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Fabrication and characterization of
Cite this: DOI: 10.1039/d4fb00359d
methylcellulose/chitosan active films incorporated
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with L-arginine and their potential in the green
packaging of grapes
Suhasini Madihalli,a Saraswati P. Masti, *a Manjunath P. Eelager,a
Manjushree Nagaraj Gunaki,a Ravindra B. Chougale,b Nagarjuna Prakash Dalbanjanc
and S. K. Praveen Kumar c
Active biodegradable films are in great demand as green packaging materials for extending the shelf life of
food. In this study, methylcellulose (MC)/chitosan (CS) active films (AMC) were fabricated by incorporating
different weight percentages of L-arginine. The fabricated active films were investigated for their
physicochemical, mechanical and functional properties. FTIR, SEM and XRD results confirmed the
intermolecular hydrogen bonding interaction and compatibility of L-arginine with the MC/CS film matrix,
improving the mechanical properties, UV light blocking ability, water vapor barrier and oxygen barrier
properties of the AMC active films. The inclusion of L-arginine improved the antimicrobial, antioxidant
and packaging efficiency of the films. Compared with the L-arginine-free MC/CS film (control), the AMC
active film containing 7.5% of L-arginine exhibited strong DPPH radical scavenging activity (72.28% ±
0.28) and displayed potent antimicrobial activity against E. coli, S. aureus, B. subtilis and C. albicans.
Received 30th November 2024
Accepted 29th April 2025
Grapes packed with the AMC active film containing 7.5% L-arginine showed a limited weight loss
percentage of 13.35% ± 1.07 and a restricted browning degree of 0.87 ± 0.01 over 17 days of storage.
DOI: 10.1039/d4fb00359d
These findings suggest that the fabricated active films meet the essential prerequisites of green food
rsc.li/susfoodtech packaging materials.
Sustainability spotlight
The signicance of the present study lies in its multifaceted contribution to sustainable and biodegradable food packaging applications. This study addresses
the growing concern of environmental problems resulting from synthetic food packaging materials by utilizing L-arginine and natural polysaccharides like
methylcellulose and chitosan, offering possibilities for signicant advancements in the food packaging sector. The prepared active lms exhibited excellent
mechanical, water vapor barrier, UV barrier, antibacterial, antioxidant, and biodegradable properties. Furthermore, the active lms extended the shelf life of
grapes to over 17 days of storage. Hence, these fabricated bioactive lms have the potential to reduce food waste while supporting eco-friendly practices, making
a valuable contribution to sustainable food packaging.
bioaccumulation and biomagnication in the tissues of
1. Introduction organisms owing to their resistance to degradation, which has
Plastics are the most adaptable components of everyday life caused microplastics to become an intrinsic component of our
owing to their ease of processing, resilience, and performance.1 food chain with increasing production. It has been reported
However, these plastics end up in landlls or get incinerated that this plastic waste also facilitates the colonization of toxin-
aer their use and remain in the environment for a long time. producing microorganisms by providing a bioadhesion
The extensive use of plastics has negatively impacted the substratum for these microbes, which are detrimental to pelagic
ecosystem and endangered life. They are also proven for their and human life.2 Globally, over 340 million tonnes of plastic
waste are generated, with the packaging sector accounting for
46% of the total plastic waste. To address these problems,
a
Department of Chemistry, Karnatak Science College, Dharwad-580 001, Karnataka, current research and scientic progress have been promoting
India. E-mail: dr.saraswatimasti@yahoo.com
bio-based food packaging materials, which can be converted
b
P.G. Department of Studies in Chemistry, Karnatak University, Dharwad-580 003,
into CO2, H2O and biomass within six months aer their end
Karnataka, India
c
P.G. Department of Biochemistry, Karnatak University, Dharwad-580 003, Karnataka,
use.3 This strategy is emerging as a sustainable option, espe-
India cially for short-term applications such as food packaging.4
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Food packaging is crucial in the food supply chain and safety Furthermore, active components that inhibit oxidation and
as it is a protective layer that can inhibit unfavorable biological microbial growth are incorporated into the polymer matrix to
and chemical changes. As a result, packaging materials should improve its functional properties. L-arginine is one such active
serve as a barrier to moisture, oxygen, dust, and chemicals. It component that imparts both of these properties. It is an
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should also inhibit the growth of harmful bacteria, odor- essential amino acid in plants, and is vital for developmental
causing fungi and other microorganisms that cause food- and cellular activities. It is crucial in minimizing inhibition
borne diseases to maintain food quality over a long period.5–7 driven by plant exposure to stress conditions. It signicantly
Foodborne diseases and bacterial contamination are signicant prevents the browning of fruits and vegetables by suppressing
concerns for the food industry. Many physical and chemical the oxidation of polyphenols found within them.22 L-arginine
processing methods have been used to control these bacterial was also reported to maintain the quality of mango fruits by
contaminations. Physical processing methods like thermal imparting antioxidant properties.23 Hence, it was selected as an
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inactivation, high-pressure processing and UV treatments can antimicrobial, antioxidant and antibrowning agent to improve
lead to nutrient loss and changes in the appearance of food the functional characteristics of the active lms.
which reduces its overall quality. Additionally, some chemical Polysaccharide-based active lms with enhanced functional
preservatives have been used in the food industry. However, properties are desirable for food packaging applications.
their use has failed to meet consumer demands due to their Several studies have been reported on the combination of L-
unpleasant taste and potential health problems. It has been arginine and chitosan. Many researchers evaluated the anti-
reported that various countries have banned the use of some bacterial activity of L-arginine-modied chitosan in detail, and
food preservatives, such as boric acid, tartrazine, sunset yellow reported that L-arginine-modied chitosan has desirable
(azodye) and amaranth (trisodium salt), due to their toxicity and biocompatibility, antibacterial activity and anticoagulant prop-
potential carcinogenicity.8 Thus, in response to modern erties.24,25 These qualities suggest the signicant potential of L-
consumer demands, such as nutritional and safety require- arginine as a bioactive component. However, no work has been
ments for food, biopolymers have been extensively used in the done on the L-arginine-functionalized MC/CS active lms.
development of active food packaging materials.9–11 Bioactive Hence, in the current study, MC and CS are used as green
packaging lms incorporated with active components, such as polymers, and L-arginine is used as an active component to
antimicrobial agents and antioxidants, can prolong the food enhance the lms' functional properties and packaging effi-
shelf life by inhibiting bacterial activity and maintaining the ciency. L-arginine was incorporated into the MC/CS matrix to
quality of food products.12,13 Several biopolymers have been fabricate the highly efficient antimicrobial, antioxidant and
used for fabricating biodegradable packaging materials, such as antibrowning novel active lms by employing eco-friendly
chitosan,14 pullulan,15 cellulose and cellulose derivatives,16 solvent casting techniques for food packaging applications.
gelatin,17 gums, carrageenan,18 and starch. Chitosan and cellu- Fabricated active lms were evaluated for their physicochemical
lose derivatives are oen employed for fabricating food pack- and mechanical properties. The inuence of L-arginine on
aging lms due to their attractive properties, such as functional properties (e.g., antimicrobial, antioxidant and
biodegradability, biocompatibility, abundance and non- antibrowning activity) was determined. Additionally, fabricated
toxicity.19 active lms were practically applied for grape packaging. The
Methylcellulose (MC) is the most promising hydrocolloid packaged grapes' weight loss and browning degree were also
polysaccharide, comprising (1–4) glycosidic chains with methyl evaluated to check the efficacy of the fabricated active lms for
groups. MC is derived from cellulose via the partial substitution use in green packaging applications.
of the hydroxyl group with methyl substituents. It affords lms
with potent lipid and oxygen barrier features. Because of its
abundance, easy processability, excellent lm-forming ability, 2. Materials and methods
thermal gelation capability and high strength, it has the 2.1. Materials
potential to be employed as a packaging material.18,19 However, Chitosan (MW = 20–100 kDa; degree of deacetylation: 75–85%)
most biopolymers, including methylcellulose, are susceptible to and methylcellulose (molecular weight = 454.5 g mol−1) with
atmospheric conditions due to their higher water affinity and methoxy substitution between 27.5–31.5% and degree of
poor barrier properties.20 Thus, in the current study, these substitution 1.5–1.9 were procured from the Tokyo Chemical
limitations were overcome by blending MC with chitosan, Industry (TCI), Japan. Acetic acid was received from Merck Life
which imparts a slightly antimicrobial property owing to the Sciences India. L-Arginine, glycerol (99.5% AR) and microbial
action of chitosan. growth media were obtained from Loba Chemie, Pvt. Ltd, India.
Chitosan (CS) is a cationic polysaccharide comprising (1–4)- Milli-Q water was used throughout the experiment.
2-amino-2-deoxy-D-glucan, and is an alkaline deacetylated chitin
product. In addition to its inherent antimicrobial and anti-
fungal properties, it has demonstrated biocompatible, biode- 2.2. Fabrication of the AMC control and active lms
gradable and non-toxic characteristics. Considering these Active lms were fabricated by adapting the solvent casting
properties, chitosan is used as a packaging material to preserve technique. MC and CS solutions were prepared by separately
various food products.14,21 dissolving 1.5 g of MC and 0.5 g of CS in Milli-Q water and 1%
acetic acid, respectively. Then, both solutions were blended and
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stirred for 6 h to obtain homogeneous blend solutions. analysis. The dry mass (m1) was recorded aer heating the
Furthermore, 1% (w/v) glycerol was added as a plasticizer. Then, samples at 100 °C for 24 h. Aer that, they were exposed to
different weight percentage of L-arginine solutions prepared by atmospheric conditions for 24 h and their nal mass (m2) was
dissolving the predetermined amounts of L-arginine 0%, 2.5%, recorded. The average moisture adsorption values were calcu-
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5.0% and 7.5% (w/w of the net dry mass of polymer) in Milli-Q lated using eqn (2).28
water were added to the blended solutions and stirred for 4 h. m2 m1
The resulting lm-forming solutions were cast in Petri plates, Moisture adsorptionð%Þ ¼ 100 (2)
m2
and le to dry for 48 h in a hot air oven set to 40 °C. The dried
lms were peeled off, packed in a zipper storage bag, and stored
For the water solubility, lm samples with dimensions of 20
in a desiccator for further characterization. Films were labeled
× 20 mm were taken, and their initial mass (W1) was noted aer
as AMC-1 (Control), AMC-2, AMC-3 and AMC-4 based on the
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dehydrating them at 70 °C for 24 h. The dried samples were
weight percentages of L-arginine, i.e., 0, 2.5, 5.0 and 7.5%,
soaked in 20 mL of Milli-Q water for 24 h. Then, the undissolved
respectively, before analysis. The MC/CS lm without L-arginine
samples were extracted and dried at 70 °C for 24 h to determine
was labeled AMC-1 and treated as a control.
their nal mass (W2). The average water solubility values were
calculated using eqn (3).29
2.3. Characterization of the fabricated control and active W1 W2
Water solubilityð%Þ ¼ 100 (3)
lms W1
2.3.1. Fourier transform infrared spectroscopy (FTIR). where W1 = initial weight of the sample and W2 = nal weight of
Fourier transform infrared (FTIR) spectra of the fabricated AMC the sample.
control and active lms were recorded using an FTIR spec- 2.3.7. UV-visible spectroscopy. A UV-vis spectrophotometer
trometer (Shimadzu Japan) in the 400 to 4000 cm−1 frequency (Model UV-1601) was used to inspect the optical properties of
range with 4 cm−1 sensitivity. the fabricated AMC control and active lms. The lm samples
2.3.2. Surface morphology. Surface micrographs of the with dimensions of 10 × 40 mm taken for the analysis were
control and active lms were captured using a scanning elec- directly inserted into the test cell, and the spectra were recorded
tron microscope (model JEOL JSM-IT500LA) operating at a 10 kV in the wavelength range between 200 and 800 nm using air as
acceleration voltage. Film samples were coated with a gold layer a reference. The results were expressed as a percentage of
using a sputtering method before analysis and mounted on transmittance. Meanwhile, the opacity of the lm samples was
a metal stab to capture images. calculated using eqn (4).30
2.3.3. X-ray diffraction (XRD) analysis. X-ray diffractograms
Absorbance
of the fabricated control and active lms were recorded using an Opacity ¼ (4)
c
XRD diffractometer (Rigaku Smart Lab, Tokyo, Japan) with an
acceleration of 40 kV. The samples were evaluated using a Cu Ka where X = lm thickness in mm.
(l = 1.5418 Å) lter and 30 mA current at 5° per minute scan 2.3.8. Water vapour transmission rate (WVTR). The WVTR
range over a 2q = 5° to 80° angular range. The crystallinity of the fabricated control and active lms was determined
percentage was obtained according to the previously reported according to the previously reported method, with some
method using eqn (1).26,27 modications.31,32 To summarize, lm samples with dimen-
Ac sions of 30 × 30 mm were sealed at the circular mouth of glass
Crystallinityð%Þ ¼ 100 (1) vials lled with 10 mL of Milli-Q water and tted with Teon
ðAc þ Aa Þ
tape. The initial mass of the glass vials was recorded as Wi, and
where Ac = area of the crystalline region and Aa = area of the the weighed samples were maintained in an oven at 40 °C for
amorphous region. 24 h. The glass vials were retrieved from the oven and weighed
2.3.4. Film thickness and mechanical properties. The to obtain the nal mass (Wf). The transmission rate of the water
thickness of the fabricated control and active lms was vapours through the control and active lms was then calcu-
measured using a digital micrometer (Mitutoyo, Japan) with lated using eqn (5).
0.001 mm precision. A universal testing machine (Model: UTM,
Wi Wf
DAK System, 7200-1KN) was employed to measure the WVTR ¼ g m2 h1 (5)
AT
mechanical parameters as per ASTM D882-1992. For analysis,
20 × 100 mm lm samples were inserted in the extension grip, where Wi = initial weight of the sample, Wf = nal weight of the
maintaining a grip separation of 50 mm, and were stretched at sample, A = area of the circular mouth of the glass vial, and T =
a cross-head speed of 1 mm min−1 at RT until they fractured. time employed (24 h).
2.3.5. Surface wettability test. The surface wettability char- 2.3.9. Oxygen permeability (OP). OP was determined
acteristic of the fabricated control and active lms was studied according to the previous method, with some alterations.17 In
using the sessile drop method by employing a contact angle short, 30 × 30 mm lms were secured at the tip of glass vials
analyzer (Model DMs-401, Kyowa Interface Science Co. Ltd, Tokyo). and sealed with Teon tape. Vials were weighed to record their
2.3.6. Moisture adsorption (MA) and water solubility (WS). initial weight (W1) and positioned in a desiccator containing
Film samples with dimensions of 20 × 20 mm were taken for
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anhydrous CaCl2 at room temperature. Aer that, the glass vials DPPH free radical scavenging activityð%Þ
were taken from the desiccator every 24 h, and their weight was Abc Abs
recorded for 3 days. The slopes (g h−1) were calculated using ¼ 100 (9)
Abc
linear regression (R2 > 0.982) and the oxygen permeability (OP)
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was determined by applying eqn (6) and (7). where Abc = absorbance of control and Abs = absorption of the
sample.
Slope
OPTR ¼ (6) 2.3.13. Overall migration studies. The overall migration
A
test was performed according to ASTM standard IS:9845-1998
x using three food stimulants: distilled water, 50% ethanol and
OP ¼ OPTR (7)
DP 3% acetic acid. In brief, 20 × 20 mm lms were immersed in
where A = area of the lm, DP = difference between the partial beakers containing 30 mL of food simulants, and placed in
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vapor pressure of the Milli-Q water and dry atmosphere (0.02308 a hot air oven for 10 days at 40 °C. The impact of the food
atm. at 25 °C), and x = lm thickness (m). stimulants on alcoholic, acidic and watery food items was
2.3.10. Soil burial test. The soil burial test was carried out evaluated gravimetrically, and the results were expressed in
according to the previously reported method with some modi- terms of mg dm−2.2 The amount of extractive was calculated
cations14 for 10 days. Film samples with dimensions of 20 × according to eqn (10).
20 mm taken for analysis were preheated in an oven at 70 °C for M
Amount of extract ¼ 1000 (10)
24 h, and the initial dry mass was recorded as W1. The dried lm V
samples were then buried in soil and were watered once a day where M = mass of residue in mg minus blank value and V =
with water. Aer ten days, the lms were isolated, gently washed total volume in mL of simulant used in each replicate.
with Milli-Q water and dried in an oven at 70 °C for 24 h. They
were cooled in a desiccator and the nal weight of the samples
was recorded as W2. The degradation percentage of the lm
samples was calculated using eqn (8).
W1 W2 2.4. Packaging efficiency of active lms
Degradationð%Þ ¼ 100 (8)
W1 The efficiency of the prepared AMC active lms for food pack-
where W1 = initial dry weight and W2 = dry weight of the sample aging applications was assessed using green grapes. The pack-
aer degradation. aging test was performed at room temperature for 17 days. The
2.3.11. Assessment of the antimicrobial efficacy. The anti- fresh green grapes purchased from the local market were
microbial efficacy of the control and active lms was assessed cleaned with Milli-Q water and packed in pouches made from
against E. coli (ATCC 10799), S. aureus (ATCC 6538), B. subtilis AMC active lms. Unpacked grapes were treated as a control.
and C. albicans (ATCC 24433). The sample solutions were The freshness and extension of the shelf life of green grapes
prepared by dissolving lms in 1% acetic acid (1 mg mL−1). were monitored.
Pure bacterial cultures were subcultured in Luria broth (LB) 2.4.1. Weight loss analysis. Weight loss of the grapes,
media until the absorbance at 600 nm reached 0.5. The broth unpacked and packed with AMC active lms, was determined by
culture was spread using a swab on the MHA dishes (4 mm in weighing the grapes on the 1st, 5th, 10th, 15th, and 17th days
thickness), followed by the introduction of 100 mL of the during the storage period. Findings were presented in terms of
prepared sample solutions into wells (8 mm in diameter) made the percentage of weight loss with respect to the initial weight.35
with a gel puncher. Petri dishes were incubated at 37 °C over- 2.4.2. Antibrowning analysis. The extinction value method
night. The inhibition zone was recorded using a Vernier was used to determine the browning degree of grapes.36 The test
scale.14,33 was performed aer 17 days of storage. In brief, 20% (w/v) of the
2.3.12. Antioxidant activity. The antioxidant activity of the grape sample solution was prepared by mixing the grape with
control and active lms was evaluated using the DPPH (2,2- cold steamed water. The absorbance of the sample solution was
diphenyl-1-picrylhydrazyl) free radical scavenging assay.34 In recorded at 410 nm. The browning degree was expressed as
short, standard ascorbic acid solutions of 100–500 mL and A410nm (absorbance at 410 nm).
sample solutions of 1 mg mL−1 were diluted to 1000 mL with
methanol, followed by treatment with 500 mL methanolic DPPH
(0.5 mM), and incubated in the dark at room temperature for
30 min. The absorbance at 517 nm was determined in a UV-
visible spectrophotometer (Labman, LMSP UV-1200), using 2.5. Statistical analysis
DPPH diluted with methanol as a control and methanol alone All of the tests were performed in triplicate, and the data were
as the blank solution. The DPPH free radical scavenging activity presented as an average value with their standard deviation
was calculated using eqn (9), which was expressed in terms of (average value ± SD). Statistical analysis was performed using
the percent inhibition. Origin-9 soware via one-way ANOVA. Tukey's test was per-
formed to distinguish the average values at the p # 0.05
signicance level.
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incorporated AMC active lms, is associated with the presence
of the guanidine group, indicating that L-arginine is success-
fully linked to the polymer backbone.41 In addition, the
stretching peak at 1642 cm−1 associated with the AMC-1
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(control) lm corresponds to the –C]O stretching of the
amide-I group, and decreased towards a lower wavenumber at
1637 cm−1 (AMC-2), 1636 cm−1 (AMC-3), and 1634 cm−1 (AMC-
4) with the inclusion of L-arginine, indicating physical interac-
tions between the components of the polymer matrix and L-
arginine.42 Band shis in FTIR spectra indicate physical and
chemical interactions within the polymer matrix, with slight
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variations indicating physical interactions and signicant shis
suggesting chemical interactions. In this study, the FTIR
spectra revealed slight variations in frequency, indicating
a physical interaction between L-arginine and the components
of the polymer matrix, rather than a chemical interaction.
3.2. Surface morphology
The morphological features of the active lms rely on the
interactions between the functional groups of the polymer
matrix and active ingredients, which impact the active lms'
physical, optical, and barrier properties. The SEM micrographs
of the AMC control and active lms are presented in Fig. 2. The
surface of AMC-1 (control) lm appeared slightly rough and
non-uniform but still exhibited compact and nonporous struc-
Fig. 1 FTIR spectra of CS, MC, L-arginine, AMC-1 (control) and AMC-2, ture. These observations indicate the good compatibility
AMC-3, AMC-4 active films. between the components of the MC/CS matrix.43 Incorporation
of L-arginine substantially improved the surface morphology of
the AMC active lms. Furthermore, the increase in the L-argi-
nine content presented a more regular and dense texture,
3. Results and discussions reecting that L-arginine has been well dispersed into the
3.1. Fourier transform infrared spectroscopy (FTIR) polymer matrix without any phase separation and agglomera-
FTIR spectroscopy was used to investigate the interactions tion.44 The AMC-4 active lm showed a smooth and homoge-
between the functional groups of the polymer matrix and the neous surface, indicating compatibility among the polymer
active ingredient. The resulting spectra are shown in Fig. 1. The matrix components. Overall, the surface morphology of all of
FTIR spectra of chitosan and methylcellulose show character- the AMC active lms revealed compact and continuous textures
istic peaks at 1027 cm−1 and 1049 cm−1, respectively, assigned that altered the physical and mechanical parameters of the lm.
to the C–O stretching frequency. This band was shied to Thus, including L-arginine improved the surface morphology of
1051 cm−1 in the AMC-1 (control) lm. Furthermore, the the AMC active lms.20
stretching frequency at 1638 cm−1 related to the –(NH2) group of
the CS was shied to 1642 cm−1 owing to the interaction 3.3. X-ray diffraction (XRD)
between the hydroxyl group of MC and the amine group of CS, X-ray diffraction analysis was performed to inspect the micro-
conrming the compatibility of CS and MC in the active lm.37 crystalline structure of the AMC active lms. The X-ray dif-
The absorption peaks at 1673 cm−1 and 1610 cm−1 in the FTIR fractograms of L-arginine, and the AMC control and active lms
spectrum of L-arginine are attributed to the stretching vibration are presented in Fig. 3. The AMC control and active lms
of the carboxyl carbonyl and guanidine groups of L-arginine, exhibited two major crystalline peaks at around 8° and 20°.
respectively.38,39 The slight decrease in the peak intensities of These peaks indicate the semicrystalline nature of the active
the –NH and –OH groups can be observed in the spectra of the L- lms.45 According to the results, the peak registered at around
arginine-incorporated AMC active lms, reecting the reduc- 2q = 20° was found to be the crystalline peak of CS, while the
tion in the content of hydroxyl and amine groups.39 The peaks located at around 2q = 8° and 20° corresponded to the
absorption bands at 3410 cm−1, 2921 cm−1,1642 cm−1 and crystalline peaks of MC.37 The XRD diffractogram of L-arginine
1051 cm−1 were migrated to 3390 cm−1, 2901 cm−1, 1634 cm−1, shows sharp and well-dened characteristic diffraction peaks at
and 1054 cm−1, respectively, due to the intermolecular inter- specic 2q angles, indicating its crystalline nature, which is in
action between the components of the polymer matrix through agreement with previously reported literature.46 Moreover, the
hydrogen bonding.18,40 Furthermore, the characteristic absorp- peak intensity of the AMC-1 (control) lm was increased with L-
tion peak around 1634–1637 cm−1, observed in L-arginine- arginine incorporation, indicating that the degree of
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Fig. 2 SEM images of the AMC-1 (control) and AMC-2, AMC-3, and AMC-4 active films.
crystallinity was improved by the addition of L-arginine 37,47. The
variation in the % crystallinity of the active lms as a result of
the incorporation of L-arginine is presented in Table 1. The
ndings illustrate that the AMC-1 (control) lm without L-argi-
nine has the lowest crystallinity compared to the AMC-2 and
AMC-3 active lms. Meanwhile, the crystallinity of the active
lms increased with an increase in the L-arginine content. This
could be attributed to the presence of four amine groups in the
L-arginine molecule, which facilitated the intermolecular
hydrogen bonding interaction within the polymer matrix,
thereby inuencing the crystallinity.41,47,48 Overall, the AMC
active lms exhibited increased crystallinity compared to the
AMC-1 control lm.
3.4. Mechanical properties
Fabricated active lms must have sufficient mechanical prop-
erties for food packaging applications to retain their ability to
endure stress during storage and transportation, which reduces
the risk of damage and scratches.29 Fig. 4(a and b) depicts the
results of the mechanical properties of the fabricated active
lms. The results show that the AMC-1 (control) lm exhibited
a tensile strength of 37.81 ± 0.68 MPa and 11.32% ± 0.36%
elongation at break. Both tensile strength and elongation at
break were signicantly improved by incorporating L-arginine,
and reached a maximum value of 41.11 ± 1.03 MPa and 14.11%
± 0.53%, respectively, with an L-arginine content of 5.0%. This
Fig. 3 X-ray diffraction patterns of the AMC-1 (control) and AMC-2,
improved tensile strength was attributed to the intermolecular
AMC-3, and AMC-4 active films.
hydrogen bonding interaction between the functional groups of
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Table 1 Thickness, crystallinity, water vapour transmission rate (WVTR), oxygen permeability (OP) and soil degradation rate of AMC-1 (control)
and AMC-2, AMC-3, AMC-4 active filmsa
Samples Thickness (mm) Crystallinity (%) WVTR (g m−2 h−1) OP × 10−5 (cc3$m−1$24 h$atm) Degradation rate (%)
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AMC-1 0.080 0.001a 47.05 0.50d 27.49 1.01a 11.6 0.20a 20.94 0.62b
AMC-2 0.078 0.003a 49.28 0.33c 26.64 0.57a 10.4 0.12b 27.53 0.72a
AMC-3 0.077 0.001a 51.56 0.22b 26.21 0.92a 10.1 0.07bc 28.23 0.43a
AMC-4 0.081 0.004a 54.09 0.16a 25.45 1.55a 9.6 0.19c 28.88 0.89a
a
Data are presented as mean ± SD a–d
. The superscript letters in every datapoint indicate statistically signicant differences (P < 0.05).
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the MC/CS matrix and the –NH2 of L-arginine and the cooper- ndings are consistent with the results reported by Hiremani
ative hydrogen bonding effect of L-arginine. The incorporated L- et al. (2021),29 indicating that both tensile strength and elon-
arginine facilitated interchain linkage by forming hydrogen gation at the break values of the chitosan lm were decreased at
bonds with the functional groups of components of the polymer a higher content of Curcuma zedoaria powder.
matrix, which hindered the mobility of the polymer network.39,49
These ndings are also consistent with FTIR studies, which
indicated that the wavenumber associated with –OH groups has 3.5. Surface wettability
decreased. Water contact angle (WCA) measurements were carried out to
In contrast, the percentage elongation at break was also inspect the surface wettability of the fabricated control and
slightly enhanced. The increased elongation at break, ascribed active lms. The images and degree of contact angle values are
to the plasticizing effect of glycerol, led to increased free volume displayed in Fig. 5. Generally, lms with a WCA < 65° are
in the polymer matrix.50 These ndings were supported by the considered hydrophilic, whereas lms with a WCA > 65° are
results reported by Narasagoudr et al. (2020),26 that elongation considered hydrophobic.43 The AMC-1 (control) lm exhibited
at break values of the rutin-induced CS/PVA lm were enhanced surface hydrophobicity with a WCA of 90.6° ± 0.21 owing to the
with rutin content. However, at a higher weight percentage of L- hydrophobic domain of the CS. It was found that the WCA of the
arginine (AMC-4), the tensile strength and elongation at break AMC active lms was decreased by incorporating L-arginine
values decreased, which might be due to the signicant impact compared to the AMC-1 (control) active lm. Furthermore,
of the lm thickness on the mechanical properties, as the a decrease in the WCA degree of the active lms was observed as
thickness has an inverse relationship with the tensile proper- the weight percent of L-arginine increased. This resulted in
ties. Hence, the mechanical properties decreased with a decreased surface hydrophobicity of the active lms, which
increased lm thickness. Abdel-Mohti et al. (2015)51 reported might be attributed to the free polar moieties available on the
that the mechanical characteristics of the lms increased with lm surface, facilitating the interaction with the water mole-
a decrease in the lm thickness, showing that the mechanical cules.21 The AMC-4 active lm with the highest L-arginine
properties are strongly reliant on the thickness of the lms. The content exhibited a lower water contact angle. This decrease in
Fig. 4 Mechanical properties: (a) stress–strain curve, (b) tensile strength and elongation at break of the AMC-1 (control) and AMC-2, AMC-3,
AMC-4 active films.
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Fig. 5 Water contact angle images of the AMC-1 (control) and AMC-2, AMC-3, and AMC-4 active films.
water contact angle is due to the hydrophilic nature of L- that the moisture adsorption capacity of the AMC-1 (control)
arginine.52 lm was 2.53% ± 0.05, which was increased by the addition of L-
arginine.54 Furthermore, the moisture adsorption capacity of
the AMC active lms increased from 2.95% ± 0.11 to 3.03% ±
3.6. Moisture adsorption and water solubility 0.07 as the concentration of L-arginine was raised. The
The majority of biopolymers are sensitive to moisture. Hence, improved MA values were attributed to the greater solubility
the moisture absorption study is accepted as a fundamental and hydrophilic character of L-arginine, which resulted in free
characteristic for food packaging applications.53 The moisture polar sites that facilitated the clustering of water molecules on
adsorption capacity of the lms was evaluated, and the results the lm surface.55 Among all the lms, AMC-4 with a high
are depicted in Fig. 6(a). The obtained results demonstrated content of arginine (7.5 wt%) exhibited the highest moisture
Fig. 6 (a) Moisture adsorption, and (b) water solubility values of the AMC-1 (control) and AMC-2, AMC-3, and AMC-4 active films.
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adsorption value (3.76% ± 0.09), which may be related to the increased. Furthermore, AMC active lms containing different
fact that water molecules occupied the free sites that were weight percentages of L-arginine exhibited higher absorption
available on the lm's surface.56 and UV light barrier qualities than the AMC-1 (control) lm. L-
Water solubility signicantly impacts the biodegradability of arginine incorporation restricted the transmission of ultraviolet
This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
lms when the lms are employed as a packaging material. light below 370 nm, and lowered the % light transmittance
Higher lm solubility inuences the degradation of lms, across all the spectral regions.61 This may be attributed to
whereas partial or low solubility is best suited for storage. The secondary interactions between the polymer matrix and L-argi-
outcomes of the percentage of water solubility of the fabricated nine that resulted in a compact molecular structure, as evi-
active lms are presented in Fig. 6(b). The AMC-1 (control) lm denced by SEM and FTIR studies, which altered the light
showed a low WS of 37.75% ± 1.47, compared to other active transmission rate through active lms.
lms, owing to the insolubility of chitosan in the blend lms at The UV barrier characteristics of the active lms were eval-
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neutral pH. The incorporation of L-arginine into the MC/CS uated between the wavelength range of 315–400 nm (UV-A),
matrix improved the solubility of the AMC active lms in 280–315 nm (UV-B), and 200–280 nm (UV-C), and the graph is
water. This is due to the hydrophilic nature and positively presented in Fig. 7(b). Lipid oxidation occurs most frequently in
charged guanidium side chain of L-arginine, which possesses the 200–315 nm wavelength range.59 With the addition of
a high pKa of 12.48 in a neutral pH environment.54,57 Addition- (7.5 wt%) L-arginine, the % transmittance of the AMC-1 (control)
ally, the lm solubility was enhanced from 38.26% ± 1.51 to lm dropped from 10.03% to 1.67% at 315 nm, and from
50.57% ± 0.70 as the concentration of arginine was raised from 50.21% to 21.98% at 400 nm. Thus, adding L-arginine to the
2.5 to 5.0 (wt%). The AMC-4 active lm with 7.5 wt% of arginine polymer matrix enhanced the barrier property through the
exhibited the highest water solubility of 58.27% ± 0.79. This absorption of UV light.60,61 The UV barrier property of the AMC
could be explained by the interaction between the carboxylic active lms is superior to that observed in a study published by
acid groups of L-arginine and an amine on the glucosamine unit Gasti et al. (2020),62 demonstrating that Solanum nigrum leaf
of chitosan with an increased degree of substitution, which led extract-added CS/PVA lms exhibited good ultraviolet (310 nm)
to a reduction in the –NH2 groups and an increase in the barrier properties. The opacity values of the AMC active lms at
number of hydroxyl groups.58 These accessible free hydroxyl 250 nm, 300 nm and 350 nm with respect to the UV-A, UV-B, and
groups interact with water molecules through hydrogen UV-C regions, respectively, are shown in Fig. 7(b). The opacity of
bonding, enhancing the lm's water solubility.14 the L-arginine-incorporated AMC active lms was higher than
that of the AMC-1 (control) lm, which could be attributed to
3.7. UV-visible spectroscopy analysis a reduction in the % light transmittance. However, the
improved opacity values of the active lms help to prevent lipid
One of the most desirable parameters of packaging lm is that it
oxidation and preserve the nutritional qualities of packaged
should safeguard the food from ultraviolet radiation. UV radi-
food.63 In the present study, the opacity value of the active lms
ation promotes numerous detrimental activities that diminish
increased as the weight percentage of L-arginine was increased.
the nutritional quality of food products.59,60 As illustrated in
The AMC-4 active lm with a high L-arginine content exhibited
Fig. 7(a), the percentage transmittance of the AMC-1 (control)
a maximum opacity value of 27.64 ± 0.13 at 250 nm in the UV-C
lm dropped with the incorporation of L-arginine, and
region, in contrast to the AMC-1 (control) lm. The increments
continued to decline as the weight percentage of L-arginine
Fig. 7 Optical parameters: (a) % transmittance, (b) opacity of the AMC-1 (control) and AMC-2, AMC-3, AMC-4 active films.
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in the opacity values might be due to the compact structure of which was decreased by incorporating L-arginine. Furthermore, it
the active lms induced by the secondary intermolecular was observed that with increased L-arginine content, the OP of the
interaction between the polymer matrix and L-arginine that AMC active lm was signicantly reduced. Meanwhile, the AMC-4
decreased the interchain gap, thereby permitting less light to active lm showed strong oxygen barrier performance with
This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
transmit through the active lms.64 a lower OP of 9.6 × 10−5 ± 0.19 cc$m−1$24 h atm compared to the
AMC-1 (control) lm. The enhanced oxygen barrier performance
3.8. Water vapour transmission rate (WVTR) of the active lms was due to the more compact, tightened
structure resulting from the compatibility of the L-arginine with
Examining water barrier properties is essential for packaging the lm matrix, which made it more difficult for nonpolar oxygen
applications, as it provides information on the diffusivity rate of molecules to pass across the lm, as shown in Fig. 8.
water vapors from the external atmosphere to packed food
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products via the packaging.65 Proper water vapor barrier prop-
erties of the packaging lm can preserve the quality and prolong 3.10. Soil burial test
the life span of the food products. The presence of moisture The degradation of active lms in soil takes place in two steps.
content in the food may promote a variety of enzymatic and In the initial step, swelling of polymer lms is caused by the
chemical reactions, diminishing the functional qualities of food penetration of water into the lms, which inuences the growth
and reducing the food products' lifespan.66 The WVTR results of of microorganisms. The second step includes disruption of
the fabricated control and active lms are shown in Table 1. polymer lms, followed by weight loss as a result of enzymatic
Initially, the AMC-1 (control) lm showed a WVTR of 27.49 ± and secreted degradation.68 In the present work, the degrada-
1.01 g m−2 h−1. However, including L-arginine in the MC/CS tion rate of the fabricated active lms in soil was examined, and
polymer matrix signicantly lowered the diffusivity rate of the resulting data are illustrated in Table 1. All of the active
water vapors to 26.64 ± 0.57 g m−2 h−1 through the AMC-2 lms showed more than 20% soil degradation within 10 days.
active lm. When the weight percentage of L-arginine The AMC-1 (control) lm showed 20.94% ± 0.62 degradation,
continued to increase, the rate of water vapour transmission while the degradation rate of the L-arginine-incorporated AMC
through the AMC-3 and AMC-4 active lms declined to 26.21 ± active lms is greater. The AMC-4 active lm exhibited 28.88%
0.92 g m−2 h−1 and 25.45 ± 1.55 g m−2 h−1, respectively, ± 0.89 soil degradation within 10 days. In comparison to the
compared to the AMC-1 (control) lm. The decrease in the AMC-1 (control) lm, this faster soil degradation might be due
WVTR was due to the complex and dense surface morphology, to the decay of small units of active lms by the action of
as witnessed by the SEM micrographs. This compact molecular microorganisms owing to the high water solubility of lms, as
structure introduced the tangled path, which lengthened the evidenced by the water solubility results. These results were
permeation path of water vapors and reduced the diffusivity rate supported by Carissimi, Flores and Rech (2018),69 who reported
of vapors through active lms,49,67 as shown in Fig. 8. that lms exhibiting high water solubility were likely to undergo
fast biodegradation.
3.9. Oxygen permeability (OP)
The oxygen barrier performance of packaging materials is essen- 3.11. Antimicrobial efficacy
tial for food packaging applications since oxygen transmission The antimicrobial efficacy of the control and active lms was
within the material promotes food oxidation, leading to food assessed against Gram-negative bacteria E. coli, and Gram-
spoilage. Table 1 presents the oxygen permeability values of the positive bacteria S. aureus, B. subtilis, and fungi C. albicans.70
control and active lms. An oxygen permeability of 11.6 × 10−5 ± The antimicrobial activity and respective inhibition zones are
0.20 cc$m−1$24 h$atm was noted for the AMC-1 (control) lm, displayed in Fig. 9 and Table 2. All of the prepared active lms
Fig. 8 Representation of the plausible scheme for the decreased WVTR and OP.
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exhibited potent antimicrobial efficacy against S. aureus and B. Table 2 Antimicrobial zone (zone of inhibition in mm) of the AMC-1
subtilis, and showed less activity against E. coli. The antimi- (control) and AMC-2, AMC-3, AMC-4 active filmsa
crobial efficacy of the control and active lms is due to the
Zone of inhibition (mm)
microbial inhibiting ability of CS. However, the precise mech-
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anism of the intrinsic antimicrobial efficacy of the CS is Samples B. subtilis S. aureus E. coli C. albicans
unknown.26 It was noticed that the AMC active lms suppressed
AMC-1 16.0 0.17 d 16.0 0.28 b 8.5 0.17 c 11.5 0.11 c
the activity of B. subtilis and S. aureus more efficiently than E.
AMC-2 17.5 0.28 c 17.5 0.40 a 9.5 0.23 b 13.0 0.23 b
coli. This signicant difference in microbial inactivation is AMC-3 19.5 0.11 b 18.0 0.34 a 10.0 0.28 b 13.5 0.17 b
attributed to the variance in the cell wall compositions of the AMC-4 21.0 0.23 a 18.5 0.23 a 11.0 0.11 a 14.5 0.28 a
Gram-positive and Gram-negative bacteria. The outer layer of a
Data are presented as Mean ± SD, a–d. The superscript letters in every
Gram-negative bacteria comprises phospholipids and lipo-
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data point indicate that there is a signicant difference (P < 0.05).
polysaccharides, which can hinder antibacterial activity,
making Gram-negative bacteria resistant to the antimicrobial
compound. The Gram-positive bacteria lack this essential outer
lipid layer, and are less resistant to antimicrobial ingredients.71 3.12. Antioxidant activity
Also, considerable antifungal activity was observed against C.
Food packaging materials must mitigate the oxidation of food
albicans. Incorporating L-arginine in the MC/CS polymer matrix
ingredients, such as proteins, lipids and fatty acids, which
further improved the antimicrobial properties of the AMC active
promote food spoilage. Hence, antioxidant packaging materials
lms. This improved antimicrobial activity is due to the posi-
prolong the shelf life of packaged foods. It was found that the
tively charged amino and guanidyl groups in L-arginine that
antioxidant efficacy is predominantly attained by suppressing
attack the anionic constituents of the bacterial cell wall via
the production of free radicals. Thus, radical scavenging activity
electrostatic interaction. Disturbing the physiological activity of
may play a signicant role in the antioxidant process.73,74 The
microbes and disrupting the microbial membrane through
results of the antioxidant response of AMC active lms are
a lytic mechanism that releases cell constituents eventually
depicted in Fig. 10. The AMC-1 (control) lm showed negligible
causes cell death.38,72
antioxidant activity, whereas incorporating L-arginine into the
Fig. 9 Demonstration of the antimicrobial activity of the AMC-1 (control) and AMC-2, AMC-3, and AMC-4 active films against B. subtilis, S.
aureus, E. coli and C. albicans.
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Fig. 10Antioxidant activity profile of the AMC-1 (control) and AMC-2,
Fig. 11 Results of the overall migration of the AMC-1 (control), and
AMC-3, and AMC-4 active films.
AMC-2, AMC-3, AMC-4 active films.
MC/CS polymer matrix improved their antioxidant potential.
and the food stimulants. In the present study, the active lms
The free radical scavenging activity was promoted as the weight
exhibited the highest overall migration rate in distilled water
percentage of L-arginine was increased. This improved radical
compared to 3% acetic acid and 50% ethanol, owing to the high
scavenging activity may be attributed to the interaction of the
solubility of L-arginine in water. Among other food stimulants,
incorporated L-arginine with free radicals via the donation of
the migration rate is higher in 3% acetic acid due to chitosan's
electrons and the development of the free radical chain process.
solubility in acetic acid. It has been found that the overall
The AMC-4 active lm with a higher L-arginine weight
migration of active lms increased as the content of L-arginine
percentage (7.5%) displayed greater antioxidant potential,
in the polymer matrix increased. This could be attributed to the
possibly related to the higher electron density of functional
swelling behavior of the active lms in all three food stimulants,
moieties on the active lms. Increased electron cloud density
which tends to increase the free volume of the MC/CS polymer
accelerates the donation of electrons, promoting the free radical
matrix and inuences the release of L-arginine from the polymer
scavenging process.73 Therefore, it was evident that the incor-
matrix.18
poration of L-arginine enhanced the accessibility of potential
electron-donating groups in the AMC active lms, resulting in
more signicant antioxidant activity in comparison to the AMC- 3.14. Packaging efficiency of the active lms
1 (control) lm.
3.14.1. Visual appearance. The visual appearance of the
green grapes unpacked and packed with AMC active lms over
3.13. Overall migration rate the storage period of 17 days is shown in Fig. 12(a). Initially, all
An overall migration test was carried out using three food the grape samples packed with AMC active lms, including the
stimulants, such as water, 50% ethanol and 3% acetic acid, to unpacked ones, had a good appearance and were green. When
evaluate the compatibility of the active lms with aqueous, the storage period was extended up to the 5th day, the unpacked
alcoholic beverages and acetic food products, respectively. The grapes shriveled, began to turn brown and worsened on the 15th
migration of packed material into the food stimulants leads to day of storage. The shrinkage and browning might be due to the
contamination of the food packed in it, and causes health dehydration and oxidation of polyphenols present in the grapes,
problems due to consuming these packed food products. The respectively. The grapes packed in the AMC-1 lm began to turn
Bureau of Indian Standards (IS:9845-1998) declared that the brown at the tip aer the storage period of 10 days. In contrast,
overall migration of the packing material should be less than the grapes packed in the AMC-4 active lm retained their
10 mg dm−2. Fig. 11 displays the results of the overall migration physical appearance and colour even aer the 15th day of
rate. The ndings revealed that the overall migration values of storage. This is due to the enhanced antioxidant ability of the
active lms in all three food stimulants were within the AMC-4 active lm, which successfully mitigated the oxidizing
permissible limit of 10 mg dm−2, suggesting their food activity of free radicals emitted on the surface of the grape and
compatibility. The overall migration values rely on thermody- maintained the quality of the grape. Incorporating L-arginine
namic properties like the swelling polarity, lm solubility and into the MC/CS matrix improved the barrier characteristics, and
the interaction between the components of the polymer matrix the antimicrobial and antioxidant efficacy of the active lms,
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Fig. 12(a) Monitoring freshness and shelf life, (b) percentage weight loss, and (c) antibrowning activity of unpacked and packed grapes in the
AMC-1 (control), AMC-2, AMC-3 and AMC-4 active films.
which resulted in preserving the grape quality for a longer time degrades the nutritional quality, shortens the shelf life and
period.75 Hence, the L-arginine weight percentages in the lms lowers the market value. Thus, browning is linked with anti-
directly correlate to the efficacy of packaging grapes. Similar microbial and antioxidant activity.78 Fig. 12(c) illustrates the
ndings were published by S. Kumar et al. (2019); as the browning degree of an unpacked grape and grapes packed with
concentration of nanoparticles in the agar-ZnO NP matrix AMC active lms. The ndings showed that the browning
increased, the shelf life of green grapes was extended.76 degree of the unpacked grape was signicantly higher than that
3.14.2. Weight loss. The weight loss of unpacked green of the grapes packed with AMC active lms, which might be due
grapes and those packed in AMC active lms are illustrated in to the oxidation of polyphenols and lipids present in the grapes.
Fig. 12(b). Generally, the weight loss of the fruits occurs due to the Meanwhile, the grape packed in the AMC-4 active lm exhibited
dehydration process caused by water loss through evaporation only the slightest browning. This suppressive action of the
and cellular respiration throughout storage.77 The weight loss AMC-4 active lm on the browning of the grape is attributed to
results suggested that the weight loss of green grapes unpacked several reasons. Firstly, it has an antibacterial property that
and packaged with AMC active lms increased as the storage mitigates the bacterial growth on the surface of the grapes.
period increased. It was observed that the unpacked grape Secondly, the antioxidant efficacy of the AMC-4 active lm
exhibited the highest weight loss, while the grape packed with the safeguards the polyphenolic components in grapes against
AMC-4 active lm exhibited the most negligible weight loss oxidation.35 In addition, the lm's UV barrier property prevents
during storage compared to those packed with other AMC active lipid oxidation, which in turn lowers the browning rate.
lms. The reduction in weight loss of grapes packed with the
AMC-4 active lm was attributed to the improved barrier prop-
erties due to the presence of a higher weight percentage of L- 4. Conclusions
arginine, as evidenced by the results of the WVTR, OP, UV barrier
In the present work, active lms of MC/CS were fabricated by
and antioxidant property, which delayed the water loss by modi-
incorporating L-arginine as an active ingredient using
fying the internal atmosphere and prevented the weight loss.35,77
a sustainable solvent casting technique for green food pack-
3.14.3. Antibrowning analysis. Colour is a crucial sensory
aging applications. FTIR analysis evidenced the hydrogen
parameter affecting the quality and visual appearance of grapes.
bonding interaction between L-arginine and the MC/CS lm
However, browning occurs naturally due to enzymatic oxidation
matrix. SEM results unveiled the dense and compact surface
and microbial development throughout storage, which
morphology with the addition of L-arginine. Incorporating L-
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