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1 s2.0 S0141813023009704 Main PDF
1 s2.0 S0141813023009704 Main PDF
PII: S0141-8130(23)00970-4
DOI: https://doi.org/10.1016/j.ijbiomac.2023.124076
Reference: BIOMAC 124076
Please cite this article as: H. Shiva, D. Supriya, K. Roopa, et al., Biodegradable hybrid
biopolymer film based on carboxy methyl cellulose and selenium nanoparticles with
antifungal properties to enhance grapes shelf life, International Journal of Biological
Macromolecules (2023), https://doi.org/10.1016/j.ijbiomac.2023.124076
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Biodegradable hybrid biopolymer film based on carboxy methyl cellulose and selenium
nanoparticles with antifungal properties to enhance grapes shelf life
Hadimani Shivaa, Dodamani Supriyaa, Koliwad Roopaa, Shivanna K. Soujanyaa, Vandakuduri
Rakshataa, Avaradi Netravatia, Vijayakumar Akshaykumar a, Savitha De Brittob and Sudisha
Jogaiaha,c*
a
Laboratory of Plant Healthcare and Diagnostics, P.G. Department of Biotechnology and
Microbiology, Karnatak University, Dharwad-580003, Karnataka, India.
b
Division of Biological Sciences, School of Science and Technology, University of Goroka,
Goroka - 441, Papua New Guinea.
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c
Department of Environmental Science, Central University of Kerala, Tejaswini Hills, Periye
(PO) – 671316, Kasaragod (DT), Kerala, India.
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*
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Corresponding author: Dr. S. Jogaiah; Email: jsudish@kud.ac.in
ORCID: 0000-0003-2831-651X
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Tel: +91-836-2779533; Fax, +91-836-2747884
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Abstract
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In the current study, cellulose was extracted from sugarcane bagasse and further converted into
carboxy methyl cellulose. The morphological, chemical, and structural characterization of
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synthesizeed carboxy methyl cellulose was performed. Further, the biopolymer was fabricated
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with mycogenic selenium nanoparticles and used to develop the biofilms. The developed
biofilms were examined for the fruit shelf life stability, antifungal activity, and biodegradation
potential. The results revealed that grapefruits wrapped with biofilms showed enhanced shelf life
of fruit at all storage time intervals. The study also witnesses the antifungal activity of biofilms
with a remarkable inhibitory action on the spores of Fusarium and Sclerospora graminicola
phytopathogens. Lastly, the biofilms were significantly degradable in the soil within two weeks
of incubation. Thus, the developed biofilms exhibit multifaceted properties that can be used as an
alternative to synthetic plastics for fruit packaging and also helps in protecting against fungal
contaminants during storage with naturally degradable potential.
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1. Introduction
The extensive use of plastic products and their improper disposal in the environment have
exacerbated ecological problems [1]. In the past, numerous efforts have been made to solve the
challenges faced by plastic trash using bioplastics that are environmentally friendly and naturally
degradable [2–4]. The majority of the efforts are focused on substituting petroleum-based
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polymers with biodegradable alternatives derived from gelatin, cellulose, plant oil, corn starch,
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and starch polymers that have similar properties and are economically feasible [5,6]. Hence,
there is growing interest among researchers in producing eco-friendly bioplastics or films as an
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alternative for reducing plastics by using biopolymers as 'building blocks of nature' as they are
cheap, malleable, and affordable [7]. They may be chemically synthesized from biological
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materials or biosynthesized by organisms. These materials have the potenatial to replace up to
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for biopolymer films for their improved practical applications [10,11]. It has been stated
elsewhere that metal or metal oxide nanoparticles present on the surface of biopolymers increase
their catalytic activity, leading to the production of effective catalysts for various enhanced
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mechanical and biological applications [12]. In this context, producing biopolymers, mainly
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carboxy methyl cellulose (CMC) from renewable sources has gained more attraction owing to
their broad range of sustainable applications, including biodegradability and renewability.
CMC is a modified type of cellulose white powder that is odourless, tasteless, and non-
toxic [13]. It is biodegradable, biocompatible, hydrophilic, and works well as a film-forming
agent with exogenous molecule alteration compared to native cellulose [14]. Sugarcane
(Saccharum officinarum L.) is a popular crop in tropical and subtropical climates and accounts
for 70% of the total sugar in the world [15]. Bagasse is an important agricultural residue that is
produced more than 1287 million tonnes per year from sugarcane globally [16]. Sugarcane
bagasse has a rich source of cellulose content that can be utilized for CMC synthesis, and
biopolymers made of such material can biodegrade under normal conditions for composting [17].
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Selenium is a trace element that is present in the body and is crucial for sustaining
healthy physiological processes. The nano-form of selenium, selenium nanoparticles (SeNPs),
exhibits less toxicity and more biocompatibility than their analogs [18]. SeNPs have a variety of
enticing biological properties, such as antibacterial [19,20], antifungal [21,22], and antiviral
properties [23]. Fungi mediate the mycogenic synthesis process for SeNPs [24], offering a quick,
secure, and environmentally friendly approach to making SeNPs. SeNPs have remarkable
antifungal activity against P. chrysogenum, F. oxysporum, B. cinerea, A. parasiticus, A. niger,
and food-borne pathogenic bacteria [25]. Also, the mycogenic-based SeNPs have been utilized as
antimicrobial agents against broad-spectrum phytopathogens, and to induce systemic resistance
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in host plants to suppress the incidence of disease [26]. SeNPs are remarkable nanocarriers for
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anti-inflammation, ant-infection, and anti-cancerous immunomodulatory agents [27].
The primary goal of this research is to extract cellulose from sugarcane bagasse and
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synthesize CMC by chemical method. The synthesized CMC is then characterized using various
microscopic and analytical techniques. The biofilm was developed by blending the biopolymer
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with myogenic selenium nanoparticles (SeNPs), and investigated for its potential to enhance fruit
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The leftover sugarcane bagasse (SCB) was collected from a Dharwad-local sugarcane
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juice vendor and the extraction of cellulose was carried out following the procedure of Gupta et
al. [28]. Briefly, two kilograms of bagasse was soaked in distilled water for 36 hours to remove
extractives which were readily soluble, and dried in an hot-air-oven at 70 ºC for three days to
remove moisture and ensure size diminution. Sugarcane bagasse was finely powdered by using a
pestle and mortar, and then sieved through a 50-mesh screen. Alkalization was achieved by
adding 2% NaOH to 50 g of bagasse. The fibres were dried for 16 hours at 70 oC in a hot air
oven after being washed with deionized water and filtered by muslin cloth. Then 50 mL of 4%
HCl (v/v) was added to dried fibres to remove lignin for 3 hours at 80 ºC, and once the acid
hydrolysis was achieved. The fibres were brought to pH 7 by rinsing with water and re-filtered
and dried in a hot air oven at 70 ºC overnight. During the bleaching process, 50 mL of 4%
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sodium chlorite (w/v) was used, and the pH was calibrated by using 1M CH3COOH and was
kept at 80 °C for incubation. Once the fibres turned white, they were filtered using a muslin
cloth. The filtrate consisting of cellulose was dried for 24 hours at 60 °C for further processing.
Ten grams of cellulose was taken into a 250 mL beaker containing 100 mL of NaOH
(20%) and isopropanol in 1:1 ratio. This mixture was stirred for 4 hours at 60 ºC, and the alkaline
fibers were cleaned and maintained in an oven for 4 hours at 70 ºC after being rinsed and filtered
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with a suitable quantity of deionized (di) water to attain mercerized pH 6.5. The fibers were
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further immersed in 150 mL of 20% sodium monochloroacetate solution and agitated for 4 hours
at 60 ºC. Further, the material was washed and filtered using a 1:1 methanol and HCl
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combination. CMC was obtained as the residue on the filter paper, and was dehydrated overnight
in a 70 ºC oven for further characterization. The yield of CMC was calculated by using the below
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formula Mondal et al. [13].
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CMC yield=
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Dried CMC (1.5 g) was combined in 100 mL of 80% of methanol-water. This solution
was mixed and held for 15 minutes before being filtered [29]. The cake on filter paper was
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cleansed with freshly prepared 100 mL of 80% of 1:1 methanol-water solution, and dried at 70
°C for 24 hours, and the purity of the CMC content was calculated using the equation below
[34].
CMC content (%) =
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N=
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2.4.1 Fourier-transform infrared spectroscopy (FTIR)
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The disc was prepared after crushing the CMC with KBr, and spectra were recorded
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on the Nicolet Summit FTIR Spectrometer (CAT: 912A0971; ThermoFisher, USA) in the
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range of 4000-400 cm.
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The crystallinity and distribution of the CMC powder was analyzed by using an X-ray
diffractometer (DteX250H) with an X-ray generator of 40 kV and 30 mA with a Cu_K-beta_1D
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For the preparation of biopolymer film, two solutions were used, solution A consisted of
1g of CMC + 50 mL of distilled water and Solution B contained 1.15 g of gelatin and 0.55 g of
agar in 50 mL of deionized water. which were separately prepared in 100 mL screw cap bottles.
Both the solutions were autoclaved at 15 lbs of pressure for 15 minutes. Later, the solutions were
mixed together thoroughly with the addition of 2% glycerol and different concentrations of
myogenic selenium nanoparticles (SeNPs) (50, 100, 150 ppm) in three separate containers [22],
and kept on the magnetic stirrer at 70 °C at 1200 rpm for 20 minutes. The froth was allowed to
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settle, and the solution was cast on a glass plate. The obtained biopolymer film was allowed to
dry in a hot air oven at 40 °C for 36 hours.
The biopolymer films were cut into appropriate sizes and fixed onto a clean glass slide
without forming air bubbles. For the AFM, NANOSURF EZ2- Flex equipment was used to
determine the surface texture of the biopolymer films.
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2.6.2 Scanning electron microscopy (SEM)
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The topography of fabricated biopolymer films was analyzed by SEM (JSM-IT500
InTouchScope™; JEOL, Japan) at a landing voltage of 8.0 kV in high vacuum mode of 3.0 nm
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(30 kV) to 15.0 nm (1.0 kV). During this procedure, the sample was blot-dried and thoroughly
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A thermogravimetric analyzer was used to determine the film sample's thermal stability.
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Under a 50 mL/min nitrogen flow, the film sample was heated from 40 ºC to 700 ºC at a rate of
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24 ºC /min. The maximum degradation temperatures were calculated using the TGA (DTG)
curves in derivative form.
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The contact angle meter was used to test the wettability of the surfaces of the fabricated
biopolymer films (DMS 401; Kyowa interface, Japan). The fabricated biopolymer films were cut
into rectangular pieces measuring 3 cm long by 7 cm wide and positioned on the instrument's
platform. A micropipette was then used to drop 10 μl of water onto the film surface, and images
of both sides of the biopolymer films were taken with the face in interface with the plate (lower)
and the side facing the air (upper).
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2.6.6 Thickness determination of biopolymer film
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A digital micrometer (Thorlabs, NJ, USA) was used to determine the thickness of the
produced biopolymer films with a precision of 0.001mm. The mean thickness of the film sample
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was determined by gauging five random spots [32].
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The opacity of the fabricated biopolymer films was measured by placing the rectangular
biopolymer films strip (4 cm in length and 1 cm wide) in a UV- Vis spectrophotometer (Agelent,
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Carry 60) at 600 nm using air as a reference. The below equation was used to compute the film's
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opacity value.
Opacity =
where A600 is the absorbance value at 600 nm, and S is the thickness of the film.
The biopolymer films were sliced into a rectangular section (2×2 cm), and subjected to
flame, and the results were recorded. Likewise, the odour was checked by smelling the
biopolymer films for any foul or pungent smell.
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Five milliliters of vegetable oil and water were taken in two separate test tubes and sealed
with biopolymer films of known size. Both the tubes were placed in over turned position on the
paper towel of known weight for 24 hours, and after the incubation period, the weight of the
tissue paper was measured. The coefficient of permeability was estimated by using the formula.
PC =
Where PC is the oil permeability coefficient (g·mm/(m2·day), ∆FPW difference in the filter
paper weight (g); T is the thickness of the biopolymer film (mm), A is the area of the biopolymer
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film (m2), and t is incubation time (in days) [17].
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2.6.10 Swelling Test
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The swelling experiments were carried out in 30 mL screw cap vials. Completely dried
biopolymer films were cut into rectangular shapes and weighed. The experiment was carried out
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in three vials containing 20 mL solvents of water, methanol and chloroform, respectively. Three
pre-weighed biopolymer films were placed in these solvents for one day at room temperature.
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This analysis was carried out to check whether the synthesized biopolymer films were
soluble, partially soluble or completely soluble in ethanol, methanol, acetic acid, chloroform and
water. For this, air-dried biopolymer films were sliced into square pieces and weighed on the
precision balance. Twenty milliliters of the tested solvents were taken in five separate vials, and
the pre-weighed biopolymer films were immersed for 24 hours. After the incubation period
again, the biopolymer films were dried,their weight was measured, and the below formula was
used to calculate the solubility percentage.
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For the preservation studies, equal and uniform sizes of grape fruits (Vitis vinifera) cv,
Thompson seedless were procured from the local market of Dharwad. Forty fruits of uniform
size were selected and washed with 2% sodium hypochlorite solution, then in water, dried and
weighed, of which five fruits were wrapped with the fabricated biopolymer films, and the rest
five unwrapped served as untreated control. The biopolymer film film-wrapped and unwrapped
samples were kept at room temperature for 14 days and monitored at regular intervals. The decay
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percentage was calculated by calculating the weight loss and colour change (green to brown) in
the fruit at regular time interval as, Slightly brown- ―+‖, moderately brown- ―++‖ and completely
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brown- ―+++‖. The weight loss was calculated according to the below formula.
About 10 grams of wrapped and unwrapped grape fruits were homogenized and filtered
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separately with distilled water. Titratable acidity was evaluated using phenolphthalein as an
indicator and comparing filtrate to a standard (0.1 M) NaOH. The following formula was used to
determine titratable acidity, which was measured in terms of the percentage of tartaric acid.
Spores of Fusarium sp. were harvested from 9-day-old culture grown on potato dextrose
agar media by flooding the petri dish with 15 mL of sterile distilled water. The final
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concentration was adjusted to 1.4 ×107 spores/mL using a hemocytometer. For Sclerospora
graminicola, the sporangium produced on host plant leaves were harvested into sterile distilled
water (SDW) in the early hours, and the concentration of sporangia was adjusted to 1.5 ×106
sporangia/mLusing a haemocytometer [33].
A total of 100 µL of these suspensions were treated with dissolved 1g of biopolymer
(CMC + SeNPs) in 1mL sterile water for 15 minutes, and Fusarium sp. spores suspension was
incubated under light conditions, and sporangium suspension of S. graminicola was stored in the
dark at room temperature. Under identical positive and negative control conditions, copper
oxychloride (100 ppm) or 100 µL of metalaxyl XL and sterile distilled water were administered
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to a 100 µL spore solution. Under a light microscope, the likely effect of the dissolved
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biopolymer on the spores was visualized [22,34].
capacity with different soil samples (black soil from agricultural fields, garden soil, black soil
mixed with sand and silt in the ratio of 1:1:1 and soil + vermicompost in 1:1 ratio). The
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fabricated biopolymer films were aseptically cut into 2 cm × 2.5 cm size using a sterile blade and
weighed on a precision balance to record the initial weight (initial). Then the biopolymer films
were placed in the beakers and covered with the respective soil, and 15 mL of H2O was splashed
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over it to boost the microbial activity. At regular intervals, the biopolymer films were removed
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carefully, and dirt from the surface was cleaned using a brush. Then the biopolymer films were
weighed to measure the final weight (final), and the below formula was employed to calculate
the percentage of degradation.
The percentage of biodegradation of biofilm was performed using four replicates and the
average means and F values (P < 0.001) significance of each treatment were analyzed using
SPSS Inc. Chicago Il, USA 20.0 version. Means followed by the same letter(s) (a-e) within the
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column are not significantly different according to Tukey’s honestly significant differences
(HSD) test.
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Magaraphan, [35]. In a separate study, bleached CMC with different concentrations of hydrogen
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peroxide produced a higher yield ranging from 135.52% to 142.41% [36]. In the past, studies
have shown an increased CMC yield from waste materials [5,37,38]. Such variations in the CMC
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yield may be due to the raw material used for synthesis and also differing conditions [39–41].
Additionally, the increase in CMC yield might be because of the large amount of carboxymethyl
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groups present in the synthesized CMC [41].
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Further, we recorded 97.4% purity of synthesized CMC after methanol treatment (Fig. 1).
This data is in agreement with the recent findings of Celikci et al. [42], who documented 92.1%
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CMC purity. Similar findings were also documented in the previous year by Gupta et al. [28],
who also reported 98.7% purity of CMC, followed by 98.23% purity of CMC from the
agricultural waste as investigated by Adinugraha et al. [43]. The enhanced purity of synthesized
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CMC mainly depends on the NaOH concentration used for the mercerization and etherification
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of cellulose [44,45].
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Fig. 1. A purified carboxymethyl cellulose (CMC) powder by 20% of NaOH used for
mercerization and etherification of cellulose.
In the present study, the degree of substitution (DS) of the CMC obtained was 1.134,
which is significantly higher than the earlier reports [28,29]. The DS is affected by the
concentration of NaOH, temperature, and time. A lower NaOH concentration and room
temperature increased the corresponding DS value [30]. The solubility of the CMC increases
proportionately with the DS value [46]. Similarly, Suriyatem et al.[36] reported that with
different concentrations of H2O2, the DS value ranged between 0.23–0.53. Likewise, Rahman et
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al. [47], showed that the DS value obtained was 1.83. It is also observed that a greater CMC DS
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causes a wider crystallinity peak and a reduction in crystallinity [48].
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3.2 Characterization of carboxymethyl cellulose (CMC)
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The results of the FTIR spectrum of carboxymethyl cellulose (CMC) demonstrated the
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groups were observed in the spectra. A typical peak at 894.54 confirmed the presence of
cellulose. Water-bound stretching was seen at 1638.19 cm-1. Our FTIR results are consistent with
the earlier reports, which confirm the presence of major carboxymethyl groups
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[28,31,49,50](Fig. 2a).
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In this study, the XRD pattern of synthesized CMC powder showed five major diffraction
peaks, of which three higher peaks at 12.28, 20.15° and 22.06° show crystalline nature (Fig. 2b),
a similar pattern of XRD peaks was also noticed by other workers [49,50]. Moreover, two
smaller peaks towards the right at 34.66° and 45.51° of 2θ corresponded to the amorphous nature
of CMC, which is in correlation with the previous report [49]. It has been reported that hydrogen
bonding in an alkaline environment is disrupted during mercerization, resulting in the amorphous
nature of CMC. This data reveals that the crystalline peak in CMC XRD spectra is displaced due
to the breakdown of β-1,4 glycosidic bonding, causing a rise in the space between cellulose
molecules [28].
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(a) (b)
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Transform Infrared (FTIR) spectral bounds representing functional groups in synthesized CMC.
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(b). X-ray diffractionograms (XRD) from 5° to 90° of synthesized CMC powder from 20%
NaOH showing its amorphous nature.
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3.3 Characterization of fabricated biopolymer films
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The current study reveals that fabricated biopolymer films had an average thickness of
0.116 mm, and an opacity of 3.948%, with a tensile strength of 1.167 MPa and elongation of
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26.539% (Fig. 3a). These positive features of the biopolymer films help to ensure high bioplastic
quality and safety as a packaging material [51,52].
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Further, the fabricated biopolymer films were analyzed for their surface topography
using an atomic force microscope at the magnification of 2.07, 5, and 10µm on the X, Y, and Z-
axes. Under visualization with AFM, we confirmed the presence of an uneven surface due to the
blending of SeNPs nanoparticles and amorphous CMC (Fig. 3b). Moreover, our data
demonstrated that the nanoparticles are evenly distributed, with no cracks, pores, or air bubbles
in the biopolymer films. Previously, similar mechanical properties of biopolymer films were also
observed and documented [49].
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The SEM analysis of fabricated biopolymer film observed under different magnifications
of 5, 10, and 100 µM field showed that the nanoparticles are evenly distributed on the
biopolymer films (Fig. 3c). We also witnessed the biopolymer films had a clean surface without
any disturbances as observed by Li et al. [50].
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of degradation. The initiation was recorded at 66.54 to 84.42 °C, which is attributed to the
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moisture removal, decline at 84.42 to 198.86 °C with 7.46% degradation, and significant decline
at 198.86 to 288.90 °C with 80% degradation, where glycerol and CMC polymer matrix
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breakdown occurs [53]. The breakdown is attributed to the removal of carbon dioxide from the
polysaccharide chain, and the saccharide ring of CMC is degraded. Also, there was an increase in
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residual particles due to the complete degradation of the fabricated biopolymer films at 788.68
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°C (Fig. 3d). This analysis shows that the biopolymer films produced can withstand higher
temperature [54].
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The sample had two separate endothermic peaks within the studied temperature range
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when analyzed by DSC. The initial endothermic peak was exhibited approximately at 82.81 °C
with an enthalpy of 157.36 J/g, corresponding to the transition glass temperature (Fig. 3e). The
observed glass transition temperature is slightly higher than that of pure CMC [55]. This might
be due to the blending or fabrication of CMC with agar, glycerol, and selenium nanoparticles. At
247.89 °C with an enthalpy change of 273.2 J/g, the second endothermic peak demonstrated
thermal breakdown or melting temperature of CMC [50].
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The synthesized biopolymer films were also evaluated for flame analysis, and it was
observed that the films burnt with a yellow sooty flame with black carbon residues at the end.
This result confirms the presence of unsaturated hydrocarbons in fabricated biopolymer films,
enhancing flame resistance considerably [56]. After smelling the biopolymer films, it is
concluded that it is odorless. In support of this data, it is referred as polysaccharide-based
biofilms produced by agricultural wastes are odorless, colorless, and exhibit resistance to flame
[57]. Collectively, it may be stated that flame resistance and odorless properties observed in this
study may be due to the use of sugarcane bagasse as raw material.
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3.3.6 Contact Angle
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This technique was employed to evaluate the wettability or hydrophilic properties of the
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biopolymer films. The fabricated biopolymer films had a contact angle of 66.2°, which is higher
than that of the pure CMC [58]. Due to the addition of SeNPs, the hydrophilic nature of the film
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is reduced, and generally, a hydrophilic contact angle below 90° is formed [59]. The strong
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hydrogen bonding conferred by the functional groups renderd hydrophilic nature to the
biopolymer fims (Fig. 3f).
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Fig. 3. Characterization of fabricated biopolymer film. (a). Change in tensile strength (MPa)
and elongation at break point (%) of fabricated (CMC and selenium nanoparticles) biopolymer
film. (b). An atomic force microscope imaging in dynamic mode showing the uniform surface
topography of fabricated biopolymer films at the magnification of 2.07 μm × 10 μm of the
scanning area. The green scale mark on the right side of the image represents the cantilever tip
surface scanning possession scale range at 251 nm. (c). Scanning electron microscopic images at
5, 10, 50 and 100 µm magnifications depicting the uniform distribution of selenium
nanoparticles on the surface of the fabricated biopolymer film. (d). Thermogravimetric curves
display the percent weight of the thermal stability with increased temperature of the fabricated
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biopolymer film. (e). Differential Scanning Calorimetry exhibiting two endothermic peaks at
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82.81 °C with an enthalpy of 157.36 J/g and 247.89 °C with an enthalpy change of 273.2 J/g,
corresponding to the fabricated biopolymer films demonstrating its melting temperature. (f).
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Surface topology affects the fabricated biopolymer film’s contact angle (66.2°).
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3.3.7 Swelling and solubility test of biopolymer
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It is clear that the biopolymer films immersed in water havemore swelling by exhibiting
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0.67 g compared to chloroform and methanol, which recorded 0.033 and 0.009 g, respectively.
This experiment demonstrated that the fabricated biopolymer films absorb water readily due to
their hydrophilic nature.
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(a)
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(b)
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The biopolymer immersed in methanol loses its weight and became more dry and rigid.
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While, the biopolymer film immersed in water showed 79.069% solubility, followed by 71.15,
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62.74 and 60% in acetic acid, methanol, and ethanol, respectively. In contrast,the lowest
solubility of the biopolymer film was observed in chloroform with 12.82% solubility during the
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immersion study (Fig. 4a, b). This increased level of solubility shows that the fabricated
biopolymer films are readily biodegradable in water [60]. Due to the water-soluble nature of the
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biopolymer films, they can be easily degraded by soil microbial enzymes. In fact, the microbes
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The oil and water permeability coefficient of fabricated biopolymer films was 11.6 and
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672.8, respectively. This result demonstrate that oil has a lower permeability coefficient than
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water because there are fewer or no lipophilic groups in the biopolymer films (Fig. 5). Studies
have reported that CMC-based composite films have lipid and oxygen barrier qualities [61,62].
Overall, based on the physicochemical properties of the developed biopolymer films, it can be
ascertained that it serves a suitable material for food packaging with rapid biodegradability.
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Fig. 5. Determination of the oil and water permeability coefficient of fabricated biopolymer films
showing oil has a lower permeability coefficient than water, when performed on inverted test
tubes.
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3.4 Determination of shelf life of Grapes by biopolymer films
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During fourteen days of preservation, the unwrapped control grapes turned brown and
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shrunk in size after 7 days of incubation (Fig. 6). The decay percentage increased to 40% in
unwrapped control fruits when compared to the grapefruits wrapped with biopolymer, which
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recorded 15% decay. In addition, the control fruits developed a foul odor, and a maximum of
43.37% of weight loss was observed. The fruits wrapped in fabricated biopolymer films did not
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brown till the 14th day of preservation and resulted in a low shrinkage or weight loss of 21.7%,
and the fruit color remained fresh (Fig. 6). This pattern of fruit storage results has also been
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reported in various studies [63–65]. It can be correlated with higher water permeability
properties of the biopolymer films, which play a vital role in extending the shelf life of fresh
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fruits.
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Fig. 6. Demonstration of Grapes shelf life. Preservation of grapes wrapped with biopolymer
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film showing remarkable shelf life of the grapes incubated from day 1 to 14. The control grapes
turned brownish and shrunken in its size after 7 days of incubation.
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The study also focused on the titratable acidity in biopolymer films wrapped and
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unwrapped grapes on the 7th and 14th days of preservation. The percentage of tartaric acid in
fruits wrapped with biopolymer films was 0.27%, and in unwrapped fruits it was 0.195% on the
7th day of preservation. While the unwrapped fruits preserved for 14 days recorded a maximum
decrease of 0.3% tartaric acid, the wrapped fruits recorded 0.105%. The same trend in the
accumulation of tartaric acid in preserved fruits was documented in many studies across the
world [65–67]. It is suggested that the acidity level in the fruit majorly determines the freshness
of the fruits, the higher the organic acids present, the more acidic the fruits [68]. During storage,
the moisture present in the environment accelerates the enzyme activity in the fruit to degrade
the organic acids (tartaric acid), and in the due course, the fruits start to ripen and later wholly rot
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[69]. Thus, coating such films is essential as they control the moisture level and increase the
shelf life of the fruit.
This study aimed to explore the biodegradation of biopolymer films in different soil
samples. An average of 67% degradation of biopolymer films was observed on the 9th day in
tested soil samples. Among the soil samples, black soil with vermicompost exhibited 99.711%
degradation on the 15th day, which was comparatively higher than the other samples (Fig. 7a, b;
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Table 1). The degradation of the fabricated biopolymer films took more time when compared to
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the earlier report by Yaradoddi et al. [17]. It might be due to the incorporation of selenium
nanoparticles that would initially hinder the degradation due to its toxicity over the microbes.
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However, after the 5th day, a sharp increase in degradation was observed due to the breakdown of
the side carbonyl chain of the fabricated films. From the above comparison, it is proved that the
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developed biopolymer films are environmentally compatible and can be utilized for food
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packaging.
(a)
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(b)
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Table 1. Percentage rate of biodegradation of the biopolymer films incubated in soil samples.
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Numbe Black soil from Garden soil Black soil mixed with Black soil mixed
r of agricultural field sand and slit with vermicompost
days Weigh % of Weigh % of Weigh % of Weigh % of
t Degradatio t Degradation t Degradation t Degradation
(mg) n (mg) (mg) (mg)
0 1000 0±0e 1000 0±0e 1000 0±0e 1000 0±0e
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c c c
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7 622.8 37.72±1.1c 629.79 37.021±1.0c 621.37 37.863±0.74 619 38.1±1.0c
c
Values are the average means of four independent replicates. Means followed by the same
letter(s) (a-e) within the column are not significantly (P < 0.001) different according to Tukey’s
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HSD.
3.6 Inhibitory action of fabricated biopolymer films against Fusarium sp. and Sclerospora
graminicola.
The antifungal activities of the fabricated biopolymer films were evaluated under in vitro
conditions using the spores of Fusarium sp. and S. graminicola which causes major disease in
crop plants. The Fusarium spore suspension treated with dissolved fabricated biopolymer film
(CMC+100 ppm SeNPs) showed burst and shrinkage of spore compared to the control, where
spores were typically normal in their growth and development (Fig. 8).
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Fig. 8. In vitro inhibition of Fusarium sp. spores. The above microscopic image showing
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extensive spores shrinkage upon treated with 100 µl of water containing diffused biopolymer
films compared to untreated suspension, which showed typical spores formation as observed
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under a light microscope at 40X magnification (bar scale – 5 µm).
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In the case of Sclerospora graminicola sporangia treated with fabricated biopolymer film
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(CMC+100 ppm SeNPs), the zoospores were intact with the sporangia and some sporangia
underwent bombardment leading to nonmotile zoospores within 15 min of incubation under 40X
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magnification of a light microscope (Fig. 9). However, in the control empty sporangium freely
releasing motile zoospores leading to pathogenesis was observed.
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Fig. 9. Inhibitory action of oomycetes pathogen. The effect of biofilms on sporangial growth
and formation of Sclerospora graminicola in suspension culture. The percent inhibition was
calculated by assessing the empty and intact sporangia with the release of zoospores as observed
under a light microscope at 40X magnification (bar scale – 5 µm).
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This inhibitory action of fungal spores may be due to the incorporation of nanoparticles
reinforced with biopolymer films [22,70,71]. Out of three different concentrations of SeNPs,
biopolymer film incorporated with 100ppm of SeNPs showed excellent fungal spore inhibitory
properties. This inhibitory action of fungal spores may be due to the incorporation of
nanoparticles reinforced with biopolymer films. The adhesive antifungal properties in
biopolymer films helps in food preservation and can potentially be used to increase the shelf-life
of food stuff [72].
4. Conclusion
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In conclusion, the ubiquitous use of plastics and toxic chemicals in their production has
led to extreme global environmental pollution. Considering the emerging environmental issue,
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manufacturers should embrace biodegradable polymer films that can reduce plastic trashes and
clean the environment. Hence, this study explores the utilization of cellulose from agricultural
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wastes for the synthesis of CMC. The purity of the biopolymer CMC was confirmed by
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fabricating with selenium nanoparticles (SeNPs) as active agents to develop biopolymer films,
resulting in significant antifungal properties. Moreover, the fabricated antifungal biopolymer
films showed a remarkable coating material for grapes by extending their shelf life. Lastly, we
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have also provided evidence of biodegradation of the biopolymer films in various soil types that
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will ensure the replacement of synthetic plastic with the developed non-toxic and antifungal
biopolymer films for their better usage in sustainable food packaging materials in the coming
years.
Acknowledgements
All the authors would like to acknowledge Vision Group of Science and Technology for
providing CISEE programme for the Laboratory of Plant Healthcare and Diagnostics, PG
Department of Biotechnology and Microbiology, Karnatak University, Dharwad with the
financial aid No. GRD-327, Government of Karnataka, India. The authors also acknowledge the
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University Scientific and Instruments Center (USIC) facilities provided by Karnatak University,
Dharwad.
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Author Contributions
Research design and supervision: S.J. Performed the experiments: H.S., D.S., K.R., S.K.S., V.R.,
A.N and V.A. Compilation of data: H.S., S.K.S and V.A. Manuscript writing: H.S., S.D.B. and
S.J. Edited the revised manuscript: S.J. Read and approved by all the authors.
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Declaration of interests
X The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐ The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
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Graphical abstract
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