitosan with GLE ranging from 1 % to 3 %. The since they can affect how well packaged goods are received by con­
results were compared to the control sample (uncoated). Before the sumers. The color characteristics (L*, a*, and b* values), total color
coating process, fresh bananas and tomatoes were immersed in sodium difference (ΔE) and hue angle (H) of the films are displayed in Table 2.
hypochlorite (1 %) for 15 min, rinsed with tap water, and dried. The As can be seen from the table, the lightness (L*) of the C2G35E0 was
coated and uncoated fruits and vegetables were kept at room tempera­ highest and subsequently dropped as the GLE was added, indicating a
ture (T = 25 ± 1 ◦ C and relative humidity of 50 %) [31]. propensity towards darkness. The results demonstrated that the pro­
duced films have the potential to be used to wrap and preserve food
3. Results and discussion because it can help to lessen the effect caused by oxidation in packaged
foods [23]. On the opposite, the films’ a* (redness/greenness) and b*
3.1. Total polyphenol in Guava leaves extract (yellowness/blueness) values grew along with the addition in GLE
content, indicating that the films were becoming more red and yellow
From the equation of the standard gallic acid curve (y = 0.0603x − while a decline in the hue angle (H) was observed when GLE was
0.0021; R2 = 0.9994), the total phenolic content (TPC) of GLE was incorporated into CS films. The polyphenolic compounds in the GLE may
calculated as 25.67 ± 1.49 mg GAE/g. However, Julio César Camarena- be the reason for the change in color in the blended films [38]. When
Tello [32] observed a different TPC of guava leaf extract while utilizing a GLE was added to the films, the overall color difference (E) also
different extraction process and several kinds of extraction solvents increased, demonstrating more colored films. Lijun Sun et al. [39] and
(aqueous, acetone, and chloroform). This indicated that the extraction Dawei Yun et al. [8] also reported the same trend when thinned young
solvent and purification technique impacted the TPC [33]. The hydroxyl apple polyphenols and rambutan peel extract are added to chitosan
groups in plant extracts have redox properties that facilitate free radical films. Fig. 1 below illustrated the physical appearance of the film
scavenging, hence increasing the film’s antioxidant function [34]. samples.
Different concentrations of GLE will be applied to CS film in this Optimizing material thickness is essential to simultaneously achieve
experiment to test and compare the scavenging activity of each sample. desirable visual aesthetics and optimal barrier function against the
The total flavonoid content (TFC) of GLE was determined as 7.19 ± transfer of water and vapor. The diffusion rate is reduced as the edible
0.12 mg RE/g using the Rutin’s calibration equation. Lu Wang et al. [35] film thickness increases [40]. The thickness of the film material is pro­
have investigated the TFC of the guava leaf tea extract with and without portional to the procedures used to prepare it, such as solvent evapo­
the fermentation treatment and employing two different extraction ration time, drying, relative humidity, and dish surface [41]. Adding
processes and obtained the result ranging from 5.5 to 28.0 mg RE/g other substances, such as plasticizers, antioxidants, and antimicrobial
extract. This showed that the extraction process and treatment can also agents, may also boost the film thickness [42]. In this study, the control
altered the TFC of the sample. film samples in Table 3 showed lower thickness (0.028 ± 0.948 mm)
The TPC and TFC of guava leaf extract using water as the extraction than other tested samples. C2G35E3.0 film sample was recorded as the
solvent investigated by previous studies are illustrated in Table 1 below. highest with a thickness of (0.038 ± 0.159 mm), followed by C2G35E2.5
According to Julio César Camarena-Tello [32], there is a major variation (0.036 ± 0.820 mm), C2G35E2.0 (0.031 ± 0.251 mm), C2G35E1.5
between the TPC and TFC of two different varieties of guava leaves (0.030 ± 0.895 mm) and C2G35E1.0 (0.029 ± 1.034 mm) film samples.
(Calvillo Siglo XXI and Hidrozac). The results suggested that the TPC The thickness of those films increased (p < 0.05) as the GLE content
and TFC can be different based on the types of guava leaf in each region. increased, which coincides with a prior study in which chitosan film was
Besides, Jongkwon Seo et al. [19] have looked into how extraction supplemented with tea polyphenol [43]. The interactions between
solvents affect the amount of TPC and TFC in GLE. The results showed polyphenols and chitosan (including hydrogen bonding and hydropho­
that the TPC of the water extract is higher than that of the ethanol and bic force) increased as total phenolic concentration rose, resulting in
methanol extracts, while the TFC of the water and ethanol extracts is stronger binding of chitosan and polyphenols [43]. A similar outcome
identical and higher than methanol extract. The polarity of the poly­ was also discovered by Lijun Sun et al. [39]. Owing to the presence of
phenolic and flavonoid compounds, which made them more soluble in polyhydroxyl groups in the molecular structures; polyphenols are
water than in organic solvents may be the reason for this phenomenon. thought to operate as a bridge, bonding with more than one chitosan
According to the research of Quang-Vinh Nguyen et al. [36] into the molecule; as a result, the distance between chitosan molecules became
relationship between drying temperature and the amount of TPC and shorter, causing the film structure to become more compact and hence
TFC in GLE, the TPC and TFC are significantly impacted by drying cir­ increased thickness.
cumstances, including temperature and the moisture content of the As shown in Table 3, the C2G35E0 film displayed the highest mois­
material. In conclusion, there are numerous variables that affect the TPC ture content (MC) of 32.97 ± 1.06 %. The strong hydrogen bonding
and TFC of guava leaf extract, including the varieties of guava leaf, the between the functional groups of the CS and the water molecules may be
extraction solvent as well as purification method, the drying conditions, the reason for this phenomenon [44]. The MC decreased continuously (p
and weather conditions. < 0.05) to 25.23 ± 0.80 %, 22.46 ± 1.34 %, 19.11 ± 1.43 %, 17.91 ±
1.40 %, 15.54 ± 1.39 % along with adding 1.0, 1.5, 2.0, 2.5 and 3 % GLE
extract respectively. Dawei Yun et al. [8] and Dongsheng Hu et al. [45]
Table 1
also found that with increased rambutan peel extract and persimmon
Comparison of TPC and TFC in guava leaves extract with previous studies.
peel extract, the MC reduced gradually and reached its lowest point at
Extraction solvent TPC (mg GAE/g) TFC (mg RE/g) Refs. the highest concentration of extract. This could be due to the replace­
Water 25.67 ± 1.49 7.19 ± 0.12 This work ment of the interaction between CS and water by the intermolecular
Water 142 50 [19] interaction between hydrogen bonds with the GLE’s hydroxyl groups
Ethanol 118 50 [19] [45].
Methanol 90 35 [19]
Water (Calvillo Siglo XXI) 242.10 ± 13.33 108.54 ± 3.54 [37]
Water vapor permeability (WVP) is an essential parameter in the
Water (Hidrozac) 187.01 ± 9.24 77.83 ± 6.65 [37] food packaging industry since it indicates the barrier capabilities against
Ethanol (sun drying) 99.45 ± 0.41 14.90 ± 0.42 [36] water transfer [39]. Table 3 shows that the film incorporated with GLE
Ethanol (hot air 50 ◦ C) 145.38 ± 2.01 21.06 ± 0.21 [36] had WVP lower than pure chitosan film (p < 0.05). The more GLE there
Ethanol (hot air 80 ◦ C) 116.17 ± 1.39 17.74 ± 0.35 [36]
is, the lower the WVP. Mainly, pure chitosan film had a significantly

4
T.A. Dinh et al. Progress in Organic Coatings 183 (2023) 107772

Table 2
Color parameters and hue angle values of the film samples.
Film samples L* a* b* ΔE H

C2G35E0 43.84±0.35 3.00±0.03 2.74±0.06 42.16±0.34 137.65±0.39

C2G35E1.0 38.94±0.08 2.36±0.08 3.10±0.04 47.02±0.08 127.22±0.53
C2G35E2.0 38.21±0.55 2.26±0.04 3.47±0.76 47.76±0.59 123.66±5.74
C2G35E3.0 37.77±0.12 2.25±0.04 3.65±0.99 48.22±0.19 122.31±7.45

Fig. 1. Physical appearance of the film samples; a) C2G35E0, b) C2G35E1.0, c) C2G35E2.0, and d) C2G35E3.0.

higher WVP than C2G35E3.0 by 3.58 g Pa− 1 .s− 1 .cm− 1 .10− 13 . The
Table 3
interaction of chitosan, glycerol, and GLE can improve moisture resis­
Thickness, moisture content, water vapor permeability of C2G35E0, C2G35E1.0,
tance by generating a dense network structure that makes water mole­
C2G35E1.5, C2G35E2.0, C2G35E2.5, C2G35E3.0 films.
cules more challenging to penetrate [45].
Films Thickness Moisture WVP Swelling The swelling capacity of a film reflects the preservation of quality
(mm) content (g.Pa− 1 .s− 1 .cm− 1 .10− 13 degree
during food packaging and storage. The swelling degree of polymeric
)
(%) (%)
films is an instrumental parameter associated with the degree of cross-
0.028 32.97 ± 164.76 ±
linkage that occurs in the polymer network and consequently impacts
±
C2G35E0 0.948 1.06 11.6 ± 0.05 3.03
0.029 ± 25.23 ± 113.71 ± the waterproofing of the film; the lower the swelling degree of polymeric
C2G35E1.0 1.034 0.80 11.2 ± 0.07 1.53 films, the more excellent the water resistance of the film [46]. Indeed, as
0.030 ± 22.46 ± 69.32 ± is well known, the amount and character of intermolecular chain con­
C2G35E1.5 0.895 1.34 11.0 ± 0.13 2.60 nections substantially influence the degree of swelling of a polymeric
0.031 ± 19.11 ± 30.25 ±
C2G35E2.0 0.251 1.43 9.19 ± 0.53 2.72
substance [47]. In this investigation, the incorporation of guava leaf
0.036 ± 17.91 ± 29.68 ± extract in chitosan-based films drastically decreased the swelling ca­
C2G35E2.5 0.820 1.40 8.67 ± 1.01 3.07 pacity of the film. The control (C2G35E0) film had the lowest water
0.038 ± 15.54 ± 28.29 ± resistance capability compared to the other bioactive films, as indicated
C2G35E3.0 0.159 1.39 8.02 ± 1.06 1.47
in Table 3. In comparison to the pure chitosan film, the chitosan film

5
T.A. Dinh et al. Progress in Organic Coatings 183 (2023) 107772

treated with the highest concentration of (3 %) guava leaf extract since there is no presence of pores as well as wrinkles, which also
exhibited the lowest swelling capacity (28.29 ± 1.47 %), followed by showed that guava leaf extract had decent compatibility with films’
C2G35E2.5 (29.68 ± 3.07 %), C2G35E2.0 (30.25 ± 2.72 %), components (CS and glycerol).
C2G35E1.5 (69.32 ± 2.60 %) and C2G35E1.0 (113.71 ± 1.53 %) films. Tensile strength (TS) and elongation at break (EAB) are two crucial
These declines can be explained by enhancing crosslinking between the film qualities. Tensile strength, which reflects the greatest strength that
chitosan matrix and plant extract, indicating that the incorporation of a film can resist, is essential to preserve the structural integrity and
GLE promoted interactions with the polar groups of the formulation barrier property of films. At the same time, elongation measures a film’s
components, resulting in fewer polar groups available to interact with capacity to stretch. In general, the mechanical behavior of composite
water molecules, resulting in a lower swelling degree [28]. The addition film is determined by the type and proportion of the incorporated active
of 1 % GLE to 1.5 % GLE results in an abrupt decrease in swelling degree compounds, the kind of polymeric matrix, and the particular in­
of the films. This could be explained better by the formation of strong teractions between different components. The tensile stress-strain
hydrogen bonds between the CS matrix and the extract’s polyphenolic curves of pure chitosan and blended films are shown in Fig. 4. These
components to form a rigid and compact structure in a thin films [48]. materials exhibit comparable behavior, with an initial stress that in­
However, the slight decrease in swelling degree from 1.5 % to 3 % GLE creases with strain, defining an obvious elastic region before plastic
concentration shows that the extract’s components may achieve satu­ deformation. The chitosan film exhibits increased plasticity, as seen by
ration or have limited interaction with the chitosan matrix at higher the mechanical parameters demonstrated in Fig. 3. When compared to
concentrations. The extract’s influence on chitosan film swelling
behavior becomes less evident, resulting in a smaller decrease in
swelling degree.
The microstructure provides information about the structural
morphology of each film and the distribution of different components in
these edible films. According to Fig. 2, CS film without guava leaf extract
had a dense structure with some pores owing to the formation of
intermolecular and intramolecular hydrogen in the combination of the
CS chain [49]. In the recently published research [50], the authors
utilized quantum chemistry calculation to prove that when they added
glycerol to CS, the CS molecules were plasticized entirely because CS
and glycerol shared a hydrogen-bond position, which made hydropho­
bic C-H ending groups to prevent the formation of intermolecular
hydrogen bonding networks. Therefore, the plasticizer with a single
hydrogen bonding position and hydrophobic ending groups was utilized
to break down the intermolecular hydrogen bonding networks and free
the chitosan molecular movements. Moreover, the pores diminished as
the concentration of guava leaf extract increased from 1 % to 2 %, which
made the surface of C2G35E2.0 smoothest, without any wrinkles or
pores. This might reveal that C2G35E2.0 had a homogeneous solution
Fig. 3. Tensile Strength and Elongation at Break of film samples [51].

Fig. 2. SEM of samples [51]; a) C2G35E0, b) C2G35E1.0, c) C2G35E1.5, d) C2G35E2.0, e) C2G35E2.5, and f) C2G35E3.0.

6
T.A. Dinh et al. Progress in Organic Coatings 183 (2023) 107772

chitosan films combined with GLE, the better plasticity of pure chitosan
film can be indicated by a larger elongation at a lower applied tensile
stress. The presence of GLE improves tensile strength while simulta­
neously decreasing elongation. The addition of additives causes an in­
crease in stress and a decrease in elongation, which is known as the
“anti-plasticization effect” and has been found in several chitosan
blended systems [52]. Although not clearly understood, it is accepted
that the anti-plasticization occurs when small content of the additive
favors more compact hydrogen bonds between the components which
decreases free volume and molecular polymer mobility [53]. The TS and
EAB figures of pure chitosan film were measured at 18.89 ± 0.14 MPa
and 37.85 ± 0.24 %, respectively. These TS and EB values were more
significant than those recorded by Ubonrat Siripatrawan et al. [54] (TS
= 6.2 MPa and EAB = 5.3 %) for glycerol-plasticized chitosan films.
Various aspects such as molecular weight, deacetylation degree, plasti­
cizers, phase interaction, film production circumstances, and storage
determine this. The films’ mechanical properties were highly connected
to their internal microstructures and intermolecular interactions be­
tween film components [45]. Conversely, the TS value was dropped in
the sample with the lowest GLE amount (1 %). This could imply that Fig. 4. Stress strain curve of the films.
weaker hydrogen bonds between polymer matrix and extract composi­
tions partially replaced stronger polymer-polymer linkages, potentially
causing interchain distances in the polymer network to expand and
structural discontinuities to form, reducing the resistance to fracture of
the film material [46,55]. Furthermore, as the GLE concentration
increased from 1.5 % to 2 %, the TS and EAB of the CS-GLE films
increased significantly (p < 0.05). This phenomenon was attributed to
the hydrogen bond interaction between CS and GLE, which resulted in a
tightly continuous internal structure that enhanced the TS and EAB of
the film [56]. However, the TS and EAB values of CS-GLE films
decreased slightly after adding 2.5 % and 3 % GLE. This result was
similar to that investigated by Yun et al. [8] and Luo et al. [57], who
concluded that the addition of incorporated extract at higher content
produces excessive hydrogen linkages between polyphenol compounds
and chitosan matrix, which may induce polymer aggregation and hence
reduce chain mobility and flexibility under compressive stress.
Increasing the GLE content from 2.5 % to 3 % resulted in a significant
reduction in EAB value. It was postulated that the formation of hydrogen
linkages between chitosan and phenolic compositions might decrease
free volume and drive the decline of film plasticity [55]. Based on
Table 4, despite a noticeable deterioration in fracture resistance, the TS Fig. 5. FT-IR spectra of samples; a) C2G35E0, b) C2G35E1.0, c) C2G35E1.5, d)
and EAB values of chitosan films containing GLE (2.5–3.0 %) were still C2G35E2.0, e) C2G35E2.5, and f) C2G35E3.0.
better than several previously reported chitosan-based films containing
different types of extracts (TS = 15 MPa and EAB = 3.11 %) [58], (TS = molecules of CS, glycerol, and water [8]. In addition, CS exhibited bands
15.3 MPa and EAB = 17.2 %) [54] and (TS = 16.0 MPa and EAB = 21.0 at frequencies of 1644 cm− 1 for amide-I, C–
– O stretching, 1555 cm− 1 for
%) [59]. In this research, adding 2 % GLE to chitosan exhibited the amide-II, N–H stretching, 1410 cm− 1 for CH2 bending vibration of
maximum mechanical property. As a result, CS film with 2 % GLE may glycerol in the CS film, 1381 cm− 1 for amide III, C–N stretching and
be efficient in safeguarding packaged food during transportation and 1026 cm− 1 for C–O skeletal stretching. The presence of these bonds was
storage. similar to the finding of Dawei Yun [8] and Behlul Koc et al. [61]. There
FT-IR spectroscopy is used to identify the intermolecular interactions was little change in the position of CH2 bending, C–N vibration, and
between the functional groups in CS and GLE. As shown in Fig. 5, the CS C–O skeletal stretching, along with the addition of GLE. Lijun Sun et al.
film displayed characteristic peaks around 3347 cm− 1, attributed to the [39] also found that the incorporation of thinned young apple poly­
vibration of O–H stretching [60], while the peak at 2920 cm− 1 was phenols into CS barely showed any remarkable shift in position
assigned to C–H stretching [48]. The O–H stretching band of the CS compared with the CS film. The addition of GLE altered the O–H
film was caused by intermolecular hydrogen bonding between the stretching wavelength in the range of 3301–3364 cm− 1. Dawei Yun et al.
[8] also reported the change in position and intensity of O–H broad­
band when rambutan peel extract was incorporated into CS. Adding GLE
Table 4 shifted the wavelength to a higher frequency regarding the N–H band.
Comparison of TS and EAB of different chitosan-based films. Moreover, the band of C– – O was shifted to 1635–1654 cm− 1 with the
Film samples Tensile (MPa) EAB (%) Refs. addition of GLE. These displacements suggested that the hydroxyl
Pure CS (C2G35E0) 18.9 ± 0.14 37.9 ± 0.24 This work groups in GLE formed intermolecular hydrogen bonds with the amino
Chitosan-GLE (C2G35E2.0) 27.0 ± 0.46 42.2 ± 1.35 This work and hydroxyl groups in CS molecules [45].
Pure CS 6.2 5.3 [54] Thermogravimetric analysis (TGA) was performed to assess the in­
CS-Propolis extract 15.3 17.2 [54]
fluence of Guava leaf extract at various concentrations (0 %, 1 %, 2 %,
CS-Pistacia terebinthus 15.0 3.11 [58]
CS- Grape seed extract 16.0 21.0 [59]
and 3 %) on the thermal stability of chitosan films. The weight losses of

7
T.A. Dinh et al. Progress in Organic Coatings 183 (2023) 107772

chitosan and chitosan-GLE films were shown in TGA profiles in Fig. 6 to

have three primary degradation stages between 30 and 800 ◦ C. The first
stage (40–150 ◦ C) corresponds to the volatilization of both adsorbed
water and residual acetic acid [62]. The second stage (150–250 ◦ C)
could be attributed to glycerol the decomposition [58]. The major
weight loss from 250 to 400 ◦ C (approximately 50.9 % breakdown) was
attributed to depolymerization and pyrolysis decomposition of the chi­
tosan polymer’s main chain [63]. In the first stage, the weight loss
percentage of bounded water was recorded at 13.57 %, 12.6 %, 10.18 %
and 8.58 %, respectively for pure chitosan, C2G35E1.0, C2G35E2.0,
C2G35E3.0. The weight loss of chitosan-extract films decreased as GLE
content was increased in the chitosan matrix, presumably owing to
hydrogen interaction between chitosan and phenolic in GLE [9]. In the
figure of C2G35E2.0, C2G35E3.0, the second stage linked to heat
degradation of glycerol was not detected about 150–250 ◦ C. It could be
ascribed to the interaction of glycerol with chitosan, phenolic sub­
stances, and as a consequence, glycerol molecules were enclosed by such
a cross-linking network, resulting in a shift in thermal degradation to
higher temperatures [64]. The maximum weight loss of neat chitosan,
CH- C2G35E1.0, C2G35E2.0, C2G35E3.0 at 275–296 ◦ C was observed
about 49.11 %, 45.87 %, 41.36 % and 37.42 %, respectively. Notably,
chitosan-GLE films presented relatively lower weight losses than plain
chitosan film, suggesting the incorporation of GLE could enhance the
thermal stability of chitosan film [49]. The residual mass at the final
stage of thermal degradation was determined to be 30.63 %, 35.70 %,
41.44 %, and 42.89 % for pure chitosan film, C2G35E1.0, C2G35E2.0,
and C2G35E3.0, respectively. In general, chitosan films blended with
GLE outperformed pure chitosan films in terms of thermal stability. A
similar trend was observed with the addition of banana peel extract,
which was similarly shown to have better thermal stability over the
chitosan film [9].
Morphological approaches can determine the miscibility of mole­
cules in a polymer. The DSC thermograms of all chitosan films are shown
in Fig. 7. The thermograms of C2G35E0, C2G35E1.0, C2G35E2.0,
C2G35E3.0 showed wide endothermic peaks around 100–150 ◦ C,
240–275 ◦ C and an exothermic peak of approximately 300 ◦ C. The
evaporation of the solvent traces (acetic acid and water) employed in
Fig. 6. TGA (a) and DSC (b) diagrams of film samples.
preparing chitosan films was related to the endothermal effects corre­
sponding to weight loss at a low temperature of the first endothermic
peak, and the second endothermic peak at 240–275 ◦ C showed that
glycerol and guava leaf extracts were denatured [58]. On the other
hand, the higher temperature exothermic peaks were because of poly­
mer decomposition [65]. It can be observed in the graph that there is an
increase in the peak temperature of the CS incorporated with GLE
sample compared to the C2G35E0 sample. This suggested that adding
GLE to CS films could improve their thermal stability [66]. Increased
melting points and temperature of deterioration following GLE incor­
poration in films suggest that these films may have more regular struc­
tures, better packing capabilities, or greater inter-chain interaction [66].
This conclusion supported the results of TGA above, which also
demonstrate that the incorporation of GLE into CS increase the thermal
stability of the blended films.

3.3. Antioxidant activity

Eliminating free radicals were frequently utilized to minimize the

oxidative stress of fruits and vegetables. In general, our results show that
the increase in the concentration of guava leaf extract in chitosan so­
lution might be useful for some commercial applications, such as the
food that are prone to oxidation [67]. An imbalance between the pro­
duction and accumulation of reactive oxygen species (ROS) in cells and
Fig. 7. Scavenging activities of C2G35E0, C2G35E1.0, C2G35E1.5, C2G35E2.0, tissues resulted in the phenomenon known as oxidative stress. There­
C2G35E2.5, C2G35E3.0 films. fore, it could damage the cells and tissues because of the overproduction
of ROS which was caused by environmental stressors (i.e., ionizing ra­
diations UV, heavy metals, as well as pollutants) [68]. One of the finest
characteristic properties of natural plants was their natural antioxidant

8
T.A. Dinh et al. Progress in Organic Coatings 183 (2023) 107772

Fig. 8. Antibacterial mechanism of chitosan-based film.

compounds which were also regarded as the most significant nutra­ promotes the interaction of phenolics with the cell membrane by
ceuticals for treating many diseases via scavenging free radicals. As hydrogen bonding. Depending on how hydrophobic they are, phenolics
depicted in Fig. 7, the CS without guava leaf extract exhibited the lowest can build up at the cell envelope’s surface, even cross their membrane to
percentage of scavenging DPPH activity. Moreover, as compared to the enter the microbial cells’ cytoplasm, where they can interact with
CS control, the antioxidant activity of CS coatings incorporated with various cell components or change the pH inside the enclosure [72].
guava leaf extracts (concentrations ranging from 1 % to 3 %) was Also, the interaction between the positively charged amino (–NH3)
approximately doubled. This might reveal that guava leaf extracts which groups of chitosan and the negatively charged carboxylate (–COO–)
were an abundant source of polyphenols as well as quercetins [69] were groups on the surface of bacterial cell membranes should be the critical
considered to be one of the best natural antioxidant compounds for mechanism for chitosan’s antibacterial activity [73], and when com­
increasing the percentage of DPPH scavenging activity as well as perfect bined with extraction, it can enhance antibacterial activity [74]. As
extractions that could complement with chitosan coatings. First, we shown in Fig. 10, these electrochemical binding has the potential to
move on to the research made by Ubonrat Siripatrawan et al. [23] which weaken and disrupt cell membranes. Chitosan has been widely
illustrated the DPPH scavenging activity between chitosan films and researched for its edible film-forming ability and natural antimicrobial.
green tea extract (GTE). There was a dramatic difference between the Table 5 and Fig. shows that C2G35E0 and C2G35E2.0 have antimicro­
antioxidant activity of both highest concentration extracts combined bial activity on 5 typical bacteria. The diameter of the inhibition zone of
with chitosan films, the 20 % GTE with around 50 % DPPH scavenging C2G35E2.0 on P. aeruginosa was higher than C2G35E0 [75]. Also, the
activity compared to 88.58 ± 0.54 % antioxidant activity of the diameter of inhibition zone C2G35E2.0 of E. faecalis was more signifi­
C2G35E3.0. The next research discussed the antioxidant property of cant than that of C2G35E0 by 2 mm. Fig. 9 depicts the MIC value,
chitosan films incorporated with pine needles extract [70]. The DPPH determining the pathogen’s sensitivity or resistance to a specific anti­
scavenging activity of C2G35E3.0 was 88.58 ± 0.54 %, which was twice biotic [76]. As a result, bioactive phenolic compounds can cause phys­
as much as the antioxidant activity of the chitosan films incorporated iological changes in microbial cell membranes, disrupt the cytoplasm’s
with pine needles extracts (41 %). In the previous studies, the DPPH DNA and RNA, and lead to bacterial mortality [77]. GLE base chitosan
scavenging activity of chitosan with green tea extracts and pine needles film can be used in packaging materials to extend the shelf life of food
extracts was lower than chitosan incorporated with guava leaf extracts, (Fig. 8).
but they were still considered good. Therefore, this might be disclosed
that the polyphenol in guava leaf extract had good compatibility with
chitosan films. 3.5. Coating application

Tomatoes and bananas were coated using the dip coating technique.
3.4. Antibacterial activity These fruits and vegetables were coated by CS with GLE, and the results
were evaluated in comparison to the chitosan solution and the control
Due to hygienic, environmental, regulatory, and marketing concerns, sample. For the convenience of viewing, the results of tomatoes and
natural plant-based antibacterial compounds have been considered bananas were chosen to compare every 7 days and 2 days, respectively.
against food pathogens [71]. The antimicrobial mechanism was based According to Fig. 11, uncoated tomatoes begin to mold after 14 days,
on the presence of OH groups in the structure of phenolics, which and the weight loss is 35.62 % after 21 days, which is the highest. The

9
T.A. Dinh et al. Progress in Organic Coatings 183 (2023) 107772

banana had too many black spots due to the oxidation of phenolic. The
peel of the banana contains a high degree of phenolic; therefore, using
GLE 3 %, which includes a high polyphenol content, causes the banana
to ripen faster and induce brown discoloration [79]. Overall, increasing
the GLE concentration to 3 % resulted in the lowest weight loss in to­
matoes, but this did not make a significant difference when compared to
the C2G35E2.0 sample’s weight loss (about 1.33 % only), and the weight
loss of the C2G35E2.0 sample performed the best when coated on ba­
nanas. In terms of visual perception to draw customers’ attraction,
C2G35E2.0 samples were smoother and fresher than C2G35E3.0 after a
specific number of days of storage in both tomatoes and bananas. In this
study, the C2G35E2.0 sample is better appraised and more promising for
consumption as an edible food wrapping since fewer chemicals are
employed, and production expenses are reduced.

3.6. Biodegradation

Fig. 13 displayed the weight loss (WL) of the CS film sample and CS
film incorporated with GLE samples after 7 days. According to the
figure, as the GLE concentration rose from 1 % to 3 %, the weight loss of
the film samples gradually increased (p < 0.05). While the addition of 1
% and 1.5 % GLE showed a moderate increase to 24.67 ± 0.55 % and
25.93 ± 0.96 %, respectively, in comparison with the CS film with 22.38
± 0.47 %, the incorporation of GLE from 2 % to 3 % illustrated a dra­
matic rise to 30.65 ± 1.20 % for the C2G35E2.0 sample, 35.31 ± 0.83 %
for the C2G35E2.5 sample before reaching its peak at 38.92 ± 0.91 % for
the C2G35E3.0 sample. The biodegradability of these films was higher
than those of Asad Riaz et al. [48], who observed a similar trend in the
biodegradability of chitosan-based films using Chinese chive (Allium
tuberosum) root extract and recorded the highest value of approximately
25 % in the first 7 days. The reason for this phenomenon is that the
compounds in the CS and GLE serve as the microorganisms’ sources of
carbon and nitrogen. When water is added, the soil bacteria become
activated and break the interactions between the polymers, enhancing
the degradation rate [80]. This study revealed that incorporating GLE
Fig. 9. Inhibition zone of C2G35E0 and C2G35E2.0 samples [51]. into CS film improved biodegradability by modifying the polymeric
matrix of the CS, transforming it into a rapidly degraded and ecologi­
appearance of the coated tomato was superior to that of an uncoated cally friendly material [48]. As a result, it could eventually be utilized as
tomato. When GLE was included, the visual impression was significantly environmentally friendly food packaging.
enhanced. With GLE 3 %, the tomato’s appearance is still smooth, and
the weight loss is 20.42 %. As a result, the more concentration of GLE, 4. Conclusions
the longer the time required to store [78]. In the case of bananas,
additional GLE causes the banana to ripen slowly. The banana with GLE The utilization of guava leaf extract (GLE) as a source of natural
2 % was found as best for dipping techniques. The weight loss of polyphenols for the fabrication of antioxidant and antibacterial films for
C2G35E2.0 is 7.45 %, shown in Fig. 12. In the case of GLE 3 %, and the food preservation was thoroughly examined. The extract was effectively
incorporated into a chitosan matrix, which improved physical

Fig. 10. The minimum inhibitory concentration (MIC) of C2G35E0 (a) and C2G35E2.0 (b) samples against E: Escherichia coli ATCC 25922, P: Pseudomonas aeruginosa
ATCC 27853, Ef: Enterococcus faecalis ATCC 29212, MS: Staphylococcus aureus ATCC 29213, MR: Staphylococcus aureus against methycilin ATCC 43300.

10
T.A. Dinh et al. Progress in Organic Coatings 183 (2023) 107772

Fig. 11. Physical appearance of tomatoes and bananas coated by C2G35E0, C2G35E1.0, C2G35E2.0, C2G35E3.0 samples.

Fig. 12. Weight loss of tomatoes and bananas uncoated and coated C2G35E0, Fig. 13. Biodegradability of film samples.
C2G35E1.0, C2G35E2.0, C2G35E3.0 films.
significant decline in water vapor permeability and swelling degree,
characteristics, antioxidant activity, and antibacterial activity. implying that the film’s water barrier can be reinforced. Furthermore,
C2G35E2.0 is the most prominent sample. It has the most uniform sur­ the C2G35E2.0 sample exhibited a high inhibitory effect against five
face in this study and several desired mechanical properties, including a food-borne pathogens. Applying the blend films containing 2 % GLE can

11
T.A. Dinh et al. Progress in Organic Coatings 183 (2023) 107772

Table 5 [9] W. Zhang, X. Li, W. Jiang, Development of antioxidant chitosan film with banana
Diameter of inhibition zone and the minimum inhibitory concentration (MIC) of peels extract and its application as coating in maintaining the storage quality of
apple, Int. J. Biol. Macromol. 154 (2020) 1205–1214.
C2G35E0 and C2G35E2.0 samples. [10] A. González-Saucedo, L.L. Barrera-Necha, R.I. Ventura-Aguilar, Z.N. Correa-
Parameter Bacteria C2G35E0 C2G35E2.0 Pacheco, S. Bautista-Baños, M. Hernández-López, Extension of the postharvest
quality of bell pepper by applying nanostructured coatings of chitosan with
Diameter of inhibition zone (mm) E. coli 16 16 Byrsonima crassifolia extract (L.) Kunth, Postharvest Biol. Technol. 149 (2019)
P. aeruginosa 16 22 74–82.
E. faecalis 16 18 [11] A.N. Adilah, M. Noranizan, B. Jamilah, Z.N. Hanani, Development of polyethylene
S. aureus 18 18 films coated with gelatin and mango peel extract and the effect on the quality of
MRSA 16 16 margarine, Food Packag. Shelf Life 26 (2020), 100577.
The minimum inhibitory concentration E. coli 10 10 [12] H.C. Wang, J.L. Brumaghim, Polyphenol compounds as antioxidants for disease
(MIC) (%) P. aeruginosa 10 10 prevention: reactive oxygen species scavenging, enzyme regulation, and metal
E. faecalis 5 5 chelation mechanisms in E. coli and human cells, in: Oxidative Stress: Diagnostics,
Prevention, and Therapy, ACS Publications, 2011, pp. 99–175.
S. aureus 5 5
[13] R. Bisht, S. Chanyal, P.K. Agrawal, Antimicrobial and phytochemical analysis of
MRSA 5 5
leaf extract of medicinal fruit plants, Asian J. Pharm. Clin. Res. 9 (2016) 131–136.
[14] R.A. Stein, D.E. Katz, Escherichia coli, cattle and the propagation of disease, FEMS
Microbiol. Lett. 364 (2017).
extend bananas’ shelf life and mold resistance by up to 7 days and to­ [15] Z. Xu, J. Xie, T. Soteyome, B.M. Peters, M.E. Shirtliff, J. Liu, J.M. Harro,
matoes by up to 21 days. Following that, biodegradability is a vital Polymicrobial interaction and biofilms between Staphylococcus aureus and
Pseudomonas aeruginosa: an underestimated concern in food safety, Curr. Opin.
factor to consider. The weight loss of edible film increases exponentially Food Sci. 26 (2019) 57–64.
after 7 days when the concentration of GLE is increased. Based on the [16] B. Caballero, Encyclopedia of Human Nutrition, Elsevier Sci., 2005.
current results, the edible blend films developed might be proposed as a [17] S. Chakraborty, N. Afaq, N. Singh, S. Majumdar, Antimicrobial activity of Cannabis
sativa, Thuja orientalis and Psidium guajava leaf extracts against methicillin-resistant
feasible alternative to synthetic polymers for usage in the food pack­ Staphylococcus aureus, J. Integr. Med. 16 (2018) 350–357.
aging field. [18] K.B. Narayanan, G.T. Park, S.S. Han, Biocompatible, antibacterial, polymeric
hydrogels active against multidrug-resistant Staphylococcus aureus strains for food
packaging applications, Food Control 123 (2021), 107695.
CRediT authorship contribution statement [19] J. Seo, S. Lee, M.L. Elam, S.A. Johnson, J. Kang, B.H. Arjmandi, Study to find the
best extraction solvent for use with guava leaves (Psidium guajava L.) for high
Dinh Thiet Anh, Le Yen Nhi, Ton That Phu, Pham Nhat Quyen, Van antioxidant efficacy, Food Sci. Nutr. 2 (2014) 174–180.
[20] V.L. Singleton, J.A. Rossi, Colorimetry of total phenolics with phosphomolybdic-
Xuan Thinh, Thanh Gia-Thien Ho: Methodology, Resources, Writing- phosphotungstic acid reagents, Am. J. Enol. Vitic. 16 (1965) 144–158.
original draft, Validation, Investigation, Formal analysis. Tri Nguyen, [21] N. Jaradat, F. Hussen, A. Al Ali, Preliminary phytochemical screening, quantitative
Ha Huynh Ky Phuong: Conceptualization, Methodology, Writing - re­ estimation of total flavonoids, total phenols and antioxidant activity of Ephedra
alata Decne, J. Mater. Environ. Sci. 6 (2015) 1771–1778.
view & editing, Supervision. All authors contributed equally to this
[22] N. Pitak, S.K. Rakshit, Physical and antimicrobial properties of banana flour/
work. chitosan biodegradable and self sealing films used for preserving fresh-cut
vegetables, LWT 44 (2011) 2310–2315.
[23] U. Siripatrawan, B.R. Harte, Physical properties and antioxidant activity of an
Declaration of competing interest active film from chitosan incorporated with green tea extract, Food Hydrocoll. 24
(2010) 770–775.
[24] T. Bourtoom, M.S. Chinnan, Preparation and properties of rice starch–chitosan
The authors declare that they have no known competing financial blend biodegradable film, LWT Food Sci. Technol. 41 (2008) 1633–1641.
interests or personal relationships that could have appeared to influence [25] R. Priyadarshi, B. Kumar, Y.S. Negi, Chitosan film incorporated with citric acid and
glycerol as an active packaging material for extension of green chilli shelf life,
the work reported in this paper.
Carbohydr. Polym. 195 (2018) 329–338.
[26] M. Jouki, F.T. Yazdi, S.A. Mortazavi, A. Koocheki, Physical, barrier and antioxidant
Data availability properties of a novel plasticized edible film from quince seed mucilage, Int. J. Biol.
Macromol. 62 (2013) 500–507.
[27] A.C.K. Bierhalz, M.A. da Silva, T.G. Kieckbusch, Natamycin release from alginate/
Data will be made available on request. pectin films for food packaging applications, J. Food Eng. 110 (2012) 18–25.
[28] L. Wang, H. Guo, J. Wang, G. Jiang, F. Du, X. Liu, Effects of Herba Lophatheri
extract on the physicochemical properties and biological activities of the chitosan
Acknowledgements
film, Int. J. Biol. Macromol. 133 (2019) 51–57.
[29] T. Dang-Bao, T.G.-T. Ho, B.L. Do, N. Phung Anh, T.D.T. Phan, T.B.Y. Tran, N.
We acknowledge the support of time and facilities from Ho Chi Minh L. Duong, P. Hong Phuong, T. Nguyen, Green orange peel-mediated bioinspired
synthesis of nanoselenium and its antibacterial activity against methicillin-resistant
University of Technology (HCMUT), VNU-HCM for this study.
Staphylococcus aureus, ACS Omega 7 (2022) 36037–36046.
[30] C. Medina-Jaramillo, O. Ochoa-Yepes, C. Bernal, L. Famá, Active and smart
References biodegradable packaging based on starch and natural extracts, Carbohydr. Polym.
176 (2017) 187–194.
[31] N.S. Aghayan, J. Seyfi, M.J. Asadollahzadeh, S.M. Davachi, M. Hasani, Developing
[1] Y. Xing, Q. Xu, Z. Che, X. Li, W. Li, Effects of chitosan-oil coating on blue mold
multicomponent edible films based on chitosan, hybrid of essential oils, and
disease and quality attributes of jujube fruits, Food Funct. 2 (2011) 466–474.
nanofibers: study on physicochemical and antibacterial properties, Int. J. Biol.
[2] Y. Xing, Q. Xu, L. Jiang, D. Cao, H. Lin, Z. Che, Y. Ma, X. Li, Y. Cai, Effect of
Macromol. 164 (2020) 4065–4072.
different coating materials on the biological characteristics and stability of
[32] J.C. Camarena-Tello, H.E. Martínez-Flores, M.G. Garnica-Romo, J.S. Padilla-
microencapsulated Lactobacillus acidophilus, RSC Adv. 5 (2015) 22825–22837.
Ramírez, A. Saavedra-Molina, O. Alvarez-Cortes, M.C. Bartolomé-Camacho, J.
[3] X. Meng, J. Han, Q. Wang, S. Tian, Changes in physiology and quality of peach
O. Rodiles-López, Quantification of phenolic compounds and in vitro radical
fruits treated by methyl jasmonate under low temperature stress, Food Chem. 114
scavenging abilities with leaf extracts from two varieties of Psidium guajava L,
(2009) 1028–1035.
Antioxidants 7 (2018) 34.
[4] L. Zimmermann, A. Dombrowski, C. Völker, M. Wagner, Are bioplastics and plant-
[33] A. Dailey, Q.V. Vuong, Effect of extraction solvents on recovery of bioactive
based materials safer than conventional plastics? In vitro toxicity and chemical
compounds and antioxidant properties from macadamia (Macadamia tetraphylla)
composition, Environ. Int. 145 (2020), 106066.
skin waste, Cogent Food Agric. 1 (2015) 1115646.
[5] K. Ogawa, T. Yui, K. Okuyama, Three D structures of chitosan, Int. J. Biol.
[34] M.A. Al-Mamary, Z. Moussa, Antioxidant activity: the presence and impact of
Macromol. 34 (2004) 1–8.
hydroxyl groups in small molecules of natural and synthetic origin, in:
[6] R. Huang, Y. Du, J. Yang, L. Fan, Influence of functional groups on the in vitro
Antioxidants—Benefits, Sources, Mechanisms of Action, 2021, pp. 318–377.
anticoagulant activity of chitosan sulfate, Carbohydr. Res. 338 (2003) 483–489.
[35] L. Wang, Y. Luo, Y. Wu, Y. Liu, Z. Wu, Fermentation and complex enzyme
[7] J.G. Caroni, A.V. de Almeida Mattos, K.R. Fernandes, D.T. Balogh, A.C.M. Renno,
hydrolysis for improving the total soluble phenolic contents, flavonoid aglycones
M.H. Okura, A.C.G. Malpass, C. Ferraresi, L.A. Garcia, R.C. Sanfelice, Chitosan-
contents and bio-activities of guava leaves tea, Food Chem. 264 (2018) 189–198.
based glycerol-plasticized membranes: bactericidal and fibroblast cellular growth
[36] Q.-V. Nguyen, B. Huyen, B. Thi, M.-D. Tran, M.-T. Nguyen, M.-D. Doan, A.-
properties, Polym. Bull. 78 (2021) 4297–4312.
D. Nguyen, T. Minh Le, V.-C. Tran, T.-N. Pham, Impact of different drying
[8] D. Yun, Y. Qin, J. Zhang, M. Zhang, C. Qian, J. Liu, Development of chitosan films
temperatures on in vitro antioxidant and antidiabetic activities and phenolic
incorporated with rambutan (Nephelium lappaceum L.) peel extract and their
application in pork preservation, Int. J. Biol. Macromol. 189 (2021) 900–909.

12
T.A. Dinh et al. Progress in Organic Coatings 183 (2023) 107772

compounds of wild guava leaves collected in the Central Highland of Vietnam, Nat. [59] M. Moradi, H. Tajik, S.M.R. Rohani, A.R. Oromiehie, H. Malekinejad, J. Aliakbarlu,
Prod. Commun. 17 (2022), 1934578X221095349. M. Hadian, Characterization of antioxidant chitosan film incorporated with Zataria
[37] J.C. Camarena-Tello, H.E. Martínez-Flores, M.G. Garnica-Romo, J.S. Padilla- multiflora Boiss essential oil and grape seed extract, LWT-Food Sci. Technol. 46
Ramírez, A. Saavedra-Molina, O. Alvarez-Cortes, M.C. Bartolomé-Camacho, J. (2012) 477–484.
O. Rodiles-López, Antioxidants, 2018. [60] P. Kanmani, J.-W. Rhim, Development and characterization of carrageenan/
[38] J.F. Rubilar, R.M. Cruz, H.D. Silva, A.A. Vicente, I. Khmelinskii, M.C. Vieira, grapefruit seed extract composite films for active packaging, Int. J. Biol. Macromol.
Physico-mechanical properties of chitosan films with carvacrol and grape seed 68 (2014) 258–266.
extract, J. Food Eng. 115 (2013) 466–474. [61] B. Koc, L. Akyuz, Y.S. Cakmak, I. Sargin, A.M. Salaberria, J. Labidi, S. Ilk, F.
[39] L. Sun, J. Sun, L. Chen, P. Niu, X. Yang, Y. Guo, Preparation and characterization of O. Cekic, I. Akata, M. Kaya, Production and characterization of chitosan-fungal
chitosan film incorporated with thinned young apple polyphenols as an active extract films, Food Biosci. 35 (2020), 100545.
packaging material, Carbohydr. Polym. 163 (2017) 81–91. [62] J. Sun, H. Jiang, H. Wu, C. Tong, J. Pang, C. Wu, Multifunctional
[40] G. Qiao, Z. Xiao, W. Ding, A. Rok, Effect of chitosan/nano-titanium dioxide/thymol bionanocomposite films based on konjac glucomannan/chitosan with nano-ZnO
and tween films on ready-to-eat cantaloupe fruit quality, Coatings 9 (2019) 828. and mulberry anthocyanin extract for active food packaging, Food Hydrocoll. 107
[41] V. Siracusa, S. Romani, M. Gigli, C. Mannozzi, J.P. Cecchini, U. Tylewicz, N. Lotti, (2020), 105942.
Characterization of active edible films based on citral essential oil, alginate and [63] A. Riaz, S. Lei, H.M.S. Akhtar, P. Wan, D. Chen, S. Jabbar, M. Abid, M.M. Hashim,
pectin, Materials 11 (2018) 1980. X. Zeng, Preparation and characterization of chitosan-based antimicrobial active
[42] D. Lin, Y. Zhao, Innovations in the development and application of edible coatings food packaging film incorporated with apple peel polyphenols, Int. J. Biol.
for fresh and minimally processed fruits and vegetables, Compr. Rev. Food Sci. Macromol. 114 (2018) 547–555.
Food Saf. 6 (2007) 60–75. [64] T.T. Nguyen, U.T. Thi Dao, Q.P. Thi Bui, G.L. Bach, C.N. Ha Thuc, H. Ha Thuc,
[43] Y. Peng, Y. Wu, Y. Li, Development of tea extracts and chitosan composite films for Enhanced antimicrobial activities and physiochemical properties of edible film
active packaging materials, Int. J. Biol. Macromol. 59 (2013) 282–289. based on chitosan incorporated with Sonneratia caseolaris (L.) Engl. leaf extract,
[44] Z. Jiang, J. Wang, D. Xiang, Z. Zhang, Functional properties and preservative effect Prog. Org. Coat. 140 (2020), 105487.
of P-hydroxybenzoic acid grafted chitosan films on fresh-cut jackfruit, Foods 11 [65] S. Mathew, M. Brahmakumar, T.E. Abraham, Microstructural imaging and
(2022) 1360. characterization of the mechanical, chemical, thermal, and swelling properties of
[45] D. Hu, X. Liu, Y. Qin, J. Yan, J. Li, Q. Yang, A novel edible packaging film based on starch–chitosan blend films, Biopolymers 82 (2006) 176–187.
chitosan incorporated with persimmon peel extract for the postharvest [66] A. Augusto, J.R. Dias, M.J. Campos, N.M. Alves, R. Pedrosa, S.F.J. Silva, Influence
preservation of banana, Food Qual. Saf. 6 (2022) fyac028. of Codium tomentosum extract in the properties of alginate and chitosan edible
[46] T.T. Nguyen, U.T.T. Dao, Q.P.T. Bui, G.L. Bach, C.H. Thuc, H.H. Thuc, Enhanced films, Foods 7 (2018) 53.
antimicrobial activities and physiochemical properties of edible film based on [67] M. Moghadam, M. Salami, M. Mohammadian, M. Khodadadi, Z. Emam-Djomeh,
chitosan incorporated with Sonneratia caseolaris (L.) Engl. leaf extract, Prog. Org. Development of antioxidant edible films based on mung bean protein enriched
Coat. 140 (2020), 105487. with pomegranate peel, Food Hydrocoll. 104 (2020), 105735.
[47] P. Di Pierro, B. Chico, R. Villalonga, L. Mariniello, A.E. Damiao, P. Masi, R. Porta, [68] C. Menzel, C. González-Martínez, F. Vilaplana, G. Diretto, A. Chiralt, Incorporation
Chitosan–whey protein edible films produced in the absence or presence of of natural antioxidants from rice straw into renewable starch films, Int. J. Biol.
transglutaminase: analysis of their mechanical and barrier properties, Macromol. 146 (2020) 976–986.
Biomacromolecules 7 (2006) 744–749. [69] S. Naseer, S. Hussain, N. Naeem, M. Pervaiz, M. Rahman, The phytochemistry and
[48] A. Riaz, C. Lagnika, H. Luo, Z. Dai, M. Nie, M.M. Hashim, C. Liu, J. Song, D. Li, medicinal value of Psidium guajava (guava), Clin. Phytoscience 4 (2018) 1–8.
Chitosan-based biodegradable active food packaging film containing Chinese chive [70] A.A. Kadam, S. Singh, K.K. Gaikwad, Chitosan based antioxidant films incorporated
(Allium tuberosum) root extract for food application, Int. J. Biol. Macromol. 150 with pine needles (Cedrus deodara) extract for active food packaging applications,
(2020) 595–604. Food Control 124 (2021), 107877.
[49] X. Wang, H. Yong, L. Gao, L. Li, M. Jin, J. Liu, Preparation and characterization of [71] N. Oulahal, P. Degraeve, Phenolic-rich plant extracts with antimicrobial activity:
antioxidant and pH-sensitive films based on chitosan and black soybean seed coat an alternative to food preservatives and biocides?, in: Natural Compounds and
extract, Food Hydrocoll. 89 (2019) 56–66. Novel Sources of Antimicrobial Agents for Food Preservation and Biofilm Control,
[50] M. Chen, T. Runge, L. Wang, R. Li, J. Feng, X.-L. Shu, Q.-S. Shi, Hydrogen bonding 2022.
impact on chitosan plasticization, Carbohydr. Polym. 200 (2018) 115–121. [72] D. Altiok, E. Altiok, F. Tihminlioglu, Physical, antibacterial and antioxidant
[51] T.A. Dinh, Y.N. Le, N.Q. Pham, P. Ton-That, T. Van-Xuan, T.G.-T. Ho, B.L. Do, properties of chitosan films incorporated with thyme oil for potential wound
T. Nguyen, H.H.K. Phuong, Fabrication of antibacterial-edible food wrap from healing applications, J. Mater. Sci. Mater. Med. 21 (2010) 2227–2236.
chitosan and guava leaf extract, Vietnam, Trade Ind. Rev. 4 (2023) 316–321 [73] Y. Xing, Q. Xu, X. Li, C. Chen, L. Ma, S. Li, Z. Che, H. Lin, Chitosan-based coating
(Vietnamese). with antimicrobial agents: preparation, property, mechanism, and application
[52] N.E. Suyatma, L. Tighzert, A. Copinet, V. Coma, Effects of hydrophilic plasticizers effectiveness on fruits and vegetables, Int. J. Polym. Sci. 2016 (2016).
on mechanical, thermal, and surface properties of chitosan films, J. Agric. Food [74] C. Amankwaah, J. Li, J. Lee, M.A. Pascall, Antimicrobial activity of chitosan-based
Chem. 53 (2005) 3950–3957. films enriched with green tea extracts on murine norovirus, Escherichia coli, and
[53] C.A. Breda, D.L. Morgado, O.B.G. Assis, M.C.T. Duarte, Processing and Listeria innocua, Int. J. Biol. Macromol. 2020 (2020).
characterization of chitosan films with incorporation of ethanolic extract from [75] S.K. Pankaj, C. Bueno-Ferrer, N. Misra, L. O’Neill, B. Tiwari, P. Bourke, P. Cullen,
“pequi” peels, Macromol. Res. 25 (2017) 1049–1056. Physicochemical characterization of plasma-treated sodium caseinate film, Food
[54] U. Siripatrawan, W. Vitchayakitti, Improving functional properties of chitosan Res. Int. 66 (2014) 438–444.
films as active food packaging by incorporating with propolis, Food Hydrocoll. 61 [76] B. Kowalska-Krochmal, R. Dudek-Wicher, The minimum inhibitory concentration
(2016) 695–702. of antibiotics: methods, interpretation, clinical relevance, Pathogens 10 (2021)
[55] Y. Shahbazi, Characterization of nanocomposite films based on chitosan and 165.
carboxymethylcellulose containing Ziziphora clinopodioides essential oil and [77] K.A. Shiekh, M. Liangpanth, S. Luesuwan, R. Kraisitthisirintr, K. Ngiwngam,
methanolic Ficus carica extract, J. Food Process. Preserv. 42 (2018), e13444. S. Rawdkuen, P. Rachtanapun, T. Karbowiak, W. Tongdeesoontorn, Preparation
[56] J. Zeng, X. Ren, S. Zhu, Y. Gao, Fabrication and characterization of an economical and characterization of bioactive chitosan film loaded with cashew (Anacardium
active packaging film based on chitosan incorporated with pomegranate peel, Int. occidentale) leaf extract, Polymers 14 (2022) 540.
J. Biol. Macromol. 192 (2021) 1160–1168. [78] A. El Ghaouth, R. Ponnampalam, F.o. Castaigne, J. Arul, Chitosan coating to extend
[57] Y. Luo, H. Liu, S. Yang, J. Zeng, Z. Wu, Sodium alginate-based green packaging the storage life of tomatoes, HortScience 27 (1992) 1016–1018.
films functionalized by guava leaf extracts and their bioactivities, Materials 12 [79] P.S. Gooding, C. Bird, S.P. Robinson, Molecular cloning and characterisation of
(2019) 2923. banana fruit polyphenol oxidase, Planta 213 (2001) 748–757.
[58] M. Kaya, S. Khadem, Y.S. Cakmak, M. Mujtaba, S. Ilk, L. Akyuz, Asier [80] T. Gasti, S. Dixit, S.P. Sataraddi, V.D. Hiremani, S.P. Masti, R.B. Chougale, R.
M. Salaberria, J. Labidi, A.H. Abdulqadir, E. Deligöz, Antioxidative and B. Malabadi, Physicochemical and biological evaluation of different extracts of
antimicrobial edible chitosan films blended with stem, leaf and seed extracts of edible Solanum nigrum L. leaves incorporated chitosan/poly (vinyl alcohol)
Pistacia terebinthus for active food packaging, RSC Adv. 8 (2018) 3941–3950. composite films, J. Polym. Environ. 28 (2020) 2918–2930.

13