International Journal of Biological Macromolecules 123 (2019) 210–220

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Review

Chitosan-functionalized nanofibers: A comprehensive review on

challenges and prospects for food applications
Bruna Silva de Farias, Tito Roberto Sant'Anna Cadaval Junior, Luiz Antonio de Almeida Pinto ⁎
School of Chemistry and Food, Federal University of Rio Grande (FURG), km 8 - Itália Avenue, 96203-900 Rio Grande, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Chitosan exhibits outstanding properties, which allow a wide range of applications. For this reason, chitosan-
Received 9 October 2018 based biomaterials have been developed over the years and, among these biomaterials, chitosan-based
Received in revised form 5 November 2018 nanomaterials may significantly change the material properties, which could result in some exceptional features.
Accepted 8 November 2018
Indeed, chitosan-based nanofibers have a larger surface area:volume ratio than the bulk materials at macro scale.
Available online 09 November 2018
Moreover, chitosan-based nanofibers could lead to enhanced porosity and mechanical properties, which could
Keywords:
also improve surface functionalities, and consequently, the range of applications. However, the diversity in
Biopolymer sources of raw materials and the production processes for the development of chitosan might provide distinct
Molecular orientation physicochemical characteristics. Because the varieties of chitosan have been limited in the most part the nanofi-
Nanobiomaterial bers synthesis, the current review describes an extensive research concerning the development of chitosan-
based nanofibers and summarizes the different techniques for the nanofibers production; in addition to point
out the effects of chitosan characteristics on the spinnability of the solution. Furthermore, the present review ex-
plores some potential studies in relation to the chitosan-based nanofibers applied to food technology, including
active food packaging, nanofood carrier and enzyme immobilization.
© 2018 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
2. Remarkable properties of chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
3. Nanofibrous materials production: advantages and drawbacks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
4. Chitosan nanofibers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
5. Composite and nanofibers blends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
6. Characterization of chitosan-based nanofibers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
7. Chitosan-based nanofibers applied in food technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
7.1. Chitosan-based nanofibers for active food packaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
7.2. Chitosan-based nanofibers for nanofood carrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
7.3. Chitosan-based nanofibers for enzyme immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
8. Final remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

1. Introduction

Chitosan is a renewable biopolymer, which has attracted consider-

able attention due to its remarkable properties, such as polycationic na-
⁎ Corresponding author.
ture, mucoadhesive and gelling properties, antibacterial and antifungal
E-mail addresses: brunafarias@furg.br (B.S. de Farias), dqmpinto@furg.br activity, nontoxicity and biodegradability. The polycationic character
(L.A. de Almeida Pinto). of chitosan has been widely used in several applications, among them,

https://doi.org/10.1016/j.ijbiomac.2018.11.042
0141-8130/© 2018 Elsevier B.V. All rights reserved.
 B.S. de Farias et al. / International Journal of Biological Macromolecules 123 (2019) 210–220
in enzyme immobilization with negatively charged enzymes [1], in bio-
medical devices with negatively charged proteins [2]; in wound healing
with negatively charged cell membranes [3]; in gene delivery with neg-
atively charged DNA molecules [4]. Moreover, chitosan is one of the
most widely studied polymers in oral drug delivery system because of
its mucoadhesive property [5].
The gelling property enables the production of chitosan hydrogels,
which are widely studied in tissue engineering [6], drug delivery [7]
and dye adsorption [8]. The chitosan antimicrobial property associated
with its biocompatibility makes chitosan widely used in the biomedical
application, such as tissue engineering, wound dressing, hemodialysis
and drug delivery carrier [9,10]. Despite the fact that chitosan has
been mostly studied for biomedical applications, there is still plenty of
potential for the food industry, which will be discussed over this review.
Although all the outstanding properties associated to chitosan, the
low surface area of its usual physical forms (powder, flakes and
biopolymeric film) can restrict their potential. For this reason,
chitosan-based nanomaterials have been drawn attention to this new
range of possibilities [11,12]. Indeed, nanomaterials comprehend a vari-
ety of morphologies, among them, the nanofiber morphology shows
flexibility in surface functionalities, easy separation and higher mechan-
ical and porosity properties [13–15]. However, the spinnability of chito-
san solution to yield nanofibers has been regarded as a challenge,
especially because chitosan can be obtained by a variety of raw mate-
rials, as well as different production processes. Therefore, this review
intended to describe the different techniques that can be used to de-
velop chitosan nanofibers, exploring the impact of the different chitosan
characteristics on the spinnability of its solution. Furthermore, this re-
view also aimed to highlight some trends and future research opportu-
nities using chitosan-based nanofibers on food field.
2. Remarkable properties of chitosan
Chitosan is a cationic biopolymer constituted by 2 acetamide-
2 deoxy-β-D-glucopyranose and 2 amino-2 deoxy-β-D-glucopyranose
residues. Its polymeric structure should have at least 50% of 2 amino-
2 deoxy-β-D-glucopyranose residues to be considered chitosan. This
biopolymer is mainly obtained by chitin alkaline deacetylation reaction
or by fermentation process, which the main sources are crab/shrimp
shells and fungal mycelia [16,17]. Chitosan has been reported to exhibit
many properties, such as its polycationic character. Due to the fact that
the pKa value of the amino group of glucosamine residues is around 6.3,
then chitosan is positively charged in acid solutions. Thus, the low pH
treatment protonates the NH2 groups along the chains, leading to the
formation of (NH3)+, which allows electrostatic interaction between
chitosan and the negatively charged molecules [18,19].
The cationic character of chitosan, associated with the possibility of
hydrogen and hydrophobic bonds between its polymeric chains, can at-
tribute mucoadhesive and gelling properties to the polysaccharide [20].
The mucoadhesive property is related to the chitosan adhesion to muco-
sal tissues. The mucosal membranes are covered by single-layered
membranes that secrete mucus [21]. Indeed, the major mechanism for
the chitosan mucoadhesion is due to the electrostatic attraction be-
tween the positively charged amino groups and negatively substruc-
tures of the mucus; because the major components of mucus are
mucin glycoproteins, which are negatively charged due to the presence
of sialic acid and ester sulfate. But this property is also accomplished by
contributions from hydrogen and hydrophobic effects [22,23].
The gelling properties of chitosan enable hydrogel formation due to
the chitosan hydrophilic groups, which allow the hydrogel to adsorb
water and watery fluids [24]. Furthermore, the film-forming property
is considered one of the most important biopolymer's characteristics,
and this ability is obtained by both mucoadhesive and gelling proper-
ties. The film-forming property is required to produce films [25], mem-
branes [26] and fibers [27]. Then, as new physical forms are developed,
the range of chitosan applications could be enhanced.
211
Chitosan has been demonstrated to inhibit the growth of a wide va-
riety of bacteria and fungi. Among the mechanisms, due to its
polycationic nature, the biopolymer interacts with the negatively
charged residues by adsorption onto the macromolecules at the bacteria
surface. Chitosan also alters cell permeability by chelating with metal
ions through microorganism's cell membrane. In addition, chitosan
binds with fungal DNA to inhibit RNA synthesis [19,28].
Chitosan properties, as well as chitosan-based materials, are highly
dependent on many important factors, including deacetylations degree,
molecular weight, crystallinity, particles size and surface area
[19,29–31]. For this reason, it is important the study of all chitosan's
characteristics to better comprehend and optimize its applications.
3. Nanofibrous materials production: advantages and drawbacks
Nanofibers have a large surface area:volume ratio, corresponding to
approximately two times higher than the nanoparticles of the same di-
ameter, based on an equal amount of material. Nanofibers have many
competitive advantages over nanoparticles. They are easier to separate
from the solution medium, which allows its reuse. Nanofibers can also
achieve small diameters (ranging from submicron to several nanome-
ters), high porosity, interconnectivity, microscale interstitial space,
small interfibrous pores size, flexibility in surface functionalities and
higher mechanical properties. All these characteristics show that nano-
fibers are multifunctional materials for a wide range of applications
[32–34].
There are several methods for nanofibers production. However, it is
important to point out the characteristics of the method to identify
which one is more suitable for each material and application, consider-
ing operating parameters, costs and production rates. Therefore, novel
strategies concerning nanofibers production have emerged, as summa-
rized throughout this topic, and some of them may be more suitable to
develop chitosan-based nanofibers.
Electrospinning is one of the most established techniques for syn-
thesizing nanofibers. This technique occurs when the applied electro-
static field is strong enough to overcome the surface tension of the
liquid. As the intensity of the electric field is increased, the mutual
charge repulsion on the drop surface is increased, altering the droplet
shape to form a Taylor cone at the tip of the spinneret. Eventually, the
charge repulsion exceeds the surface tension and the jet is ejected
from the Taylor cone to the grounded target. During the acceleration,
the jet undergoes a whipping process, which stretches the fiber and sig-
nificantly reduces fiber diameter. Then, the jet stream can be collected
as a nanofiber (Fig. 1). Moreover, the viscosity of the solution must be
sufficiently high, because low-viscosity liquids cannot achieve sufficient
resistance against the Rayleigh instability, and tend to break up into
droplets, which is not desired. The morphology of the electrospun fibers
depends on several factors, such as solution properties, processing con-
ditions and ambient conditions. The solution properties include the
polymer type, polymer molecular weight, surface tension, conductivity
and viscosity. Among the processing conditions, there are the electric
field strength applied, spinneret diameter, distance between the spin-
neret and the collecting substrate and feeding rate. The ambient param-
eters, particularly, humidity and temperature, can influence the fiber
morphology and the productivity of the electrospinning process
[35–38]. Electrospinning has the advantage to produce nanofibers of
different materials in various fibrous assemblies with diameters
b100 nm, even up to 5 nm [32,33,39]. The electrospinning technique
has already produced several types of synthetic and natural based
nanofibrous materials, including poly(ethylene-oxide) [40], poly(vinyl
alcohol) [41], polycaprolactone [42], collagen [43] and silk fibroin [44].
Several adaptations of the electrospinning technique have been cre-
ated recently. These include co-axial electrospinning, needleless and
multi-needle. The last ones are used to increase the productivity of con-
ventional electrospinning, while the co-axial electrospinning is utilized
to synthesize multilayer composite and core-shell nanofibrous
212 B.S. de Farias et al. / International Journal of Biological Macromolecules 123 (2019) 210–220
Fig. 1. Basic scheme of electrospinning technique.
structures with some additional property and enhanced quality. Molnar
and Nagy [45] reported a needleless method which has two orders of
magnitude higher than the conventional method. Moreover, Jiang and
Qin [46] were able to produce massive production of core-shell nanofi-
bers by enhancing the development more than one hundred times over
the traditional method. Even though most nanofibers are prepared
using electrospinning, the method has some drawbacks, such as the
high electrical potential, which can impact directly on scale-up limita-
tion. Despite the electrospinning has been used to a wide range of nat-
ural polymers, the electrospinning of chitosan is still considered a
challenge. Chitosan nanofibers can be synthesized by electrospinning,
although more studies on chitosan structure are required to be able to
produce pure chitosan nanofibers.
Mechano-electrospinning is another adaptation which uses the elec-
trical forces as electrospinning, but it also utilizes mechanical forces.
Then, the combination of both forces drives the solution for the high-
resolution writing. One of the major advantages of the mechano-
electrospinning is the fact that position, writing resolution and mor-
phology can be digitally controlled by the system. The processing pa-
rameters which can control the dynamic behavior of the jet are the
translational speed, the nozzle-to-substrate distance and the applied
voltage. By controlling these parameters, plenty structures might be
produced [47].
Solution blowing process is a recent nanofiber spinning process
(Fig. 2). The technique has been developed to overcome the drawbacks
Fig. 2. Basic scheme of solution blowing technique.
of the electrospinning process, such as the need of solvents with high di-
electric constants or even a large electric field. In addition, the solution
blowing process has some similarities with industrial methods of fibers
production, which enables scale-up production [48]. The method re-
quires a polymer dissolved in a volatile solvent and a compressed gas
source. The compressed gas applies high pressure to cause acceleration,
which deforms the polymer solution into a conical shape, similarly to
the Taylor cone during the electrospinning process [49]. The method
has already been used to synthesize nanofibers with poly(methyl meth-
acrylate) [49], nylon 6 [50]; cellulose [51], gelatin [52], poly(ethylene
oxide)/soy protein [53], chitosan/PVA [54]. Similar to electrospinning,
the polymer characteristics, as well as solutions properties, play a signif-
icant role in nanofibers production. For this reason, further studies are
needed in order to improve the chitosan spinnability.
Centrifugal jet spinning was also developed to overcome the scale-up
problem of electrospinning. It was designed to synthesize nanofibers in a
larger scale by enhancing the rotational speed to achieve higher efficiency
and lower costs when compared with electrospinning [55–57]. In fact, its
fibers synthesis is estimated to be about five hundred times higher than
the electrospinning conventional method [56]. In addition, the centrifugal
spinning does not require high voltage, which reduces safety-related con-
cerns. The method is based on the controlling of centrifugal force and the
viscoelastic and mass transfer fluid properties. The polymer-solvent sys-
tem is introduced into a rotating spinneret, and then ejected, as the jet
overcomes the surface tension (Fig. 3). Some of the polymers used on
nanofibers production by centrifugal spinning are PEO [58], poly(L-lactic
acid) [55], polycaprolactone [59], ethyl cellulose/polyvinyl pyrrolidone
[60], PCL/gelatin [61], poly(3 hydroxybutyrate co 3 hydroxyvalerate)
[62]. Dotto et al. [63] were able to produce chitosan/polyamide nanofibers
by Forcespinning®; although, more studies regarding chitosan properties
are needed for predicting solution behavior, and consequently, the chito-
san spinnability.
Freeze-drying is a technique which has drawn increasing attention
for nanofibers production (Fig. 4). Indeed, the method has the advan-
tage of using water-based systems, which enhance the potential appli-
cations in the food science field. The polymer dispersion, emulsion or
solution is frozen using dry ice, refrigerator or liquid nitrogen, and the
freezing rate dramatically influences the structure of the polymer nano-
structures upon ice removal. When the freezing rate significantly over-
comes polymer's Brownian diffusion, the polymer solution is mixed
among the ice clusters, and then, occurs sublimations of the frozen solu-
tion by freeze-drying, which forms aligned micro and nanofibers [64].
The freeze-drying technique has already been used to synthesize nano-
fibers composed by polypyrrole [64], chitin [65], chitosan/sodium algi-
nate [66], chitosan/sodium hyaluronate [67]. Nevertheless, the
technique results in fibers diameter distribution relatively broad. More-
over, Wang and Wakisaka [68] were able to develop chitosan nanofibers
by using ultrasonic atomization and freeze casting. The authors verified
that in addition to higher freezing rates, a high-initial velocity
 B.S. de Farias et al. / International Journal of Biological Macromolecules 123 (2019) 210–220 213

Fig. 3. Basic scheme of centrifugal jet spinning technique.

atomization led to fibers aligned. For this reason, the freeze-drying tech-
nique associated with other synthesize process could be a promising
method to produce chitosan nanofibers.
For the carbon dioxide laser supersonic drawing (CDLSD), the poly-
mer should be submitted to an extrusion process, previously, in order to
produce fibers in microscale. Then CDLSD is applied to transform the
microfibers into nanofibers by irradiating a continuous-wave carbon di-
oxide laser on fibers while drawing it in a supersonic jet (Fig. 5). The su-
personic jet is obtained by blowing air into vacuum through an orifice
responsible to inject the fibers into a vacuum compartment. The mech-
anism of the method is based on the adiabatic expansion of air across
the orifice, which reduces the temperature of the jet. Then the fiber is
melted by the high-power laser beam that irradiates the low-
temperature supersonic jet, causing deformation by shear force in the
supersonic flow [69]. The method can be considered a green process
due to the fact it requires only carbon dioxide laser, and it does not
use any type of solvents. The carbon dioxide laser supersonic drawing
has been applied to prepare nanofibers of poly(phenylene sulfide)
[70], nylon 66 [71], ethylene tetrafluoroethylene [72], poly
(ethylene 2,6 naphthalate) [73], poly(ethylene terephthalate) (PET)
[69] and poly(L-lactic acid) [74]. However, the method might be only
used for thermoplastic polymers [75]. Since chitosan usual physical
forms are not thermoplastic, due to degradation under their melting
point, the biopolymer would just be suitable to the method if some
modification was performed to improve its thermal properties.
4. Chitosan nanofibers
The development of pure and stable chitosan nanofibers has been
considered a challenge. Although some researches have been reported
in the literature, as shown in Table 1 [68,76–85], chitosan spinnability,
as well as the reproducibility of the successful attempts, were found to
be difficulties.

Fig. 4. Basic scheme of freeze-drying technique.

Fig. 5. Basic scheme of carbon dioxide laser supersonic drawing technique.

214 B.S. de Farias et al. / International Journal of Biological Macromolecules 123 (2019) 210–220

Table 1
Summary of attempts at nanofiber formation from pure chitosan.

Method Chitosan MW (g mol−1) Chitosan DD (%) Solvent type Nanofiber diameter (nm) Refs.

Electrospinning 210,000 78.0 Trifluoroacetic acid 490.0 Ohkawa et al. [76]

Electrospinning 106,000 54.0 Acetic acid 130.0 Geng et al. [77]
Electrospinning 570,000 95.0 Trifluoroacetic acid/dichloromethane 130.0 Sangsanoh and Supaphol [78]
Electrospinning 190,000–310,000 75.0–85.0 Acetic acid 70.0 De Vrieze et al. [79]
Electrospinning 294,000 75.0–85.0 Acetic acid 140.0 Homayoni et al. [80]
Electrospinning 1000,000 80.0–90.0 Acetic acid 73.0 Terada et al. [81]
Electrospinning – – Trifluoroacetic acid 137 Haider et al. [82]
Electrospinning 311,500 70.0 Trifluoroacetic acid/dichloromethane 150.0 Wu et al. [83]
Freeze Casting 22,000 80.0 Lactic acid 103.0 Wang and Wakisaka [68]
Electrospinning 102,000 – Acetic acid 85.0 Lemma et al. [84]
Electrospinning 100,000 95.0 Trifluoroacetic acid/dichloromethane 86.0 Li et al. [85]

Ohkawa et al. [76] reported that the successful spinning of the chito-
san in trifluoroacetic acid (TFA) solution was due to the formation of
salts between amino groups and TFA over chitosan chains. Geng et al.
[77] pointed out that the enhancement of acetic acid concentration re-
duced the surface tension of the chitosan solution, which was highly im-
portant in the chitosan spinning. Terada et al. [81] studied the effect of
chitosan solution concentration and negatives voltages on nanofibers
development, and their results indicated that the electrostatic repulsion
along the chitosan chains could have decreased the solution viscosity.
Haider et al. [82] prepared chitosan solutions using a sonicator bath at
55 °C for 90 min; although the effect of the molecular weight was not re-
ported, the cavitation caused by ultrasound waves might have provoked
chain scission and, consequently, reduction in the molecular weight, as
well as in the viscosity. Lemma et al. [84] were able to produce pure chi-
tosan nanofibers with two steps; first, polyethylene oxide (PEO) was
added into chitosan solution, and after the nanofiber synthesis, PEO
was removed.
From Table 1 is possible to verify that, even though the molecular
weight and deacetylation degree are the main characteristics of chito-
san, there is a lack of pattern about these parameters range, which re-
veals the complexity of chitosan spinnability. This fact can be
associated to the different sources of raw materials, in addition to the
different reactions that can be used on chitosan production. All these
variabilities have an influence directly the chitosan characteristics and,
consequently, on the synthesis of chitosan nanofibers. For this reason,
a further deepening of chitosan characteristics could improve the repro-
ducibility of nanofibers production.
Some authors have used the Berry number, Be (Eq. (1)) to estimate
the minimum concentration, c, needed to obtain the chain entangle-
ments [86–88]. This concentration represents the transition between
the dilute to the semi-dilute regimes.
Be ¼ c ½η
where c is the solution minimum concentration (g mL−1) and η is the
intrinsic viscosity (mL g−1).
Terada et al. [81] estimated that the Berry number for the chitosan
solution used in their work, as well as in other successful attempts,
ranged from 13 to 23. This parameter can provide a starting point to
evaluate the influence of chitosan molecular weight on nanofibers syn-
thesis, which is related to concentration and intrinsic viscosity. Indeed,
the Berry number is based on Huggins equation (Eq. (2)) [89].


ηSP ¼ ½ηc þ kH ½η2 c2 þ …
where ηSP is the specific viscosity (dimensionless), [η] is the solution in-
trinsic viscosity (mL g−1), c is the polymer solution concentration
(g mL−1) and kH is the Huggins coefficient (dimensionless).
According to Huggins equation, the solution viscosity is directly de-
pendent on polymer concentration. However, this statement is only
valid for the dilute regime whereby, at low concentrations, the polymer
chains are far from each other and, consequently, the hydrodynamic in-
teractions among them could be neglected. It is known that the nanofi-
ber development occurs by chain entanglement, but at this condition,
the effect of chains interaction becomes significant. Therefore, the solu-
tion viscosity is also influenced by entanglement and excluded volume
effects [89,90]. For this reason, other approaches can be more appropri-
ated for further study about the effects of viscosity and molecular
weight on chitosan solution parameters.
Graessley [91] found five viscoelastic regimes for polymeric solu-
tions in function of concentration and molecular weight, which were
the dilute, semi-dilute not entangled, semi-dilute-entangled, concen-
trated not entangled and concentrated entangled (Fig. 6). While re-
gimes without entanglement do not have the necessary overlap of the
chain, the concentrated entanglement regime may avoid the formation
of solution jet during spinning. Therefore, the boundary concentration
between semi-dilute and concentrated regimes, c+, can be obtained
by Eqs. (3) and (4).
63=2 M w
cþ ¼ D E3=2 ð3Þ
8Na R2
 
D E 2Mw 2
R2 ¼ a2 C∞ l ð4Þ
Mo
where Mw is the polymer average molecular weight (g mol−1), Mo is the
molecular weight correspondent to the chain which is overlapped
(g mol−1), Na is the Avogadro's number (mol−1), 〈R2〉 is the mean
square end-to-end distance of the chain (m2), a is the Flory expansion
factor (dimensionless), C∞ is the characteristic ratio (dimensionless)
and l is the bond length (m).
ð1Þ Considering that intrinsic viscosities [η] values are more feasible to
obtain than chain dimensions and that chitosan can be considered a lin-
ear chain (ϕ approx. 2.5 × 1023 mol−1), it is possible to use the Fox-Flory
equation (Eq. (5)).
D E3=2
R2
½η ¼ ϕ ð5Þ
Mw
where ϕ is the Flory constant (mol−1).
Using Eq. (5) to rewrite Eq. (3), it results in Eq. (6).
ð2Þ 0:77
cþ ¼ ð6Þ
½η 
The intrinsic viscosity [η], related to the boundary between semi-
dilute regime and concentrated regime can be determined using the
Flory expansion factor, a (Eqs. (7), (8) and (9)) [92–94]. When the solu-
tion approaches the c+, the Flory expansion factor achieves unity. From
 B.S. de Farias et al. / International Journal of Biological Macromolecules 123 (2019) 210–220
Fig. 6. Viscoelastic regimes for polymeric solutions in function of concentration and molecular weight.
the plot of [η]/Mw1/2 versus Mw1/2 is possible to obtain the parameters B
and Kθ, and then obtain [η]θ and a from Eqs. (8) and (9).
½η 
¼ K θ þ 0:51BðMw Þ
Mw 1=2
½ηθ ¼ K θ M1=2
w
½η
a3 ¼
½ηθ
where Kθ is the unperturbed parameter and [η]θ is the intrinsic viscosity
(mL g−1) at θ state.
Dobrynin et al. [95] studied both entanglement and not entangle-
ment regimes for polyelectrolytes solutions, which could be applied to
chitosan solution, since chitosan behaviors as a cationic polyelectrolyte
in acid medium. From the specific viscosity, ηSP (Eq. (10)), they plotted
the specific viscosity versus concentration. The authors identified two
slopes increased at the transition concentration between the semi-
dilute not entanglement to the semi-dilute entanglement regimes and,
also, at the transition concentration between the semi-dilute entangle-
ment regime to the concentrated regime.
 
ηo −ηs
ηSP ¼
ηs
where ηo is the kinematic viscosity of the polymer solution (m2 s−1) and
ηs is the kinematic viscosity of the solvent (m2 s−1).
The viscoelastic property associated with operating parameters also
play an important role in beaded fiber formation. Indeed, this defect is
caused by the jet break-up into droplets, which occurs due to an insta-
bility provoked by the action of surface tension into the fluid through
a circular cross-section. Considering that chitosan solution is a Newto-
nian fluid, the Rayleigh instability is the major issue related to jet
break-up into beaded fibers. For instance, the competition between vis-
cous and surface tension forces can be quantified by the ratio of Weber
number (We) (dimensionless) (Eq. (11)) to Reynolds number (Re) (di-
mensionless) (Eq. (12)), which is defined as the capillary number (Ca)
(dimensionless) (Eq. (13)) [58,96]. Therefore, Ca must be above a criti-
cal value to avoid Rayleigh instability and beaded nanofiber.
ρv2 D
We ¼
γ increased Young's modulus and tensile strength, which improved the
215
ρvD
Re ¼ ð12Þ
μ
μv
ð7Þ Ca ¼ ð13Þ
γ
ð8Þ where μ is the dynamic viscosity of polymer solution (N s m−2), ρ is the
density of polymer solution (kg m−3), γ is the surface tension of poly-
mer solution (N m−1) v is the polymer jet speed (m s−1), D is the spin-
ð9Þ neret diameter (m).
Besides the viscosity and molecular weight effects of chitosan solu-
tion on nanofiber production, the process of chitosan production may
also affect the molecular weight distribution [97]. For this reason, the in-
fluence of chitosan polydispersity index (PDI) on nanofiber synthesis
could also be an interesting parameter to be evaluated.
Chitosan is considered a semi-crystalline biopolymer, which is con-
stituted with an amorphous portion and a crystalline portion. However,
chitin can present distinct crystalline structure forms depending on the
different sources of raw material: α-chitin, in anti-parallel arrangement,
β-chitin, in parallel arrangement, and ϒ-chitin, in mixed arrangement
[31,98]. Moreover, the different types of chitin associated to the differ-
ent reactions conditions may also influence the diffusion of water
through the biopolymer structure during the chitosan production,
which may result in various deacetylation degrees, as well as crystallin-
ð10Þ ity degrees. In fact, the crystallinity degree has been suggested to be di-
rectly proportional to the molecular orientation. Furthermore, during
the spinning process, the jet undergoes a bending instability, which
leads to a molecular orientation through the polymeric chains and re-
sults in the nanofiber [99–101]. Therefore, it is reasonable to argue
that further research could be carried out to evaluate whether the ex-
tension of chitosan crystallinity impacts nanofibers synthesis.
5. Composite and nanofibers blends
As discussed previously, chitosan solution spinning has been consid-
ered a challenge, and for this reason, several research groups have been
developed chitosan blends and composites with other compounds, in
attempt to improve the solution properties for the spinning process,
as well as some physicochemical properties. Table 2 shows some at-
tempts at nanofiber formation from chitosan blends and composites
[63,102–115].
Karimi et al. [109] developed poly(3 hydroxybutyrate) (PHB)/chito-
san electrospun nanofiber, and they reported that the presence of PHB
ð11Þ
216 B.S. de Farias et al. / International Journal of Biological Macromolecules 123 (2019) 210–220

Table 2
Summary of attempts at nanofiber formation from chitosan blends and composites.

Blend/composite system Chitosan MW (g Chitosan DD Method Solvent type Refs.

mol−1) (%)

- Chitosan/sericin – – Electrospinning Trifluoroacetic acid Zhao et al. [101]

- Chitosan/polyethylene oxide/silica 200,000 75.0 Electrospinning Acetic acid Toskas et al. [102]
- Chitosan/silk fibroin – 98.0 Electrospinning Trifluoroacetic acid/dichlomethane Lai et al. [103]
- Chitosan/polyamide-6 150,000 85.0 Forcespinning® Formic acid Dotto et al. [63]
- Chitosan/poly(ε-caprolactone) 80,000 80.0–90.0 Electrospinning Formic acid/acetic acid Khan et al. [104]
- Chitosan/gelatin – – Electrospinning Formic acid/acetic acid Telebian and Mansourian [105]
- Chitosan/polyethylene oxide/ɛ-polylysine 100,000–300,000 – Electrospinning Acetic acid Lin et al. [106]
- Chitosan/poly(lactic acid) 200,000 85.0 Electrospinning Formic acid Samadi et al. [107]
- Chitosan/poly(3-hydroxybutyrate) – 75.0–85.0 Electrospinning Trifluoroacetic acid Karimi et al. [108]
- Chitosan/polyvinyl alcohol – 85.0 Electrospinning Acetic acid Das et al. [109]
- Chitosan/chitin 120,000 92.0 Electrospinning Acetic acid Dobrovolskaya et al. [110]
- Chitosan/poly (ethylene oxide) 1,000,000 – Electrospinning Acetic acid Darbasizadeh et al. [111]
- Chitosan/polyethylene oxide/lauric arginate 150,000–300,000 95.0 Electrospinning Acetic acid Deng et al. [112]
- Chitosan/cobalt ferrite/titanium oxide 200,000 – Electrospinning Trifluoroacetic acid/dichlomethane Radmansouri et al. [113]
- Chitosan/xanthan gum 28,000 89.0 Electrospinning Formic acid Shekarforoush et al. [114]

mechanical properties. Dobrovolskaya et al. [111] found that the addi-
tion of chitin nanofibrils into chitosan solution increased the solution
spinnability. The interaction between chitosan molecules and chitin
nanofibrils resulted in the formation of cluster structures, thus making
the molecules become oriented under shear stresses. Darbasizadeh
et al. [112] indicated that the addition of poly (ethylene oxide) (PEO)
into chitosan solution enhanced chain entanglement and solution
spinnability, due to hydrogen bonds formed between polyether oxygen
from PEO and amine group from chitosan.
Therefore, these studies also reveal the importance of molecular ori-
entation and entanglement through the polymeric chains. Indeed, it is
also reasonable to suggest that the interaction of chitosan functional
groups with other crystalline structures could make the solution
spinnability more feasible.
crucial to consider some basic characterization, such as morphological,
structural, physicochemical, thermal and mechanical. Table 3 shows
some usual methods for the characterization of chitosan-based nanofi-
bers [116–123].
7. Chitosan-based nanofibers applied in food technology
Chitosan stands out due to its polycationic character, biodegradabil-
ity, nontoxicity, antimicrobial, chelating, mucoadhesive and gelling
properties. All these properties associated to the large surface area:vol-
ume ratio of nanofibrous structures makes chitosan-based nanofibers
useful for a variety of applications in food technology, which have not
been fully explored. Thus, the next few sessions point out some plausi-
ble studies in this field.

6. Characterization of chitosan-based nanofibers 7.1. Chitosan-based nanofibers for active food packaging

The characterization of chitosan-based nanofibers plays an impor-
tant role in providing scientific knowledge about the effect of chitosan,
as well as the interaction between chitosan and other compounds on
nanofiber quality. Moreover, in order to evaluate whether the
chitosan-based nanofibers are suitable for a particular application is
Food wastage is a challenge for the food industry. According to the
Food and Agriculture Organization of the United Nations [124], one-
third of food in the world is wasted, which directly impacts in economic
and environmental issues. In fact, the food packaging has emerged as an
alternative to improve quality and safety for food products by

Table 3
Main methods for characterization of chitosan-based nanofibers.

Characterization type Analytical instruments Main function Refs.

Morphological and Optical microscopy - Preliminary analysis. Širc et al. [116]

structural Scanning Electron Microscopy (SEM) - Fiber diameter determination; Širc et al. [116]
- Fiber homogeneity evaluation;
- Defects detection.
Transmission Electron Microscopy (TEM) - Internal structure evaluation; Shi et al. [117]
- Characterization of core-shell structure.
Physicochemical Atomic Force Microscopy (ATF) - Evaluation of atomic structure and intermolecular interactions; Leite et al. [118]
- Determination of surface and adhesion forces.
Energy Dispersive Spectroscopy X-ray (EDS or - Elemental analysis. Asmatulu and Khan
EDAX) [119]
X-ray diffraction (XRD) - Provide cell dimensions; Murthy and Minor
- Determination of crystallinity degree; [120]
- Verification of amorphous and/or crystalline regions.
Raman spectroscopy - Determination of molecular and crystal symmetry; Asmatulu and Khan
- Evaluation of bond strengths; [119]
- Verification of structural disorders and bond defects.
Fourier Transform Infrared Spectroscopy (FTIR) - Functional groups determination; Zhu et al. [121]
- Verification of chemical interaction.
Thermal Differential Scanning Calorimetry (DSC) - Evaluation of glass transition and crystallization and melting Andrady [122]
process;
- Determination of heat capacity.
Thermogravimetric analysis (TGA) - Verification of residual solvent; Shi et al. [117]
- Evaluation of thermal stability.
Mechanical Texture analysis - Measure of tensile strength, elongation and Young's modulus. Ko and Wan [123]
 B.S. de Farias et al. / International Journal of Biological Macromolecules 123 (2019) 210–220
enhancing their biological, physical and chemical stability. In order to
accomplish these properties is necessary to avoid chemical contami-
nants, oxygen, microorganism, light and moisture. Thus, active packag-
ing with antimicrobial properties has attracted significant attention
[125–127]. Chitosan-based nanofibers have great potential to be applied
in active food packaging. Chitosan has been widely reported to be effec-
tive against fungi, bacteria and virus [128–133]. Moreover, chitosan as a
renewable and biodegradable polymer becomes an interesting really
option in the development of green packaging materials as an alterna-
tive to the petroleum-based polymers.
Arkoun et al. [134] prepared chitosan/poly(ethylene oxide) (PEO)
and investigated the antibacterial action against Esherichia coli, Salmo-
nella enterica serovas Typhimurium, Staphylococcus aureus and Listeria
innocua, which are pathogenic microorganism related to food contami-
nation. The authors reported that the nanofibers had an irreversible an-
tibacterial effect, which led to a bactericidal instead of bacteriostatic
mechanism. Moreover, the bacterial growth was reduced at pH 5.8,
below pKa of amine groups on chitosan, and for this reason, the authors
suggested that the nanofibers could be applied to foods such as yogurt,
milk, cheese, meat and fish, which the lactic acid is liberated through the
storage period.
Cui et al. [135] developed chitosan/poly(ethylene oxide) loaded
with tea tree oil and applied against some food pathogenic microorgan-
isms, Salmonella enteric subsp. Enteric serovar Enteritidis and Salmonella
typhimurium. The tensile strength increased around 350% when the
concentration of tea tree liposome was enhanced to 50%. The addition
of tea tree liposomes also improved the antibacterial effect of the chito-
san/poly(ethylene oxide) nanofiber. Furthermore, the chitosan/poly
(ethylene oxide) loaded with liposomes tea tree demonstrated a stable
antibiofilm activity into chicken meat samples during four days.
Ceylan et al. [136] developed electrospun chitosan/thymol/liquid
smoke nanofibers to avoid microbial spoilage of fish fillets. The
electrospun chitosan/thymol/liquid smoke nanofibers were efficient to
reduce almost 60% of total mesophilic bacteria. Moreover, the authors
also indicated that the nanofibers were thermostable until 150 °C,
which is within the temperature range used in conventional methods
of food preservation.
The food packaging field is an emerging topic, which will probably
grow over the upcoming years. There are still many opportunities to fur-
ther research the role of chitosan-based nanofibers in other pathogenic
microorganisms, as well as in different foods types. In addition, the ef-
fect of other biopolymers and bioactive compounds on food packaging
properties can also be evaluated, such as the mechanical properties
and water vapor permeability, for example. The chitosan-based nanofi-
bers may also arise as nanosensors to control food quality, by monitor-
ing external and internal conditions.
7.2. Chitosan-based nanofibers for nanofood carrier
Bioactive compounds, volatile molecules, antioxidants and flavors
are usually unstable or even susceptible to degradation [137,138].
Thus, chitosan-based nanofibers could enhance the stability of func-
tional food compounds, besides to increase their bioavailability. Simi-
larly, to drug delivery, the mucoadhesive property of chitosan could
be used for the delivery of bioactive molecules to the body, whereby
the mucoadhesion of the functional compounds increases their adsorp-
tion through gastrointestinal tract [139,140]. All these properties, in ad-
dition to the non-toxic and biocompatibility advantages of chitosan-
based nanofibers, have been raised the interest for the application on
drug delivery carrier [108,112,141,142]. Although some studies have
been done applying different nanomaterials as food carriers
[143–145], chitosan-based nanofibers have not been adequately ex-
plored as food carrier yet.
Shekarforoush et al. [115] produced electrospun chitosan/xanthan
gum nanofiber as a delivery carrier for curcumin. The authors found
that the release of curcumin from nanofiber at pH 2.2 was lower than
217
other pH studied (6.5 and 7.4). It was suggested that at pH below pKa
of amine groups on chitosan, such as pH at 2.2, there were stronger elec-
trostatic interactions between chitosan and xanthan, which reduced the
swelling behavior of nanofiber and, consequently, the diffusion of
curcumin. Therefore, the electrospun chitosan/xanthan gum was able
to be used as a carrier for curcumin, which enhanced its stability and
bioavailability.
Chitosan-based nanofibers still have potential to be applied like food
carrier such as the several bioactive compounds, β-carotene, anthocya-
nin, probiotic bacteria, procyanidin, lignans, stilbenes, ascorbic acids,
quercetin, cinnamic acid, astaxanthin, limonene among many others.
Future research should be devoted to the development of chitosan-
based nanofiber as bioactive compounds carrier.
7.3. Chitosan-based nanofibers for enzyme immobilization
Enzymes are biocatalysts that play an important role in the food in-
dustry. But their green chemistry and substrate specificity are hindered
by their low operational stability and shelf storage life; in addition to
difficult recovery and reuse [146,147]. However, the enzyme immobili-
zation on chitosan-based nanofibers have potential to overcome these
problems. In fact, they stand out as promising supports due to their
high pore-interconnectivity, which enhances mass transfer between
substrate and enzyme and, consequently, the enzyme activity. The
surface-functionalized by chitosan's functional groups could be used
to adsorb enzymes with controlled-release.
Haghju et al. [148] developed β-D-galactosidase immobilized chito-
san/poly(vinyl alcohol) nanofibers to hydrolyze lactose. The authors
reported that the immobilized enzyme was more thermostable than
free enzyme at 50 °C. Moreover, the immobilized β-D-galactosidase
also kept 77% and 42% of its activity after 28 days of storage at 4 °C
and 25 °C, respectively. Therefore, the immobilization of β-D-
galactosidase into chitosan/poly(vinyl alcohol) nanofibers improved
thermal and storage stability.
Chitosan-based nanofibers have been emerged for enzyme immobi-
lization. Indeed, they are promising support materials to be used in the
development of hypoallergenic foods, as well as several dairy products
(baking, jam, jellies, wine, beer and juices). Therefore, substantial effort
should be devoted to the application of chitosan-based nanofibers on
immobilization of enzymes in food processing, such as amylase, trypsin,
pectinase, protease, tyrosinase, lipase, pectin lyase, pectin, laccase,
among many others.
8. Final remarks
Chitosan-based materials have many properties, such as polycationic
character, biodegradability, nontoxicity, mucoadhesion and antimicro-
bial activity. However, chitosan has the disadvantage to possess a
low specific surface area, which could compromise some of its specific
sites. Hence, chitosan-based nanofibers stands out as a promising
nanobiomaterial in the food industry, due to its large ratio of surface
area:volume and flexibility in surface functionalities. For this reason,
this review focused on the effect of chitosan characteristics on nanofibers
development. In addition, several strategies in nanofibers technology
were discussed, and this review highlighted which techniques could be
more suitable to synthesize chitosan nanofibers. Moreover, some trends
and future challenges were summarized, which should be directed to-
wards the application of chitosan-based nanofibers into active food
packaging, nanofood carrier and enzyme immobilization.
Acknowledgements
This study was financed in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance
Code 001, and the Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) - Brazil.
218 B.S. de Farias et al. / International Journal of Biological Macromolecules 123 (2019) 210–220
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