Carbohydrate Polymers 237 (2020) 116167

Contents lists available at ScienceDirect

Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Characterization of bacterial cellulose composite films incorporated with T

bulk chitosan and chitosan nanoparticles: A comparative study
Siyi Jua, Fenglun Zhangb, Jiufang Duana, Jianxin Jianga,*
a
Department of Chemistry and Chemical Engineering, MOE Engineering Research Center of Forestry Biomass Materials and Bioenergy, Beijing Forestry University, Beijing,
100083, China
b
Nanjing Institute for the Comprehensive Utilization of Wild Plant, Nanjing, 211111, China

A R T I C LE I N FO A B S T R A C T

Keywords: Ternary composite films containing bulk chitosan (CS) and chitosan nanoparticles (CSNPs) with different con-
Chitosan centrations were prepared using bacterial cellulose/poly(vinyl alcohol) as the base film and the composites films
Nanoparticles were compared. The micromorphology and mechanical, physical, chemical, antibacterial, and optical barrier
Antibacterial properties properties of the composite films were compared. CS incorporation improved the mechanical properties, as the
Bacterial cellulose
maximum tensile strength was increased to 130.55 ± 9.42 MPa. The dense structure of CSNPs prevented water
Poly(vinyl alcohol)
diffusion and lessened the water content of the composite membranes. The inclusion of CS and CSNPs both
reduced the water solubility and water vapor permeability. CS-doped films possessed good transparency, while
CSNPs had better ultraviolet-barrier properties (3.84 % of transmittance at 200–280 nm). In addition, CSNPs-
embedded membranes exhibited prominent antibacterial properties against Escherichia coli and Staphylococcus
aureus, which were much greater than those of CS composite membranes with a maximum bacteriostatic dia-
meter of 10.33 ± 1.55 mm.

1. Introduction formed from repeated dimers of D-glucose units cross-linked by β-1,4-

bonds, and each displays individual properties. BC has excellent prop-
Biodegradable polymer materials are receiving increasing attention, erties, such as a high elastic modulus, crystallinity, porosity, transpar-
and it is hoped that they can replace petrochemical-based plastics ency, biocompatibility, and high degree of polymerization (Gatenholm
partially or completely. Most research on biodegradable polymers used & Klemm, 2010; Putra, Kakugo, Furukawa, Gong, & Osada, 2008),
in food packaging has concentrated on renewable raw materials, such which is attributed to its ultrafine network and nanofiber network
as proteins and polysaccharides, which possess good film-forming structure. Therefore, BC has been applied in many fields and uses, in-
properties or contain lipid components related to film-forming agents cluding the paper and textile industries, car tires, high-quality speaker
(Bourtoom, 2008). Among the new environmentally friendly materials, diaphragms, separators, and electronic paper (Du, Zhao, Peng, Zhou, &
single biopolymers including starch, chitosan, cellulose, and their de- Han, 2018; Rani, Rastogi, & Appaiah, 2011; Torres, Commeaux, &
rivatives exhibit far lower mechanical and barrier properties than Troncoso, 2012; Zhong et al., 2013). However, BC lacks some desirable
synthetic polymers (Cazón, Vázquez, & Velazquez, 2018). As a result, characteristics including antibacterial performance, optical transpar-
their applications are limited. However, by mixing biodegradable ency and ability to withstand stress (Esa, Tasirin, & Rahman, 2014),
polymers with different structures and properties, the interactions be- which limits its potential for packing food.
tween polymers can be used to develop films with synergistically en- Poly(vinyl alcohol) (PVA) is a semi-crystalline synthetic polymer,
hanced properties (Muppalla, Kanatt, Chawla, & Sharma, 2014). which possesses remarkable film-forming properties, transparency, and
BC is a product secreted by microorganisms, particularly elasticity (Cazón, Velázquez, & Vázquez, 2019). PVA is used in various
Gluconacetobacter xylinus in the genus Acetobacter (Naghdi et al., 2019). industrial applications to enhance the mechanical properties of packa-
The chemical structure of BC is the same as that produced by plants ging films due to its compatible structure and hydrophilicity (Limpan,

Abbreviations: BC, bacterial cellulose; PVA, poly(vinyl alcohol); UV, ultraviolet; CS, bulk chitosan; CSNPs, chitosan nanoparticle; XRD, X-ray diffraction; SEM,
scanning electron microscopy; TS, tensile strength; E%, elongation percentage; WS, water solubility; WVP, water vapor permeability; E. coli, Escherichia coli; S. aureus,
Staphylococcus aureus
⁎
Corresponding author.
E-mail addresses: jusiyi0328@163.com (S. Ju), fenghuolun2000@163.com (F. Zhang), duanjiu99@163.com (J. Duan), jiangjx2004@hotmail.com (J. Jiang).

https://doi.org/10.1016/j.carbpol.2020.116167
Received 22 January 2020; Received in revised form 9 March 2020; Accepted 11 March 2020
Available online 12 March 2020
0144-8617/ © 2020 Elsevier Ltd. All rights reserved.
S. Ju, et al. Carbohydrate Polymers 237 (2020) 116167

Prodpran, Benjakul, & Prasarpran, 2012). Consequently, the combina- Firstly, chitosan was dissolved in 0.3 % v/v acetic acid to prepare
tion of PVA with other biopolymers, such as cellulose, gelatin, chitosan, 200 mL chitosan solution with the concentration of 0.5 % w/v. 0.8 g
and starch, has been studied extensively. TPP was dissolved in 107 mL deionized water. Then the solution was
Chitosan is the second most abundant polysaccharide resource in added dropwise into the above chitosan solution under magnetic stir-
nature, which usually derives from industrial shellfish waste. Chitosan ring to react until the emulsion was obtained. Finally, the solution was
has excellent properties, including film-forming ability, antibacterial centrifuged. The precipitate was washed with distilled water and
properties, and easy availability, among which antibacterial activity is ethanol, then dried in vacuum and analyzed.
the most attractive. Bacterial growth on food surfaces was inhibited
during contact with chitosan applied to packaging and coating mate- 2.3. Preparation of composite films
rials (Moreira, Roura, & Ponce, 2011; Tripathi, Mehrotra, & Dutta,
2010). PVA was dissolved in water at 90 °C for 4 h under magnetic stirring
In addition, chitosan possesses protonated amino groups and can be to obtain a PVA solution (2% w/v) which was subsequently mixed with
used to generate intermolecular and intramolecular ionic crosslinking BC suspension under stirring to give a dry weight ratio of PVA to BC of
by polyvalent polyanions; hence, chitosan nanoparticles (CSNPs) can be 2:5. The blend solution was then cast into polytetrafluoroethylene petri
prepared based on particle gelation. Sodium tripolyphosphate (TPP) is dishes (20 g/solution per dish) with a diameter of 9 cm and dried at
the most commonly studied polyanion due to its nontoxicity and ability 37 °C for 2 d. The obtained binary composite film was used as a base
to perform rapid gelling (de Moura et al., 2009). CSNPs have been film. CS and CSNPs solutions of disparate concentrations of 0.5 %, 1.0
widely developed as delivery systems of bioactive compounds due to %, 2.5 % w/v were prepared, followed by being casting into the petri
their biodegradability, biosafety, and biocompatibility applying in the dishes containing the above base film, and dried at 37 °C for 24 h.
field of biomedicine (Wang et al., 2015, 2016; Xie et al., 2017). CSNPs Finally, the films was equilibrated in a saturated K2CO3 regulated dryer
have been successfully used as fillers to develop films with reinforced (approximately 50 % RH) prior to the analyses.
properties like water vapor barrier properties and antibacterial prop-
erties (Chang, Jian, Yu, & Ma, 2010; Romainor, Chin, Pang, & Bilung, 2.4. Scanning electron microscopy (SEM) and X-ray diffraction (XRD)
2014). However, there are almost no related studies about in-
corporating CSNPs into cellulose or PVA films. In this paper, CSNPs SEM (Merlin, Carl Zeiss AG, Germany) and XRD analysis (Smartlab,
were characterized for the development of food packaging films. Rigaku, Japan) were performed to study the composite films in-
In our previous research, BC-PVA-chitosan films prepared by the corporated with CS and CSNPs.
immersion method were characterized. The obtained results showed
that there were strong interactions among the three polymers and film 2.5. Film thickness measurement
properties were improved by the combination of the polymers (Ju, Yu,
Ji, Wang, & Jiang, 2020). In this work, a BC/PVA composite film was The thickness of the film was measured with a spiral micrometer
fabricated by blending BC and PVA to improve the mechanical prop- (Nanjing, China), determined by the average of the thickness of 10
erties and visual appearance characteristics. Subsequently, CS and different points on the film.
CSNPs were incorporated into the BC/PVA film, which served as a base
film to obtain ternary composite films. 2.6. Mechanical properties
The purpose of this study was to develop novel biodegradable films
for food packaging and to compare the effects of CS and CSNPs on the The mechanical properties of the films were measured at room
micromorphology, mechanical, physical, chemical, and optical prop- temperature in accordance with ASTM D882. A universal material
erties of the composite films. Furthermore, the effect of the two chit- testing machine (Xforce HP, Zwick/Roell, Germany) was employed for
osan types on the antibacterial activity of the composite membrane tensile testing with the initial distance between the two clamps and the
against Escherichia coli (gram-negative) and Staphylococcus aureus cross head rate set as 10 mm and 5 mm/min, respectively.
(gram-positive) was also tested in vitro.
2.7. Moisture content at equilibrium
1.1. Hypotheses
A certain weight (W0, g) of the equilibriumed initial sample was
The composite films developed in this work can be used in food weighed, dried at 105 °C for 24 h, and then placed in a silica gel dryer.
packaging applications because they would have excellent properties After cooling to room temperature, the weight (W1, g) of the dried film
including remarkable tensile strength and antibacterial properties. was obtained. The moisture content at equilibrium was calculated with
the following Eq. (1):
2. Materials and methods
W0 − W1
M%= ∙ 100%
W0 (1)
2.1. Materials

Bacterial cellulose suspension, a solution of 0.5 wt%, was obtained 2.8. Water solubility (WS) and water vapor permeability (WVP)
from Qihong Technology Co., Ltd (Guilin, China). Poly(vinyl alcohol)
was provided by Aladdin Biochemical Technology Co., Ltd (Shanghai, WS was defined as the percentage of dissolved substance after a film
China) with alcoholysis degree of 98–99 %. Chitosan was obtained from was completely soaked in distilled water for 24 h. The Eq. (2) was used
the Sinopharm Chemical Reagent Co., Ltd with deacetylation degree of to calculate the WS value:
80.0–95.0 % and sodium tripolyphosphate was bought from Macklin
W0 − W1
Biochemical Co., Ltd (Shanghai, China). All the other reagents were of WS%= ∙ 100%
W0 (2)
analytical grade including acetic acid and ethyl alcohol.
Where W0 (g) is the original weight of the film, and W1 (g) is the weight
2.2. Preparation of chitosan nanoparticles (CSNPs) obtained by constant weight of the undissolved substance after the film
was soaked for 24 h.
CSNPs were prepared by ionic gelation of chitosan and TPP in the The WVP of the membrane was quantitatively analyzed by the
modified way reported by Vijayalakshmi, Gomathi, and Sudha (2014). methods in the reference (Zhao et al., 2019).

2
S. Ju, et al. Carbohydrate Polymers 237 (2020) 116167

2.9. Antimicrobial tests can form stronger intermolecular and intramolecular hydrogen bonds
(Ramya, Sudha, & Mahalakshmi, 2012). By comparing the XRD results
The antimicrobial properties of the composite films were assessed of CSNPs and pure CS, a significant change in the diffraction pattern
by the amended method of agar plate diffusion (Kanmani & Rhim, was observed, and a broad amorphous peak near 2θ = 21.38° was ob-
2014). Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) tained. This result may be due to the increased accumulation of chit-
were used for biological testing in this work. The composite films were osan chains by ionic cross-linking, which deforms the crystal region.
punched into small discs with 8 mm diameter, and sterilized by UV The morphological images obtained via scanning electron micro-
irradiation before the antibacterial experiment. The antibacterial ac- scopy (SEM) are shown in Fig. 1c and d. By comparing the microscopic
tivities of the samples were determined by the diameter of the inhibi- images of CS and CSNPs powders, it can be seen that the CSNPs showed
tion zone (circle) displayed on the plates. The diffusion assay was obvious spherical particle morphology after pure CS was made into
performed in triplicate. nanoparticles, and the distribution was uniform.
Furthermore, CSNPs were characterized by Fourier transform in-
2.10. Light barrier properties and transparency frared spectroscopy, thermogravimetric analysis, and transmission
electron microscopy. In brief, the results indicate that CSNPs were
An ultraviolet (UV) spectrophotometer was used to perform full- successfully prepared and more stable compare to CS at elevated tem-
wavelength scanning between 200−800 nm in transmission mode to perature. CSNPs displayed obvious nanoparticles and were easy to ag-
analyze the UV and visible light blocking properties and transparency glomerate between nanoparticles.
of the samples. The transparency of the films was calculated according
to the Eq. (3): 3.2. SEM analysis of films
log%T600
transparency = SEM was used to observe the surface and cross-sectional micro-
d (3)
morphologies of the films, as shown in Fig. 2. The surface of the BC/
where %T600 is the percent transmittance at 600 nm and d (mm) is the PVA-based film was uniform and smooth. After coating with CS, the
thickness of the film. composite film surface exhibited a smooth and compact structure.
Characteristically, nanoparticles can aggregate easily; therefore, the
3. Results and discussion surface of the CSNPs-coated composite film was coarse, and agglom-
erates appeared simultaneously. Similar situations were observed for
3.1. Characterization of CSNPs other bionanocomposite membranes (Kanmani & Rhim, 2014; Salari,
Khiabani, Mokarram, Ghanbarzadeh, & Kafil, 2018). According to the
As shown in Fig. 1a, the XRD pattern of CS showed obvious crys- cross-sectional images, no visible interface between the base film and
talline peaks around the 2θ values of 11.26° and 19.64°, indicating that the coating layer (CS or CSNPs) was observed, indicating that both CS
the CS sample showed semi-crystalline properties (Vijayalakshmi et al., and CSNPs could penetrate into the base-film matrix. Moreover, no ir-
2014). CS molecules can easily form crystalline regions, possibly due to regularities, such as vesicles, holes, or crevices, were detected, in-
the huge amounts of -OH and -NH2 groups in the CS molecule, which dicating that there was interaction and compatibility between BC/PVA

Fig. 1. XRD patterns of (a) bulk chitosan (CS) and (b) chitosan nanoparticles (CSNPs) and SEM images of (c) CS and (d) CSNPs powders.

3
S. Ju, et al. Carbohydrate Polymers 237 (2020) 116167

Fig. 2. SEM micrographs of the (a) surface and (b) cross-section of BC/PVA, BC/PVA/CS, and BC/PVA/CSNPs films (the concentration of both CS and CSNPs was 1.0
% w/v). CS means bulk chitosan and CSNPs means chitosan nanoparticles.

and chitosan in both forms. In addition, the cross-section of the CSNPs exhibited in Fig. 4. The addition of 1% w/v CS increased the tensile
film appeared jagged, which may be caused by the breakage of nano- strength (TS) of the film from 119.56 ± 7.56 MPa of the base film to a
particle aggregates. maximum of 130.55 ± 9.42 MPa. However, when its concentration
was further increased, the TS slightly decreased but remained higher
3.3. X-ray analysis of films than that of the BC/PVA film. The adjunction of CS increased the
polymer stiffness, probably because CS forms a strong interaction with
Fig. 3 displays the XRD spectra of composite films with different the glucon chains of interfacial BC due to the similar polysaccharide
contents of CS and CSNPs. The XRD curve of the BC/PVA-based film structures of CS and BC. Fig. 4b illustrates that CS addition led to a
showed four distinct diffraction peaks. The peaks at 14.66°, 16.96°, and decrease in the elongation percentage (E%) of the film, which was
22.42° corresponded to the (101), (101), and (002) crystal planes of obvious with the increase in CS concentration. This result was in ac-
cellulose for BC (Jung et al., 2010), while the peak at 19.40° was at- cordance with a previous report (Cazón et al., 2019).
tributed to the (101) plane of PVA crystals (Wahid et al., 2019). Pos- However, the addition of CSNPs gave rise to obvious decreases in
sibly due to the low relative chitosan content or polymer interactions, both TS and E%, and the effect became more pronounced with in-
the diffraction patterns of the composite films combining the two creasing concentration. At the corresponding concentration, CSNPs had
chitosan forms showed no significant differences, and the positions of a significantly more negative impact than CS on both TS and E%. This
the characteristic peaks were the same as those of the base film with a may be caused by nanoparticle agglomeration during the interaction
slight change in angle. Furthermore, the intensity of the corresponding between CSNPs and the polymer matrix (Chang et al., 2010). None-
peaks in the composite films decreased by increasing the concentration theless, the TS values of the films (the lowest value of
of CS or CSNPs, possibly because hydrogen bonds were formed between 45.39 ± 2.80 MPa) were greater than those of CS or CSNPs films
the polymers (Chen, Wei, Gong, Zheng, & Guan, 2018). commonly reported in the literatures (Cazón et al., 2019; Romainor
et al., 2014).

3.4. Mechanical properties

The mechanical properties of CS and CSNPs composite films are

Fig. 3. X-ray diffraction (XRD) patterns of films incorporated with (a) bulk chitosan (CS) and (b) chitosan nanoparticles (CSNPs) at different concentrations (w/v).

4
S. Ju, et al. Carbohydrate Polymers 237 (2020) 116167

Fig. 4. Tensile strength (a) and elongation (b) of films incorporated with bulk chitosan (CS) and chitosan nanoparticles (CSNPs) at different concentrations (w/v).

3.5. Moisture content at equilibrium 2010). However, a higher CS concentration led to a smaller WS de-
crease, which might be related to the higher moisture content of the
Moisture can influence the physical, chemical, and mechanical film at high CS content. The moisture increased the swelling degree of
properties of films. The moisture content of all samples is listed in the polymeric matrix, thereby promoting the instability of inter-
Table 1. Regarding the BC/PVA base film, both BC and PVA are hy- molecular interactions, and thus increased its solubility in water
drophilic components, and multiple hydroxyl groups enable it to retain (Carvalho et al., 2009). In addition, the embedding of CSNPs slightly
some amount of moisture (7.77 ± 0.48 %). Table 1 indicates that the improved the WS of the composite membrane. Unlike CS, higher CSNP
moisture contents of the composite films coated with CS were increased concentrations allowed lower membrane WS. The compact structure of
and positively correlated with the CS concentration, which could be CSNPs hindered water diffusion in the membrane, thus reducing the
ascribed to CS containing a mass of hydrophilic amino groups and water solubility.
hydroxyl groups (Cazón et al., 2019). Liu et al. (2018) also confirmed
that the composite had higher moisture content when the CS con-
centration was increased. Nevertheless, the CSNPs coating lowered the 3.7. Water vapor permeability (WVP)
moisture content of the membranes, which exhibited a prominent de-
crease when the concentration was 2.5 % w/v with a minimum value of The exchange of water between the packaged product and the film
3.94 ± 0.11 %. Compared to CS, nano-sized CSNPs are more compact used for food packaging should be minimized, meaning that WVP must
in structure, resulting in fewer of the exposed groups of chitosan chains be decreased. As shown in Table 1, the WVP of the BC/PVA-based film
being attached to water molecules (Antoniou, Liu, Majeed, & Zhong, was 0.449 ± 0.014 g mm m−2 h−1 kPa−1, and the addition of CS and
2015). CSNPs both reduced the WVP of the membrane. The WVP was declined
mildly in the case of CS, presumably because the hydrogen bonding
between CS and the base film diminished the free adsorption site of
3.6. Water solubility (WS)
water in the membrane, which led to reduced WVP. There was similar
case for sweet potato starch-based edible films (Shen et al., 2010). The
Water solubility (WS) is an important aspect of composite films. The
WVP value was significantly decreased to 0.361 ± 0.016 g mm
potential applications require composites to be as water insoluble as
m−2 h−1 kPa−1 when using 1.0 % w/v CSNPs. The permeability
possible to ensure product integrity and water resistance. The WS va-
showed no evident change when CSNPs were increased to the highest
lues of the respective composite films are exhibited in Table 1. It was
concentration (2.5 % w/v). CSNPs loading helped the water vapor to
observed that all samples maintained their intact structure after im-
pass around the dispersed nanoparticles and to create zigzag pathways
mersion for 24 h under shaking, while pure BC, PVA, and both chitosan
for water diffusion, therefore contributing to a lower WVP. However, at
forms were dispersed or dissolved into amorphous material. The WS of
higher concentration, the superabundant nanoparticles actually tended
composite films combined with both CS and CSNPs was improved to
to aggregate, leading to a decreased effective content, which promoted
varying degrees. The addition of CS resulted in a remarkable decrease
WVP (Chang et al., 2010).
in WS with a minimum of 9.34 % due to the hydrogen bonding inter-
action between CS and BC/PVA matrix (Shen, Wu, Chen, & Zhao,

Table 1
Experimental results for the thickness, moisture content, water solubility (WS) and water vapor permeability (WVP) of films incorporated with bulk chitosan (CS) and
chitosan nanoparticles (CSNPs).
Film type CS/CSNPs concentration (% w/v) Thickness (mm) Moisture content (%) WS (%) WVP (g mm m−2 h−1 kPa−1)

BC/PVA 0 0.045 ± 0.004 7.77 ± 0.48 37.89 ± 1.06 0.449 ± 0.014

BC/PVA + CS 0.5 0.053 ± 0.007 11.55 ± 0.87 9.349 ± 1.67 0.436 ± 0.017
1.0 0.056 ± 0.013 13.90 ± 0.30 20.97 ± 2.05 0.406 ± 0.018
2.5 0.067 ± 0.015 16.62 ± 0.17 35.29 ± 1.02 0.399 ± 0.015
BC/PVA + CSNPs 0.5 0.071 ± 0.006 6.74 ± 0.39 34.83 ± 2.26 0.397 ± 0.017
1.0 0.078 ± 0.009 6.70 ± 0.11 31.51 ± 1.14 0.361 ± 0.016
2.5 0.093 ± 0.016 3.94 ± 0.11 30.12 ± 1.72 0.367 ± 0.008

Data are reported as the average of three determinations ± standard deviation.

5
S. Ju, et al. Carbohydrate Polymers 237 (2020) 116167

Fig. 5. The diffusion assay results of films incorporated with bulk chitosan (CS) and chitosan nanoparticles (CSNPs) at different concentrations (w/v) against (a)
Escherichia coli and (b) Staphylococcus aureus.

3.8. Antimicrobial tests properties when the CSNPs concentration was as low as 0.5 % w/v
(minimum 7.33 ± 1.00 mm), while BC/PVA/CS showed maximum
The pictures of diffusion assay resulting from BC/PVA base film and 7.73 ± 0.20 mm when the CS concentration was 2.5 % w/v. The an-
composite films with different concentrations of CS and CSNPs are tibacterial properties are higher than those of the chitosan biocompo-
presented in Fig. 5 and the width of inhibitory zone of the films after site films reported in other literatures (Du et al., 2018; Patil, Bohara,
subtracting the sample diameter is listed in Table 2. For the control BC/ Meshram, Nanaware, & Pawar, 2019). The bacteriostatic diameter of
PVA film, the membrane area was contaminated with bacteria, in- CSNPs films was much larger than the CS films at the corresponding
dicating that it had no antibacterial properties. It can be observed from concentration, which differs from the case of previous reported films
the pictures that a composite membrane doped with a certain con- incorporated bulk CS and CSNPs (Antoniou et al., 2015).
centration of chitosan can produce a clear area of inhibition for bac- CSNPs have higher antibacterial activity than pure CS as a result of
teria. The BC/PVA/CS films showed no antibacterial properties against special properties of nanoparticles. Many studies have explored the
S. aureus at 0.5 % and 1.0 % w/v CS concentrations. The inhibitory antibacterial mechanisms related to chitosan, in which the reaction
effect of BC/PVA/CS films on both E. coli and S. aureus was dose-de- between the positive charge on the protonated ammonium group of
pendent, which increased significantly with the enhancement of CS chitosan and the negative charge on the microbial cell membrane is the
content. Du, Yang, Ye, and Li (2013) obtained similar trends in anti- most widely accepted one. The electrostatic interaction between them
microbial activity as a function of CS concentration. The BC/PVA/ could destroy the cell membrane, leading to protein leakage, which in
CSNPs composite membranes exhibited excellent antibacterial turn interferes with its metabolism and kills bacterial cells (Kanmani &
Rhim, 2014). Unlike the large volume of CS, the CSNPs system has the
Table 2 advantage of large specific surface area and can provide more charge
The diameter (mm) of inhibition zone of films incorporated with bulk chitosan sites (amino groups) for microbial interactions (Romainor et al., 2014).
(CS) and chitosan nanoparticles (CSNPs) against food pathogenic bacteria of Therefore, polycationic CSNPs with higher surface charge density in-
Escherichia coli and Staphylococcus aureus. teract more with bacteria than bulk CS. With the increase of the con-
Film type CS/CSNPs Diameter of the inhibitory zone (mm) centration of CSNPs, the antibacterial performance of the membrane
concentration (% w/ did not increase to a large extent differing from the case of CS. This may
v) Escherichia coli Staphylococcus be because higher doping levels provide more charge action points
aureus
(amino groups), thus more interaction brought about the formation of
BC/PVA 0 0 0 clusters and aggregates of among the nanoparticles. Therefore, the
BC/PVA + CS 0.5 1.50 ± 0.82 0 limited charge sites useful for attaching bacteria do not lead to a sig-
1.0 4.17 ± 0.47 0 nificant improvement in antibacterial activity (Goy, Britto, & Assis,
2.5 7.73 ± 0.20 2.50 ± 0.12
2009).
BC/PVA + CSNPs 0.5 9.08 ± 0.06 7.33 ± 1.00
1.0 9.33 ± 1.03 8.67 ± 0.59
2.5 10.00 ± 0 10.33 ± 1.55

Data are reported as the average of three determinations ± standard deviation.

6
S. Ju, et al. Carbohydrate Polymers 237 (2020) 116167

Fig. 6. UV–vis spectra of films incorporated with (a) bulk chitosan (CS) and (b) chitosan nanoparticles (CSNPs) at different concentrations (w/v).

Table 3 the BC/PVA/CS films were still very transparent, as the transparency
Optical properties of the films incorporated with bulk chitosan (CS) and chit- value was only reduced to 29.06 at 2.5 % w/v CS concentration. For
osan nanoparticles (CSNPs). %T (200–280) is the average percentage of CSNPs composite films, the addition of 2.5 % w/v CSNPs reduced the
transmittance at 200 − 280 nm region; %T600 is the percent transmittance at maximum transmittance of the composite films to a minimum of 14.80
600 nm. % while reducing the transparency value to 12.34.
Film type CS/CSNPs % T(200–280) % T600 Transparency
concentration 4. Conclusion
(% w/v)

BC/PVA 0 17.68 50.30 38.24 CS and CSNPs have been successfully incorporated into the film of
BC/PVA + CS 0.5 32.75 66.95 34.45 BC/PVA base film. CS improved the film TS, while CSNPs reduced both
1.0 42.14 81.08 34.09 TS and E%. CS possesses numerous hydrophilic amino and hydroxyl
2.5 39.06 88.46 29.06 groups, leading to a growth in membrane moisture content. The dense
BC/PVA + CSNPs 0.5 12.74 35.78 21.88
1.0 9.66 32.39 19.36
structure of CSNPs hindered water diffusion by increasing its path in the
2.5 3.84 14.04 12.34 membrane, thereby reducing water solubility and WVP. Compared with
CS, CSNPs possess a higher surface charge density and could have more
interactions with bacteria. Therefore, CSNPs showed much greater an-
3.9. Light barrier properties and transparency timicrobial properties against E. coli and S. aureus than did CS, and the
effect on E. coli was more obvious. In addition, CS-doped films showed
Optical properties can affect product appearance and therefore have higher transparency; however, CSNPs-doped films exhibited preferable
a significant impact on determining whether the films can be used on UV-barrier properties. The composite films developed in this work can
food surfaces (Pereda, Amica, & Marcovich, 2012). Fig. 6 and Table 3 be used in food packaging applications because they possess excellent
show the transmittance and transparency of all films at wavelengths of properties including high tensile strength and remarkable antibacterial
200–800 nm. Lipids are more susceptible to oxidation reactions within properties.
200–280 nm, which can lead to oxidative deterioration, nutrient loss,
and color changes in packaged foods. Hence, the UV-barrier perfor- CRediT authorship contribution statement
mance of the films at this region is particularly important (Martins,
Cerqueira, & Vicente, 2012). Siyi Ju: Conceptualization, Methodology, Investigation, Writing -
The average transmission between 200 and 280 nm was used to original draft, Writing - review & editing. Fenglun Zhang: Formal
characterize the barrier properties of the film in this region. The analysis, Resources, Data curation, Writing - review & editing. Jiufang
average transmittance of BC/PVA was 17.68 %, which was increased to Duan: Validation, Visualization, Writing - review & editing. Jianxin
32.75 % by adding 0.5 % w/v CS. However, when the CS concentration Jiang: Writing - review & editing, Supervision, Project administration,
was augmented from 1.0%–2.5% w/v, the barrier properties of the film Funding acquisition.
were not further negatively affected (i.e., increased transmittance). The
gelatin-chitosan edible films prepared by Hosseini, Rezaei, Zandi, and Acknowledgments
Ghavi (2013) also showed that CS caused an increase in transmission.
The increase in CSNPs enhanced film transmittance, and higher CSNP This research was funded by the financial support of the National
concentrations led to lower film transmittance. When 2.5 % w/v CSNPs Natural Science Foundation of China (31670579) and the Specific re-
were added, the average film transmittance decreased to 3.84 %, which search project of Guangxi for research bases and talents (AD18126005).
may be due to the compact structure of CSNPs.
Besides blocking properties, eminent visual appearance and high Appendix A. Supplementary data
transparency are also vital qualities required for the developed com-
posite films. The visual appearance can be evaluated from the trans- Supplementary material related to this article can be found, in the
mission curves in the UV–vis region (280–800 nm) and the transmission online version, at doi:https://doi.org/10.1016/j.carbpol.2020.116167.
percentage at 600 nm, as shown in Table 3. In this region, the maximum
transmission of BC/PVA is 53.90 %, and the transparency value is References
38.67. CS significantly accelerated the sample transmittance, with a
maximum value of 89.7 % at 2.5 % w/v concentration. Furthermore, Antoniou, J., Liu, F., Majeed, H., & Zhong, F. (2015). Characterization of tara gum edible

7
S. Ju, et al. Carbohydrate Polymers 237 (2020) 116167

films incorporated with bulk chitosan and chitosan nanoparticles: A comparative Muppalla, S. R., Kanatt, S. R., Chawla, S., & Sharma, A. (2014). Carboxymethyl cellulo-
study. Food Hydrocolloids, 44, 309–319. se–polyvinyl alcohol films with clove oil for active packaging of ground chicken
Bourtoom, T. (2008). Edible films and coatings: Characteristics and properties. meat. Food Packaging and Shelf Life, 2(2), 51–58.
International Food Research Journal, 15(3), 237–248. Naghdi, T., Golmohammadi, H., Vosough, M., Atashi, M., Saeedi, I., & Maghsoudi, M. T.
Carvalho, R. A.d., Maria, T., Moraes, I., Bergo, P. V. A., Kamimura, E. S., Habitante, A., ... (2019). Lab-on-nanopaper: An optical sensing bioplatform based on curcumin em-
Sobral, P. J.d. A. (2009). Study of some physical properties of biodegradable films bedded in bacterial nanocellulose as an albumin assay kit. Analytica Chimica Acta,
based on blends of gelatin and poly (vinyl alcohol) using a response-surface metho- 1070, 104–111.
dology. Materials Science and Engineering C, 29(2), 485–491. Patil, P. P., Bohara, R. A., Meshram, J. V., Nanaware, S. G., & Pawar, S. H. (2019). Hybrid
Cazón, P., Vázquez, M., & Velazquez, G. (2018). Composite films of regenerate cellulose chitosan-ZnO nanoparticles coated with a sonochemical technique on silk fibroin-
with chitosan and polyvinyl alcohol: Evaluation of water adsorption, mechanical and PVA composite film: A synergistic antibacterial activity. International Journal of
optical properties. International Journal of Biological Macromolecules, 117, 235–246. Biological Macromolecules, 122, 1305–1312.
Cazón, P., Velázquez, G., & Vázquez, M. (2019). Characterization of bacterial cellulose Pereda, M., Amica, G., & Marcovich, N. E. (2012). Development and characterization of
films combined with chitosan and polyvinyl alcohol: Evaluation of mechanical and edible chitosan/olive oil emulsion films. Carbohydrate Polymers, 87(2), 1318–1325.
barrier properties. Carbohydrate Polymers, 216, 72–85. Putra, A., Kakugo, A., Furukawa, H., Gong, J. P., & Osada, Y. (2008). Tubular bacterial
Chang, P. R., Jian, R., Yu, J., & Ma, X. (2010). Starch-based composites reinforced with cellulose gel with oriented fibrils on the curved surface. Polymer, 49(7), 1885–1891.
novel chitin nanoparticles. Carbohydrate Polymers, 80(2), 420–425. Ramya, R., Sudha, P. N., & Mahalakshmi, J. (2012). Preparation and characterization of
Chen, J., Wei, D., Gong, W., Zheng, A., & Guan, Y. (2018). Hydrogen-bond assembly of chitosan binary blend. International Journal of Scientific and Research Publications,
poly (vinyl alcohol) and polyhexamethylene guanidine for nonleaching and trans- 2(10), 1–9.
parent antimicrobial films. ACS Applied Materials & Interfaces, 10(43), 37535–37543. Rani, M. U., Rastogi, N. K., & Appaiah, K. A. (2011). Statistical optimization of medium
de Moura, M. R., Aouada, F. A., Avena-Bustillos, R. J., McHugh, T. H., Krochta, J. M., & composition for bacterial cellulose production by Gluconacetobacter hansenii UAC09
Mattoso, L. H. (2009). Improved barrier and mechanical properties of novel hydro- using coffee cherry husk extract–an agro-industry waste. Journal of Microbiology and
xypropyl methylcellulose edible films with chitosan/tripolyphosphate nanoparticles. Biotechnology, 21(7), 739–745.
Journal of Food Engineering, 92(4), 448–453. Romainor, A. N. B., Chin, S. F., Pang, S. C., & Bilung, L. M. (2014). Preparation and
Du, R., Zhao, F., Peng, Q., Zhou, Z., & Han, Y. (2018). Production and characterization of characterization of chitosan nanoparticles-doped cellulose films with antimicrobial
bacterial cellulose produced by Gluconacetobacter xylinus isolated from Chinese per- property. Journal of Nanomaterials, 2014, 1–10.
simmon vinegar. Carbohydrate Polymers, 194, 200–207. Salari, M., Khiabani, M. S., Mokarram, R. R., Ghanbarzadeh, B., & Kafil, H. S. (2018).
Du, X., Yang, L., Ye, X., & Li, B. (2013). Antibacterial activity of konjac glucomannan/ Development and evaluation of chitosan based active nanocomposite films con-
chitosan blend films and their irradiation-modified counterparts. Carbohydrate taining bacterial cellulose nanocrystals and silver nanoparticles. Food Hydrocolloids,
Polymers, 92(2), 1302–1307. 84, 414–423.
Esa, F., Tasirin, S. M., & Rahman, N. A. (2014). Overview of bacterial cellulose production Shen, X. L., Wu, J. M., Chen, Y., & Zhao, G. (2010). Antimicrobial and physical properties
and application. Agriculture and Agricultural Science Procedia, 2(1), 113–119. of sweet potato starch films incorporated with potassium sorbate or chitosan. Food
Gatenholm, P., & Klemm, D. (2010). Bacterial nanocellulose as a renewable material for Hydrocolloids, 24(4), 285–290.
biomedical applications. MRS Bulletin, 35(3), 208–213. Torres, F. G., Commeaux, S., & Troncoso, O. P. (2012). Biocompatibility of bacterial
Goy, R. C., Britto, D.d., & Assis, O. B. (2009). A review of the antimicrobial activity of cellulose based biomaterials. Journal of Functional Biomaterials, 3(4), 864–878.
chitosan. Polímeros, 19(3), 241–247. Tripathi, S., Mehrotra, G., & Dutta, P. (2010). Preparation and physicochemical evalua-
Hosseini, S. F., Rezaei, M., Zandi, M., & Ghavi, F. F. (2013). Preparation and functional tion of chitosan/poly (vinyl alcohol)/pectin ternary film for food-packaging appli-
properties of fish gelatin–chitosan blend edible films. Food Chemistry, 136(3-4), cations. Carbohydrate Polymers, 79(3), 711–716.
1490–1495. Vijayalakshmi, K., Gomathi, T., & Sudha, P. (2014). Preparation and characterization of
Ju, S. Y., Yu, H. L., Ji, L., Wang, K., & Jiang, J. X. (2020). Preparation and characterization nanochitosan/sodium alginate/microcrystalline cellulose beads. Der Pharmacia Lettre,
of bacterial nanocellulose-based composite membranes. Science of Advanced 6(4), 65–77.
Materials, 12, 1–8. Wahid, F., Wang, F. P., Xie, Y. Y., Chu, L. Q., Jia, S. R., Duan, Y. X., ... Zhong, C. (2019).
Jung, H. I., Lee, O. M., Jeong, J. H., Jeon, Y. D., Park, K. H., Kim, H. S., ... Son, H. J. Reusable ternary PVA films containing bacterial cellulose fibers and ε-polylysine with
(2010). Production and characterization of cellulose by Acetobacter sp. V6 using a improved mechanical and antibacterial properties. Colloids and Surfaces B,
cost-effective molasses–corn steep liquor medium. Applied Biochemistry and Biointerfaces, 183(110486), 1–9.
Biotechnology, 162(2), 486–497. Wang, K., Fan, X., Zhang, X., Zhang, X., Chen, Y., & Wei, Y. (2016). Red fluorescent
Kanmani, P., & Rhim, J.-W. (2014). Physical, mechanical and antimicrobial properties of chitosan nanoparticles grafted with poly (2-methacryloyloxyethyl phosphorylcho-
gelatin based active nanocomposite films containing AgNPs and nanoclay. Food line) for live cell imaging. Colloids and Surfaces B, Biointerfaces, 144, 188–195.
Hydrocolloids, 35, 644–652. Wang, K., Zhang, X., Zhang, X., Fan, X., Huang, Z., Chen, Y., ... Wei, Y. (2015).
Limpan, N., Prodpran, T., Benjakul, S., & Prasarpran, S. (2012). Influences of degree of Preparation of biocompatible and photostable PEGylated red fluorescent nano-
hydrolysis and molecular weight of poly (vinyl alcohol) (PVA) on properties of fish particles for cellular imaging. Polymer Chemistry, 6(32), 5891–5898.
myofibrillar protein/PVA blend films. Food Hydrocolloids, 29(1), 226–233. Xie, G., Ma, C., Zhang, X., Liu, H., Yang, L., Li, Y., ... Wei, Y. (2017). Chitosan-based cross-
Liu, L., Liang, H., Zhang, J., Zhang, P., Xu, Q., Lu, Q., ... Zhang, C. (2018). Poly (vinyl linked fluorescent polymer containing aggregation-induced emission fluorogen for
alcohol)/Chitosan composites: Physically transient materials for sustainable and cell imaging. Dyes and Pigments, 143, 276–283.
transient bioelectronics. Journal of Cleaner Production, 195, 786–795. Zhao, N., Chai, Y., Wang, T., Wang, K., Jiang, J., & Yang, H. Y. (2019). Preparation and
Martins, J. T., Cerqueira, M. A., & Vicente, A. A. (2012). Influence of α-tocopherol on physical/chemical modification of galactomannan film for food packaging.
physicochemical properties of chitosan-based films. Food Hydrocolloids, 27(1), International Journal of Biological Macromolecules, 137, 1060–1067.
220–227. Zhong, C., Zhang, G. C., Liu, M., Zheng, X. T., Han, P. P., & Jia, S. R. (2013). Metabolic
Moreira, M.d. R., Roura, S. I., & Ponce, A. (2011). Effectiveness of chitosan edible coat- flux analysis of Gluconacetobacter xylinus for bacterial cellulose production. Applied
ings to improve microbiological and sensory quality of fresh cut broccoli. LWT-Food Microbiology and Biotechnology, 97(14), 6189–6199.
science and technology, 44(10), 2335–2341.