polymers

Article
Functional Nanocellulose, Alginate and Chitosan
Nanocomposites Designed as Active Film Packaging Materials
Gregor Lavrič 1 , Ana Oberlintner 2,3 , Inese Filipova 4 , Uroš Novak 2 , Blaž Likozar 2
and Urška Vrabič-Brodnjak 5, *

1 Pulp and Paper Institute, Bogišićeva Ulica 8, 1000 Ljubljana, Slovenia; [email protected]
2 National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia; [email protected] (A.O.);
[email protected] (U.N.); [email protected] (B.L.)
3 Jožef Stefan International Postgraduate School, Jamova Cesta 39, 1000 Ljubljana, Slovenia
4 Latvian State Institute of Wood Chemistry, Dzerbenes Street 27, LV-1006 Riga, Latvia; [email protected]
5 Department of Textiles, Graphic Arts and Design, Faculty of Natural Sciences and Engineering,
University of Ljubljana, Snežniška 5, 1000 Ljubljana, Slovenia
* Correspondence: [email protected]; Tel.: +386-1-200-32-82

Abstract: The aim of the study was to characterize and compare films made of cellulose nanocrystals
(CNC), nano-fibrils (CNF), and bacterial nanocellulose (BNC) in combination with chitosan and
alginate in terms of applicability for potential food packaging applications. In total, 25 different
formulations were made and evaluated, and seven biopolymer films with the best mechanical
performance (tensile strength, strain)—alginate, alginate with 5% CNC, chitosan, chitosan with 3%



CNC, BNC with and without glycerol, and CNF with glycerol—were selected and investigated


regarding morphology (SEM), density, contact angle, surface energy, water absorption, and oxygen
Citation: Lavrič, G.; Oberlintner, A.;
and water barrier properties. Studies revealed that polysaccharide-based films with added CNC are
Filipova, I.; Novak, U.; Likozar, B.;
the most suitable for packaging purposes, and better dispersing of nanocellulose in chitosan than in
Vrabič-Brodnjak, U. Functional
alginate was observed. Results showed an increase in hydrophobicity (increase of contact angle and
Nanocellulose, Alginate and Chitosan
reduced moisture absorption) of chitosan and alginate films with the addition of CNC, and chitosan
Nanocomposites Designed as Active
Film Packaging Materials. Polymers
with 3% CNC had the highest contact angle, 108 ± 2, and 15% lower moisture absorption compared
2021, 13, 2523. https://doi.org/ to pure chitosan. Overall, the ability of nanocellulose additives to preserve the structure and function
10.3390/polym13152523 of chitosan and alginate materials in a humid environment was convincingly demonstrated. Barrier
properties were improved by combining the biopolymers, and water vapor transmission rate (WVTR)
Academic Editors: Cédric Delattre was reduced by 15–45% and oxygen permeability (OTR) up to 45% by adding nanocellulose compared
and Paola Scarfato to single biopolymer formulations. It was concluded that with a good oxygen barrier, a water barrier
that is comparable to PLA, and good mechanical properties, biopolymer films would be a good
Received: 14 June 2021 alternative to conventional plastic packaging used for ready-to-eat foods with short storage time.
Accepted: 29 July 2021
Published: 30 July 2021
Keywords: carbohydrate; polysaccharide; nanocellulose; alginate; chitosan; film packaging material;
functional active design; biomass-derived biomaterial nanocomposites; oxygen/air/water barrier
Publisher’s Note: MDPI stays neutral
properties; bio-based biopolymer composites for food
with regard to jurisdictional claims in
published maps and institutional affil-
iations.

1. Introduction
According to estimates by the United Nations Joint Group of Experts on the Scientific
Aspects of Marine Pollution (GESAMP), between 70 and 95% of waste will join millions of
Copyright: © 2021 by the authors.
tons already present in seas, lakes, air, rivers, groundwater, crop fields, landfills, and cities.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
Most of this waste is coming from the traditional commercial food packaging materials with
distributed under the terms and
a petroleum-based origin such as polyethylene (PE), polypropylene (PP), and polystyrene
conditions of the Creative Commons (PS). Researchers have estimated that 31.9 million tonnes of mismanaged plastic waste
Attribution (CC BY) license (https:// enter the environment every year, with 4.8–12.7 million tonnes going into the oceans and
creativecommons.org/licenses/by/ significant quantities contaminating terrestrial ecosystems [1,2]. Still, it is expected that the
4.0/). demand for plastics will continue to grow in the future to enable resource-efficient products

Polymers 2021, 13, 2523. https://doi.org/10.3390/polym13152523 https://www.mdpi.com/journal/polymers

Polymers 2021, 13, 2523 2 of 15

needed by society. One of the establishing trends is in designing mindful products from
sources including recycled chemicals and renewable raw materials, and using processes
powered by renewable energy in striving to establish an efficient circular economy [3]. To
be in line with the UN sustainable development goals and sustainable plastic strategy, the
global plastics industry’s shift from a manufacturing system based predominantly on fossil
fuels to sustainable and affordable alternatives is being envisioned [4,5].
Packaging materials are an essential part of product processing, therefore the number
of investigations on the development and use of new alternatives has increased in recent
times. That the transition towards non-virgin petrochemical and bio-based raw materials
as alternative feedstocks should include recycled chemicals from plastics waste, sustainable
biomass, industrial wastes such as CO2 , and modified biopolymers such as cellulose or
starch, is suggested mainly due to the interest in minimizing the environmental impact
caused using synthetic packaging materials. One of the most desired features expected
in packaging is the capability of decomposing into carbon dioxide, methane, water, in-
organic compounds, or biomass, the dominant mechanism of decomposition being the
enzymatic action of microorganisms and that the resulting products can be obtained and
measured in a period of a certain time [6]. The materials used to make biodegradable pack-
aging can be biopolymers of natural origin (alginate, starch, gelatine, collagen, proteins,
chitosan, (nano)cellulose, pectin) or of synthetic origins, such as polylactic acid, polycapro-
lactone, and polyvinyl alcohol [7,8]. Especially the materials of natural origin have recently
(re)gained popularity, due to their special properties, which in many industries can become
an alternative for fossil fuel-based plastic. However, high production costs, low perfor-
mance, and not less important, ethical implications, still hinder the market penetration of
plastics-free alternatives so far. One of the currently underutilized sources of feedstock for
bio-based polymers can be found in the side streams of both agricultural and forest feed-
stock, which are a good source [9]. Cellulose, which exists in the lignocellulosic biomass,
is the most abundant polysaccharide present in nature. It is encapsulated by lignin and
hemicellulose and produces a linear polysaccharide with nanometre diameter by repeating
the connection of β-d-glucose [10]. Cellulose nanofibrils (CNFs) or cellulose nanocrystals
(CNCs) can be isolated from wood and other plant sources by partial disruption of their
natural structures, which is usually achieved using chemical and/or mechanical treatments.
Besides plants, some bacteria naturally produce cellulose microfibrils, which are referred to
as bacterial nanocelluloses (BNCs) [11]. These three types of nanocelluloses (CNCs, CNFs,
and BNCs) have different morphologies (sizes and shapes) depending on their biological
origin and the processes used to isolate them [12,13]. Unlike the rigidity of the CNC, the
CNF is flexible. This is mainly because the structure of CNF is an individual or aggregated
soft and long chain, which is formed by alternately connecting crystalline regions and
amorphous regions to each other [9–11].
The second most abundant natural biopolymer is chitin, the crucial structural biopoly-
mer of crustaceans’ exoskeletons, whereby its content varies not only between different
sources but also between different species [9]. These types of polysaccharides have pre-
viously mostly been utilized and studied for biomedical applications [14]. However, in
the search for new carbon-neutral renewable resources using the biorefinery approaches,
turning cast-off shells into nitrogen-rich chemicals would benefit economies and the en-
vironment [15]. Other largely exploited sources for biopolymers are macroalgae, which
are the rich source of indigestible polysaccharides that are commonly produced by and
refined from various brown seaweed and can be developed into active food packaging
materials [16,17]. In the recent review from Zhang et. al., the effect of the incorporation of
CNC on the film’s characteristics, including thickness, optical properties, barrier properties,
water sensitivity, mechanical properties, antioxidant properties, and antimicrobial proper-
ties have been presented [18]. The main advantage of using cellulose nanoparticles as a
reinforcing part in the film is most addressed by the “tortuous theory”, where the cellulose
nanoparticles, due to their size, form a denser microstructure, which mostly leads to an
increase in the mechanical strength of the composite (bio)material. Moreover, cellulose
Polymers 2021, 13, 2523 3 of 15

nanoparticles act as a physical barrier structure in the composite film materials leading to
reducing the movement of gas molecules through the film. The films with a higher degree
of nanocellulose fibrillation absorbed more water, and had the higher contact angles for
glycerol and lower contact angles for water [19]. Recently, many studies have shown that
the incorporation of cellulose nanomaterials as an additive could improve the performance
of the food packaging films [20–22]. Further advances in nanocellulose research in biopoly-
mers film are quite promising for active packaging applications, including the controlled
release packaging and responsive packaging [22–25].
This study aimed to evaluate and compare the effect of the CNC, CNF, and BNC
cellulose nanomaterial, where the first two were obtained from lignocellulosic biomass and
the last one from bacterial origin. Altogether, 25 combinations of biopolymer films using
chitosan, alginate, or nanocelluloses as a single component or in different combinations
were prepared. Based on the determined mechanical properties in the first part of the study,
the best film in terms of mechanical properties and physical appearance were selected
and further characterized for density, contact angle, surface energy, water absorption, and
morphological examination with SEM, oxygen, and water barrier properties. Finally, some
suggestions and challenges of potential new sustainable packaging that need a further
improvement/focus to commercially exploit this (nano) material renewable bioresource
for packaging application.

2. Materials and Methods

2.1. Materials
High molecular weight (Mw = 310,000–375,000 Da) chitosan (<75% deacetylated),
sodium alginate and 85 wt% lactic acid were purchased from Sigma Aldrich (Steinheim,
Germany) and glycerol was purchased from Pharmacem Sušnik (Ljubljana, Slovenia).
Nanocellulose materials were produced as presented in the sections below.

2.1.1. CNF Production

For CNF production, bleached hardwood Kraft pulp (kindly provided by Metsä
Fibre, Ainekoski, Finland) was oxidized at 70 ◦ C for 4 h in APS (ammonium persulfate)
solution (APS: fibers amount ratio 5:1) with continuous stirring. Oxidation was stopped
by cooling the mixture to 15 ◦ C, treated fibres were washed until neutral and kept at 4 ◦ C.
Oxidized cellulose fibres were then suspended in water (1.5% w/w), sonicated (ultrasonic
homogenizer SONIC-650W, MRC Ltd., Holon, Israel) for 15 min (90% power, 9 s on, 1 s off),
and then processed in microfluidizer (LM20, Microfluidics, Quadro Engineering, Waterloo,
ON, Canada); the first 3 times through 200 µm ceramic chamber H30Z, then through
100 µm diamond chamber H10Z at 300–600–900–1500 bar, three passes at each pressure,
followed by 6 passes at 2000 bar. Sample was cooled in an ice bath during the treatment.
Semi-transparent viscous 1.5% w/w solution was obtained and kept at 4 ◦ C until used. The
yield of CNF reached ~80% from the initial amount of pulp.

2.1.2. BNC Production

The raw material for BNC production was a cellulose-rich bio-film formed after acetic
fermentation of apple juice [26] and was obtained from a local vinegar producer. Bio-film
was thermo-mechanically treated as described in the article by Lavrič, Medvešček, and
Skočaj [27]. Treatment separated the individual nanofibrils, resulting in the formation
of a homogeneous semi-transparent 0.5% w/w BNC gel. The yield of BNC from the
initial solution (mother of vinegar) was ~87%. The rest were removed impurities (mainly
brownish-colored particles of the apple pulp that had served as the raw material for the
vinegar production).

2.1.3. CNC Production

CNC was prepared in accordance with the procedure described by Kunaver, Anžlovar,
and Žagar in 2016 [28]. The liquefaction reaction, using glycols and mild acid catalysis
Polymers 2021, 13, 2523 4 of 15

(methane sulphonic acid), was applied to eucalyptus wood. The process contains four
steps: the milling, glycolysis reaction, centrifugation, and final rinsing with an organic
solvent. The yield of CNC was 63 ± 8.5% and the final product was a stable, highly
concentrated CNC suspension in water, which was diluted to 1.5% w/w before being used
in film-forming solutions.

2.2. Films Preparation

Films were prepared with different polymer matrices: chitosan, alginate, and nanocel-
lulose. Protocol commonly used in similar studies, e.g., [29], for dissolution, blending,
and casting of alginate, chitosan, and nanocellulose was used. Chitosan and alginate
film-forming solutions were prepared at concentrations 1.5% w/w by dissolving chitosan in
1 wt% aqueous solution of lactic acid and glycerol, and by dissolving sodium alginate in
ultrapure water. Dissolution was realized for approximately 24 h under constant stirring
with a magnetic stirrer (Ika, Staufen, Germany). The mixtures were then vacuum filtered
through 4 layers of medical gauze to eliminate impurities. For chitosan and alginate films
with nanocellulose additives, CNC, CNF, or BNC in amounts of 3 or 5 w/w% with respect
to main biopolymer were added to prepared chitosan and alginate solutions and then
homogenized with UltraTurrax (Ika, Staufen, Germany). To eliminate the air bubbles in the
film, the mixtures were left overnight. For nanocellulose films, corresponding solutions
BNC 0.5% w/w, CNF 1.5% w/w (or diluted to 0.5–1.0% w/w if viscosity was too high) and
CNC 1.5% w/w were used. Glycerol was used as a plasticizer in some types of films and
was added in the amount of 30 wt% with respect to the main biopolymer. The casting
volumes of FFS were chosen in a range of 50–100 mL depending on dry mass of polymers
in different solutions used and respecting the requisite final film casting weight, which was
47 ± 7 g m−2 . All films were casted into 12 × 12 cm2 polyurethane petri dishes and dried
under constant airflow in a laminar flow hood (Microbium d.o.o, Ljubljana, Slovenia) at
room temperature and RH 40% for 48 h. In the case of nanocellulose films, a silicon pad
was placed on the bottom of Petri dishes to prevent sticking.

2.3. Tensile Properties

Tensile properties of films were determined in accordance with ASTM D 882, using
the tensile testing machine Zwick Roell Z010 equipped with 20 N measuring cell (Class 0.5,
ISO 7500-1) and the testing software testXpert (Version II V3.2, Zwick GmbH & Co. KG,
Ulm, Germany). Samples with a width of 15 mm were tested at 10 mm/min testing speed.
The clamping length was set to 70 mm. Testing took place at 23 ◦ C and 50% RH. Samples
were exposed to these conditions 48 h before testing.

2.4. Water Contact Angle

Film was cut into pieces of approximately 2 × 3 cm2 in size and placed onto the micro-
scope glass. Contact angles were measured with water, employing the sessile drop method
with Tensiometer Theta T200 (Biolin Scientific, Darmstadt, Germany). The measurements
were done in triplicates.

2.5. Film Density

Films were cut into pieces of 3 × 3 cm2 in size. The thickness was measured with ABS
Digital Thickness Gauge (Mitutoyo, Japan) on three different parts of the film and then
weighed on an analytical scale. The density was calculated through the Equation (1):
m  
Density = g · cm−3 (1)
d ·S
where m is the mass of the tested sample, d is the thickness of the film in cm, and S is the
area of the sample. All measurements were done in triplicates.
Polymers 2021, 13, 2523 5 of 15

2.6. Scanning Electron Microscope (SEM)

Film surfaces, as well as pure nanocellulose, were investigated under vacuum condi-
tions by SEM SUPRA 35VP (Carl Zeiss, Jena, Germany). A small amount (approx. 50 mg) of
CNC, BNC, and CNF were solvent exchanged to acetone through successive centrifugation
steps and then placed onto a piece of microscope glass over a heating plate. This ensures
quick evaporation of the solvent and prevents aggregation of nanocellulose. Before analy-
sis, the samples were coated with 10 nm of gold. The size of nanocellulose particles was
measured using ImageJ software (Version 1.52, LOCI, University of Wisconsin, Madison,
WI, USA) on at least 10 different points. Films were placed on graphite tape before analysis.
The magnification of all samples was 10,000×.

2.7. Water Vapour Transmission Rate (WVTR) and Water Vapor Permeability
(WVP) Determination
Water vapor transmission rate was determined according to the principles of the
ISO 2528:2018 standard at 23 ◦ C and 50% RH. Since the hot wax could damage the films
during the sample preparation (according to standard procedure), special vessels with a
double-sided seal and a system of screws were used to perform the measurements.

∆m  
WVTR = g · cm−1 · day−1 (2)
A·t

where A is tested area in cm2 , t time after 24 h of testing, and ∆m the mass difference of
tested sample.
Based on WVTR, WVP values were calculated. Calculations were done according to
ASTM E96, described by Equation (3):

WVTR  
WVP = g · m−1 · s−1 Pa−1 (3)
S (R1 − R2 )

where WVTR is calculated through Equation (2), S is the saturation vapour pressure at test
temperature (21.068 mmHg at 23 ◦ C), R1 is the relative humidity in the environment (50%),
and R2 is the relative humidity in the test tube (0%).

2.8. Oxygen Permeability (OTR)

Oxygen permeability of samples was determined in accordance with ISO-2:2003 at
23 ◦Cand 50% RH using Labthink Perme OX2/230 device (Labthink, Boston, MA, USA).

2.9. Moisture Absorption

Moisture absorption was measured modifying the method proposed by Soni et al. [30].
Films were cut in pieces with dimensions 1 × 3 cm and conditioned at 0% RH (relative
humidity) for 24 h. Film samples were then weighted and placed at 85% RH for 24 h. The
relative humidity was created with a saturated solution of potassium chloride at room
temperature. The samples were weighed, and the moisture absorption was calculated
using the Equation (4):

W85 − W0
Moisture absorption (%) = × 100 (4)
W0

where W85 is the weight of the sample after 24 h at 85% RH and W0 is the initial weight
of the sample after conditioning at 0% RH. Four replicate measurements were taken for
each film.

2.10. Statistical Analysis

Statistical analysis was done using the one-way ANOVA with the confidence level of
95% (p < 0.05) in conjunction with Tukey’s honestly significant difference post-hoc test. All
Polymers 2021, 13, 2523 6 of 15

Polymers 2021, 13, 2523 2.10. Statistical Analysis 6 of 15

Statistical analysis was done using the one-way ANOVA with the confidence level of
95% (p < 0.05) in conjunction with Tukey’s honestly significant difference post-hoc test.
All experiments were done in a minimum of five parallels and the results were expressed
experiments were done in a minimum of five parallels and the results were expressed as
as the mean ± standard deviation.
the mean ± standard deviation.
3. Results
3. Results and
and Discussion
Discussion
3.1. Tensile Properties
Tensile Properties
Altogether, 25
Altogether, 25combinations
combinationsofofbiopolymer
biopolymerfilms filmsusing
using chitosan,
chitosan, alginate,
alginate, or or nano-
nanocel-
cellulose
lulose (NC)(NC)as aassingle
a single component
component or inordifferent
in different combinations
combinations werewere prepared;
prepared; how-
however,
ever, selection
selection of samples
of samples for further
for further investigation
investigation was made,
was made, based based on preliminary
on preliminary eval-
evaluation,
which
uation,was based
which was onbased
the appearance of dry films—integrity,
on the appearance surface properties,
of dry films—integrity, visual ap-
surface properties,
pearance of film homogeneity,
visual appearance presence of
of film homogeneity, cracks, of
presence performance during film
cracks, performance handling—the
during film han-
possibility
dling—the of peeling of
possibility ofthe casting
peeling dish,
of the and appropriateness
casting for testing for
dish, and appropriateness (Table S1).(Table
testing Since
tensile
S1). Sinceproperties are one of the
tensile properties are basic
one ofcriteria
the basicfor criteria
packaging materials, selection
for packaging materials, was based
selection
also on thealso
was based results
on theof measured tensile strength
results of measured tensile(TS) and strain
strength (TS) andat break
strain(E) of all films,
at break (E) of
when it was
all films, when technically possible to
it was technically perform
possible to measurements. DifferentDifferent
perform measurements. film compositions
film com-
showed
positionssignificantly different results
showed significantly different(allresults
the results areresults
(all the given as areSupplementary Files), for
given as supplementary
instance, alginate-based films showed TS from 11.7 ± 0.7 to 42.6
files), for instance, alginate-based films showed TS from 11.7 ± 0.7 to 42.6 ± 3.6 MPa ± 3.6 MPa and chitosan
and
films showed TS from 14.0 ± 2.2 to 30.9 ± 2.2 MPa depending
chitosan films showed TS from 14.0 ± 2.2 to 30.9 ± 2.2 MPa depending on the amount on the amount and typeand
of
NC
typeadded
of NC to the main
added to thebiopolymer.
main biopolymer.In the case
In theofcase
chitosan, the addition
of chitosan, of anyofNC
the addition anytype
NC
additive improved
type additive improvedthe mechanical
the mechanicalstrength of the
strength offilm; however,
the film; in the
however, in case of alginate
the case of algi-
films, the impact
nate films, depended
the impact on theon
depended type
the and
typeamount of NCofand
and amount NCwasandnegative in most
was negative in cases
most
when
cases when CNF or BNC was added. As a result of preliminary evaluation, seven were
CNF or BNC was added. As a result of preliminary evaluation, seven films films
selected for further
were selected investigation:
for further alginate,
investigation: alginate
alginate, +5% CNC,
alginate +5% CNC,chitosan, chitosan
chitosan, +3%
chitosan
CNC, BNC with and without glycerol, and CNF with glycerol.
+3% CNC, BNC with and without glycerol, and CNF with glycerol. Their properties areTheir properties are Table 1
and Figure 1.
Scheme 1 and Figure 1.

Figure 1.
Figure 1. Tensile
Tensilestrength
strengthwith
withthe
therespective
respective strain
strain forfor nanocellulose,
nanocellulose, alginate,
alginate, andand chitosan
chitosan nano-
nanocom-
composites. The composition of the films 1–7 are described in
posites. The composition of the films 1–7 are described in Table 1.Table 1.
Polymers 2021, 13, 2523 7 of 15

Table 1. Selected polysaccharide films are based on the specified properties and include the most promising candidate from
each biopolymer. These films are also subjected to a full characterization of morphological and barrier properties (values are
given as mean ± SD).

Thickness Tensile Strength Strain at Break Water Contact

Sample Name Film Composition
(µm) (MPa) (%) Angle (◦ )
Film 1 Alginate 55 ± 3 40 ± 10 22 ± 6 39 ± 2
Film 2 Alginate + 5% CNC 50 ± 2 43 ± 4 28 ± 4 58 ± 3
Film 3 Chitosan 123 ± 3 14 ± 2 51 ± 4 75 ± 3
Film 4 Chitosan + 3% CNC 70 ± 1 31 ± 2 55 ± 6 108 ± 2
Film 5 BNC 66.4 ± 0.8 60 ± 11 4.2 ± 1 46 ± 3
Film 6 BNC + 30% glycerol 56 ± 2 53 ± 5 10 ± 0.4 65 ± 4
Film 7 CNF + 30% glycerol 31 ± 3 47 ± 3 2.4 ± 0.6 23 ± 1

As it was said above, tensile properties are one of the basic criteria for packaging
materials. Namely, the mechanical behaviour of packaging films is a very important
property of the film to maintain its authenticity and to withstand the environmental impact
during the packaging application. The TS and E at break were determined for all film
samples. The TS determines the maximum load that can be sustained per cross-sectional
area of the film. Strain at break shows the extension of the film, e.g., the flexibility that
can be stretched before the breaking point. These characteristics support the correlation
of the mechanical properties of films with their compositions and chemical structures.
Samples with chitosan and alginate, with the addition of CNC, showed an increase in both
TS and E. The average tensile strength of pure CNC films is about 63 MPa, as reported
in the literature [31,32]. As expected, the mechanical properties were influenced by the
addition of CNC. Films based on alginate exhibited TS of 40 MPa, which increased by 12%
upon the addition of 5% CNC. According to the measurement results, the alginate films
of all samples showed the best TS/E ratio. Huq et al. [33] reported that the high TS of the
alginate-based bio nanocomposite films is due to a good interfacial interaction between
the nanofillers and the alginate-based matrix due to similar polysaccharide structures of
cellulose and alginate, which was also confirmed on our samples [33]. The largest increase
was found in chitosan film, where the addition of CNC improved strength by 120%. The
same increase in E was found in both samples (alginate for 30% and chitosan for 6%). It
is known from the literature that CNC has a large length/diameter ratio and very good
tensile properties. Our analysis confirmed that there are interactions between CNC and
chitosan molecules, such as electrostatic association and hydrogen bonding, which create an
interactive network and improve overall tensile properties [34]. Cellulose based films have
low flexibility, plasticizers should be added to improve this mechanical property and to
facilitate the handling of these biopolymers’ films. The most used plasticizer is glycerol due
to its stability and compatibility with hydrophilic biopolymer chains [35]. The results have
shown that bacterial nanocellulose films (BNC) have the highest TS (60.1 MPa) but a lower
E (4.2%). The addition of glycerol changed the properties of the film made of BNC. Namely,
the plasticization of BNC with glycerol, which reduced the strength of the hydrogen bonds
between adjacent cellulose chains, changed the TS of the film. The TS decreased by 11.6%.
At the same time the addition of glycerol increased the E values by about 145% (from 4.2
to 10.3%). According to the results obtained, it is predicted that the moisture absorbed
into the matrix of the film had a plasticizing effect. As a result, the TS decreased and E
increased by weakening the intermolecular forces, thus increasing the space between the
polymers and reducing the crystallinity [36,37]. Overall, by improving the strain of rather
rigid films, the glycerol improved their suitability for packaging materials.
CNF were produced from hardwood Kraft pulp by a mechanical process with previous
chemical oxidation with APS, as described previously. The results showed that the fibrils
were shorter and thinner compared to CNF produced by the TEMPO process [37], which
is caused by the fibre cleaving effect of persulfate. Similar reinforcement properties for
APS and TEMPO oxidized CNF have been proven [36]. However, in our research, CNF
Polymers 2021, 13, 2523 8 of 15

Polymers 2021, 13, 2523 8 of 15

which is caused by the fibre cleaving effect of persulfate. Similar reinforcement properties
for APS and TEMPO oxidized CNF have been proven [36]. However, in our research, CNF
apparently
apparentlyappears
appearsless
lesscross-linked,
cross-linked,resulting
resultingin inaasmaller
smallersurface
surfacearea
areaand
andpore
porevolume.
volume.
With the glycerol, the tensile properties decreased. As explained
With the glycerol, the tensile properties decreased. As explained in many studies, in many studies, the ad-
the
dition of plasticizer in biopolymers reduces crystallinity, which leads
addition of plasticizer in biopolymers reduces crystallinity, which leads to a significantto a significant de-
crease in film
decrease strength
in film strengthand and
modulus
modulus[38–40]. In ourIn
[38–40]. case,
ourthe addition
case, of glycerol
the addition also re-
of glycerol
duced the flexibility
also reduced of the films,
the flexibility of thewhich
films,is which
in contrast
is intocontrast
previous toinvestigations. The com-
previous investigations.
bination of decreasing
The combination tensile strength
of decreasing tensile and strainand
strength at break
strainisatsurprising, and the explana-
break is surprising, and the
tion
explanation could be the reduced density. The addition of glycerol lowered theof
could be the reduced density. The addition of glycerol lowered the density the film
density of
(1.29 g∙cm −3). In the −
the film (1.29 g·cm CNF films
3 ). In described
the CNF films in the literature,
described in the the film density
literature, wasdensity
the film about 1.52
was
gabout 1.52 g·cm−3 [37].
cm−3 [37].

3.2.Water
3.2. WaterContact
ContactAngle
Angle
The information
The information about about interactions
interactionsbetween
betweenfilms films and
and water
water is is very
very important
important for for
packaging. Hydrophobic or
packaging. orhydrophilic
hydrophiliccharacter
characteris is frequently
frequentlydetermined
determined by surface free
by surface
energy
free and and
energy surface morphology.
surface morphology.The contact angleangle
The contact of theofsurface with water
the surface is important
with water is im-
to characterize
portant a material
to characterize as such and
a material can and
as such give can
an impression of absorption
give an impression and adhesion
of absorption and
as well. A lower contact angle with water is an indicator that films
adhesion as well. A lower contact angle with water is an indicator that films are hydro- are hydrophilic and
hygroscopic. The most wettable surfaces have low values ( < 20 ◦ ) and the hydrophobic
philic and hygroscopic. The most wettable surfaces have low values (˂20°) and the hydro-
surfaces
phobic have high
surfaces havevalues
high of contact
values angle (>
of contact 90◦ ) [38].
angle (˃90°)In[38].
Table In1Table
and Figure
1 and 1, contact
Figure 1,
angles of polysaccharide films with water are recorded. Comparing
contact angles of polysaccharide films with water are recorded. Comparing films consist- films consisting of
onlyofone ◦
ing onlybiopolymer, chitosan
one biopolymer, exhibited
chitosan the highest
exhibited contact
the highest angleangle
contact (75 ) (75°)
and CNF with
and CNF
the addition of 30% glycerol the lowest (23 ◦ ) (Figure 2). Alginate and nanocellulose films
with the addition of 30% glycerol the lowest (23°) (Figure 2). Alginate and nanocellulose
can be
films considered
can as hydrophilic.
be considered When
as hydrophilic. combining
When combining chitosan andand
chitosan alginate withwith
alginate CNC,CNC,the
contact angle with water increased by 44% and 49%, respectively.
the contact angle with water increased by 44% and 49%, respectively. This trend was also This trend was also
confirmed by
confirmed by Mao
Mao etet al.
al. [34],
[34], where
where the
the hydrophobicity
hydrophobicity of of chitosan/CNC
chitosan/CNC film film increased
increased
compared to only chitosan film. Although cellulose consists of β-D-glucopyranose
compared to only chitosan film. Although cellulose consists of β-D-glucopyranose units units
with three hydroxyl groups, which are responsible for the hydrophilic
with three hydroxyl groups, which are responsible for the hydrophilic character of cellu- character of cellulose,
electrostatic association and hydrogen bonding bids the CNC and chitosan molecules
lose, electrostatic association and hydrogen bonding bids the CNC and chitosan molecules
closely together, which improves the hydrophobicity [34,39–41]. The higher contact angle
closely together, which improves the hydrophobicity [34,39–41]. The higher contact angle
can also be a result of changed morphology, which interrupts water spreading. CNF + 30%
can also be a result of changed morphology, which interrupts water spreading. CNF +
glycerol films are the most hydrophilic. This behaviour is an indication of the high affinity
30% glycerol films are the most hydrophilic. This behaviour is an indication of the high
of glycerol for water. Glycerol in cellulose films tends to migrate to the surface, as also
affinity of glycerol for water. Glycerol in cellulose films tends to migrate to the surface, as
confirmed by Spoljaric et al. [42]. However, this was not the case for BNC films, where
also confirmed by Spoljaric et al. [42]. However, this was not the case for BNC films, where
the addition of glycerol led to an increase (by 141%) in the contact angle. It is possible
the addition of glycerol led to an increase (by 141%) in the contact angle. It is possible that
that glycerol filled the pores between the fibres, reducing porosity, and thus decreased the
glycerol filled the pores between the fibres, reducing porosity, and thus decreased the
surface free energy. In packaging, more hydrophobic materials are generally desired as
surface free energy. In packaging, more hydrophobic materials are generally desired as
they offer a wider range of applications [40].
they offer a wider range of applications [40].

Figure
Figure 2.
2. Visualization
Visualization of
of the
the water
water contact
contact angle
angle for
for the
the selected
selected films
films are
are shown
shown in
in Table
Table 11 (ALG—alginate;
(ALG—alginate; Alg
Alg ++ 5%
5%
CNC—alginate + 5% CNC; CH—chitosan; CH + 3% CNC—chitosan + 3% CNC; BNC—BNC; BNC + 30% GLYC—BNC +
CNC—alginate + 5% CNC; CH—chitosan; CH + 3% CNC—chitosan + 3% CNC; BNC—BNC; BNC + 30% GLYC—BNC +
30% glycerol; CNF + 30% GLYC—CNF + 30% glycerol).
30% glycerol; CNF + 30% GLYC—CNF + 30% glycerol).
Polymers 2021, 13, 2523 9 of 15

3.3. Barrier Properties of the Films

The gas phase permeation through a non-porous material occurs by adsorption at the
front interface, diffusion through the material, and desorption at the rear interface, and is
often measured with transfer rate, permeance, and permeability. The transmission rate is
the volume or weight of a permeating agent (e.g., oxygen or moisture) passing through a
film per unit surface area and time in equilibrium with the test conditions.
The addition of CNC to alginate and chitosan reduced WVTR by 15% and 45%,
respectively. OTR decreased by 45% for alginate and CNC and by 38% for chitosan and
CNC, compared to pure film. It is obvious that the nanostructure of nanocomposites
created a tortuous path for oxygen, which was also demonstrated by Enescu et al. [43].
As shown in the tensile properties of BNC with added glycerol, the water absorbed
into the matrix of the films had a plasticizing effect, reducing tensile strength and increasing
strain at break. In this area, adsorbed water molecules promoted the reorganization of the
polymer chains, which was reflected in the change in water permeability in this area. The
water barrier properties decreased enormously by 198%. The same trend was observed for
oxygen permeability, which decreased for 77%.
For CNF film with added glycerol, it was impossible to measure the WVTR because
the sample was too fragile and, therefore, this test was not performed. On the other hand,
the OTR results of the sample showed that the oxygen permeability increased enormously,
compared to pure CNF from the previous research [44]. This could be the reason for the
microcracks that were present on the sample because the fibrils were very short, the film
was fragile, and the oxygen could easily pass through.
In Figure 3, the results of WVTR and OTR of the films fabricated in this study as well as
for other commercially polymer blends for comparison are presented. Pure polysaccharide
films had higher WVTR compared to most commercial packaging films, except TOCN
(TEMPO-oxidized cellulose nanofibers). When CNC was combined with alginate and
chitosan, the OTR results showed higher OTR compared to cellophane but still lower OTR
compared to bio-based polylactic acid films. Pure BNC and BNC with added glycerol
showed similar oxygen and water vapor permeability to PLA and TOCN.
 In Figure 3, the results of WVTR and OTR of the films fabricated in this study as well
as for other commercially polymer blends for comparison are presented. Pure polysaccha-
ride films had higher WVTR compared to most commercial packaging films, except
TOCN (TEMPO-oxidized cellulose nanofibers). When CNC was combined with alginate
Polymers 2021, 13, 2523 and chitosan, the OTR results showed higher OTR compared to cellophane but still10lower
of 15
OTR compared to bio-based polylactic acid films. Pure BNC and BNC with added glycerol
showed similar oxygen and water vapor permeability to PLA and TOCN.

Figure3.3.WVTR
Figure WVTRandandOxygen
Oxygentransmission
transmissionrate
rate(OTR)
(OTR)ofofsome
somesynthetic
syntheticpolymers
polymerscompared
comparedtotoour
our nanocomposites packaging films. Adapted from [45] (Black circles—Petroleum-based
nanocomposites packaging films. Adapted from [45] (Black circles—Petroleum-based Polymers, Poly-
mers, Black diamonds—commercial Biopolymers, red diamond—biopolymer nanocomposites
Black diamonds—commercial Biopolymers, red diamond—biopolymer nanocomposites tested in this
study) (HDPE—high-density polyethylene; LDPE—low-density polyethylene; PP—polypropylene;
PVC—polyvinyl chloride; PET—polyethylene terephthalate; PLA—polylactic acid; TOCN—TEMPO-
oxidized cellulose nanofibers).

The addition of CNC reduced the WVP value of alginate films from 9.36 g/(m·s·Pa)
to 7.32 g/(m·s·Pa) (Table 2). An even greater decrease in this value was observed with
3% CNC addition into the films made of chitosan. In this case, the value dropped from
10.1 g/(m·s·Pa) to 1.39 g/(m·s·Pa). The addition of glycerol drastically increased the WVP
value of BNC films (from 3.62 g/(m·s·Pa) to 9.17 g/(m·s·Pa).

Table 2. Density, moisture absorption, water vapor transmission rate (WVTR), properties of films (values are presented as
mean ± SD). Water vapor permeability results (WVP) are presented as a calculated mean value.

Film Density Moisture WVTR WVP

Sample Name Film Composition
(g·cm−3 ) Absorption (%) (g·cm−1 ·day−1 ) (g/(m·s·Pa))
Film 1 Alginate 1.87 ± 0.2 57.4 ± 1.6 239 ± 8 9.36
Film 2 Alginate + 5% CNC 1.34 ± 0.16 51.2 ± 1.9 203 ± 5 7.23
Film 3 Chitosan 2.00 ± 0.35 49.2 ± 1.5 115 ± 9 10.07
Film 4 Chitosan + 3% CNC 1.05 ± 0.10 36.5 ± 1.8 63 ± 2 1.39
Film 5 BNC 0.74 ± 0.05 9.7 ± 0.8 77 ± 4 3.62
Film 6 BNC + 30% glycerol 0.79 ± 0.10 21.3 ± 1.0 230 ± 11 9.17
Film 7 CNF + 30% glycerol 1.29 ± 0.20 20.4 ± 0.6 Not applicable Not applicable

3.4. Visualization and Morphology of the Films

CNF films appeared as fully transparent materials with a glossy surface, CNC films
were slightly whitish and pale semi-transparent material, while BNC films were slightly
brownish semi-transparent material (Figure 4a). CNC additive in amounts of 3–5% w/w did
not change the transparency or colour of chitosan or alginate films. CNC and CNF have
been known for their application in optically transparent films [46]; however, CNC can be
less transparent and haze depending on the size of crystals and thickness of film [47]. The
Polymers 2021, 13, 2523 11 of 15

brownish colour of BNC films can be explained by the influence of fermentation medium,
Polymers 2021, 13, 2523 which was apple juice and culturing bacteria used for obtaining the initial material, since
11 of 15
the growing conditions have a significant effect on the properties of bacterial cellulose [48].

Figure4.4.(a)
Figure (a)Visualisation
VisualisationofofBNC,
BNC,CNF,
CNF, and
and CNC
CNC films.
films. SEMSEM micrographs
micrographs of of
(b)(b) BNC,
BNC, (c)(c)
CNFCNF
film
film with 30 wt% glycerol, (d) CNC film with 30 wt% glycerol, (e) chitosan film with 30 wt% glyc-
with 30 wt% glycerol, (d) CNC film with 30 wt% glycerol, (e) chitosan film with 30 wt% glycerol,
erol, (g) alginate + 5% CNC, (f) chitosan + 3% CNC, and (h) alginate film with 30 wt% glycerol.
(g) alginate + 5% CNC, (f) chitosan + 3% CNC, and (h) alginate film with 30 wt% glycerol.
3.5. Inspection
Film Density
ofand
the Moisture Adsorption
films with SEM revealed that BNC and CNF were in the shape of a
Alginate network
long, fibrillary and chitosan films
(Figure have
4b,c) the
with anhighest
averagedensity,
diameterand
of it decreases
fibril of 69 ±with
24.3 the
nm addi-
and
tion
26 of nanocellulose,
± 6.5 which
nm, respectively. Oncorresponds to observations
the other hand, on the films’
CNC were rod-like morphology
shaped (Fig-
particles with
ure
an 4d,e). The
average mixing
width ± CNC
of 83of intoand
18.8 nm thelength
alginate
of or
777 ± 112 nm
chitosan matrix creates
(Figure 4d).porosity,
Chitosanwhich
and
is responsible
alginate for the
films have lower density.
a smooth Wang et.al.
surface before being[44] described
mixed that CNC has(Figure
with nanocellulose a “rice-like”
4e,g,
respectively).
structure, whichIn alginate-CNC composite,
causes the changes in the clusters of CNCs
microstructure ofare
thevisible,
films andindicating that
consequently
in the density.
Similarly, BNC and CNF have lower density compared to films with chitosan and
alginate matrix but can be slightly increased with the addition of glycerol (Table 2).
Polymers 2021, 13, 2523 12 of 15

although both alginate and CNCs are hydrophilic, CNC tends to agglomerate when mixed
into alginate matrix (Figure 4g), while in chitosan film, the CNCs are more homogeneously
dispersed (Figure 4f).

3.5. Film Density and Moisture Adsorption

Alginate and chitosan films have the highest density, and it decreases with the addition
of nanocellulose, which corresponds to observations on the films’ morphology (Figure 4d,e).
The mixing of CNC into the alginate or chitosan matrix creates porosity, which is responsible
for the lower density. Wang et al. [44] described that CNC has a “rice-like” structure, which
causes the changes in the microstructure of the films and consequently in the density.
Similarly, BNC and CNF have lower density compared to films with chitosan and
alginate matrix but can be slightly increased with the addition of glycerol (Table 2).
As it was said earlier in section about contact angle measurements, interactions be-
tween films and water are significant for packaging and hydrophobicity or hydrophilicity
properties of films can determine their application areas. Moisture absorption from air
medium was tested and all biopolymer films, regardless of composition, demonstrated
hygroscopic behaviour at high (85%) relative humidity, therefore showing their ability to
absorb water vapour from ambient air. Such a high RH number was chosen in order to
investigate the variation of absorption between films of different compositions. Although
CNC film was not selected for detailed investigation because of cracks and breakage, the
moisture absorption was measured and the comparison of nanocellulose films demon-
strated moisture absorption of 13.3 ± 0.2% in the case of CNC and slightly higher in the
cases of CNF (20.4 ± 0.6%) and BNC (21.3 ± 1.0%). Fibrillated forms of nanocellulose tend
to absorb more water due to the fibrillar structure and bigger proportion of amorphous
regions, where it is easier for water molecule to get into and to bond with hydroxyl groups
of cellulose. After 24h in high humidity, nanocellulose films became more flexible on
touch, especially BNC one; however, they retain their shape and part of their stiffness.
Cellulosic fibres, being hydrophilic in nature, absorb moisture from their environment until
equilibrium is reached [49]. Adding plasticizer to BNC increased moisture absorption by
120%, improving the highly hygroscopic behaviour of glycerol.
Chitosan and alginate films absorb more moisture than pure nanocellulose films. It
is 49.2 ± 1.5% in the case of pure chitosan and 57.4 ± 1.6% in the case of pure alginate. It
is worth noting that moisture changes the structure of chitosan and alginate films—they
become sticky, loose, lose their shape and stiffness, and stick to the surfaces. However, the
addition of 3–5% CNC decreased the moisture absorption of films. Moisture absorption
values decreased to different extents depending on the amount of nanofillers. The addition
of 3% CNC decreased the moisture absorption of chitosan films by 15.1%, however, in the
case of alginate, the addition of 5% nanocellulose decreased the moisture absorption by
10.8%. Therefore, it can be concluded that nanocellulose additives help to preserve the
structure of chitosan and alginate films in a humid environment and should be considered.
Ability of NC to prevent the absorption of moisture of NC-reinforced chitosan films were
investigated elsewhere [30].

4. Conclusions
In this research, 25 different formulations of 5 sustainable biopolymers were used to
produce and characterize thin and flexible films with potential use for packaging purposes.
Results of mechanical testing showed that the addition of 3–5% CNC, CNF, and BNC
improved the tensile strength of chitosan films, however, for alginate films, the impact of
NC depended on the type and amount of additive and was positive only when CNC was
added. From the cellulose-based films, BNC had the highest tensile strength, 60 ± 11 MPa
and 53 ± 5 MPa without and with glycerol accordingly, CNF (with glycerol) followed with
a result of 47 ± 3 MPa. CNC films appeared as slightly whitish and pale semi-transparent
material, while BNC films were slightly brownish semi-transparent material, and other
formulations were fully transparent. Seven formulations—alginate, alginate +5% CNC,
Polymers 2021, 13, 2523 13 of 15

chitosan, chitosan +3% CNC, BNC with and without glycerol, and CNF with glycerol—
were selected as the most appropriate for packaging purposes based on visual/physical
appearance and mechanical properties of films, and characterized in terms of morphological
examination with SEM, density, contact angle, surface energy, water absorption, and oxygen
and water barrier properties. SEM examination of cellulose-based films revealed typical
morphology of crystalline and fibrillar forms of NC. Investigation of mixed formulations
revealed more homogenous dispersing of CNC in chitosan than in alginate. Alginate
and chitosan films had the highest density, which decreased with the addition of CNC
because of greater porosity. Water contact angle differed among selected samples, the
lowest was detected for CNF with glycerol (23 ± 1) and the highest for chitosan with 3%
CNC (108 ± 2), the other films having results in the range from 39◦ to 75◦ and showing the
increase of hydrophobicity of chitosan and alginate with the addition of CNC. This fact was
approved also by moisture absorption results, which showed reduced moisture absorption
for chitosan and alginate films after the addition of CNC for 15.1% and 8.8%, accordingly.
Overall, chitosan and alginate films absorb more moisture than pure nanocellulose films,
however, the addition of CNC can help to preserve the structure and function of chitosan
and alginate packaging materials in humid environments. Therefore, materials with higher
hydrophobicity would be more appropriate as they offer a wider range of applications.
Results of barrier properties showed that the addition of CNC improved the WVTR of
alginate by 15% and of chitosan films by 45%, while OTR decreased by 45% for alginate
with CNC and by 38% for chitosan with CNC, compared to one component films. The
addition of glycerol to BNC films decreased WVTR almost twice and OTR for 77%.
Based on the findings of this study, it was concluded that polysaccharide-based films
with added CNC are the most suitable for packaging purposes. With good oxygen barrier,
water barrier that is comparable to PLA, and good mechanical properties, we propose
that such films would be a good alternative to conventional plastic packaging used for
ready-to-eat foods with short storage time, such as sandwiches and solid, refrigerated
vegetables (for instance cucumbers, cauliflower, broccoli).

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10

.3390/polym13152523/s1, Table S1: Overview of all the prepared biopolymers films and nanocom-
posites together with evaluated mechanical properties and moisture absorption.
Author Contributions: Conceptualization, G.L., U.N. and U.V.-B.; methodology, G.L., U.N. and A.O.;
validation, G.L. and U.V.-B.; formal analysis, I.F.; investigation, G.L., A.O. and I.F.; data curation, G.L.,
A.O., U.N. and U.V.-B.; writing—original draft preparation, G.L., U.N., A.O., I.F., B.L. and U.V.-B.;
writing—review and editing, G.L., U.N., A.O., I.F., B.L. and U.V.-B.; supervision, U.V.-B.; funding
acquisition, B.L. All authors have read and agreed to the published version of the manuscript.
Funding: Author I.F. acknowledge the European Regional Development Fund, Contract No. 1.1.1.2/
VIAA/1/16/211 (Agreement No. 1.1.1.2/16/I/001) “Study of novel method for nanocellulose
isolation from biomass and its residues”. This research was also funded by the PhD research grant
(A.O.) and Slovenian Research Agency (Program P2-0152 and Program P2-0213 Textiles and Ecology).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article and Supplementary Material.
Acknowledgments: The authors acknowledge Anže Prašnikar for preparation of SEM images and
Matej Skočaj for critical reading of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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