Chitosan

Anabelle Camarotti de Lima Batista, Weslley de Souza Paiva, and

Francisco Ernesto de Souza Neto

Contents
1 Fungi Chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1 Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2 Extraction and Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3 Purification and Production Advanced Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2 Biochemical and Biological Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.1 Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.2 Biological Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3 Biotechnology Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3.1 Applications and Uses in the Pharmaceutical Industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.2 Applications and Uses in Biomedical Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Abstract
Chitosan has inspired several researchers around the world due to its use versa-
tility, mainly due to the presence of the amine group in its structure. Initially, the
research used animal chitosan because it is cheaper to obtain. However, research
groups and companies have been investigating the advantages of using fungal
chitosan. Its production can be sustainable since it can use residues from the
agricultural sector or industry as a culture medium. There are several applications,
especially in the biomedical area, due to the standardization of production and
side effects reduction, particularly allergens and immune system sensitizers.

A. C. de Lima Batista (*)

Agriculture Department, CCHSA, Federal University of Paraiba, Bananeiras, PB, Brazil
PROFBIO, CCEN, Federal University of Paraiba, João Pessoa, PB, Brazil
W. de Souza Paiva
RENORBIO, Federal University of Rio Grande do Norte, Natal, RN, Brazil
F. E. de Souza Neto
Nova Esperança, College of Mossoro, Mossoró, RN, Brazil

© Springer Nature Switzerland AG 2021 1

J. Oliveira et al. (eds.), Polysaccharides of Microbial Origin,
https://doi.org/10.1007/978-3-030-35734-4_14-1
2 A. C. de Lima Batista et al.

Considering the advantages and descriptions of successful applications men-

tioned here, we hope that new research groups will be interested in working
with fungal chitosan for the biomedical area, among others.

Keywords
Fungal chitosan · Biotechnology apply · Zygomycetes · Deacetylation degree ·
Aspergillus niger

1 Fungi Chitosan

Among the microbial polysaccharides previously described, fungal chitosan also

appears as a biotechnological product with great potential for commercialization and
profit in the international market. Its physicochemical and biological characteristics
give it an advantage in terms of the different possibilities of application and
conjugation with other molecules, which can be natural or synthetic. Its use in
synergy also favors its use in biomedical applications and brings advantages com-
pared to chitosan produced from animal chitin.
Thus, considering its importance, this chapter will explain the production, extrac-
tion, and purification of fungal chitosan, some of its physical-chemical and biolog-
ical characteristics, and its application in areas of biotechnology directly or indirectly
related to regenerative medicine.

1.1 Production

Fungi have a cell wall composed mainly of a network of interconnected molecules

consisting of proteins, glucans, chitin, chitosan, lipids, and polyphosphates that may
have their quantity and quality altered due to the environmental conditions and
intrinsic characteristics of their species (Campos-Takaki et al. 1983; Paiva et al.
2021). Among these compounds, chitosan stands out for being associated with
increased wall integrity, favoring protection against high temperatures, and cell
inhibitors to which the fungal strain may be subjected (Baker et al. 2007). The
high degree of deacetylation in chitosan’s cell wall interferes with its flexibility, and
the low degree interferes with its solubility (Franca et al. 2011).
Chitosan production in fungi is linked to the amount of chitin and the action of the
enzyme chitin deacetylase (EC 3.5.1.41) on nascent or established chitin residues
directly on the cell wall (Fig. 1) (Campos-Takaki et al. 1983; Fesel and Zuccaro
2016). This enzymatic synthesis of chitosan was first described by Araki and Ito
(1974) in Mucor rouxii (Zygomycetes), demonstrating that about 14% of chitin
deacetylase is associated with particles from cellular fractions, 49% is associated
with a soluble fraction of the supernatant, and about 37% is extracellular (Araki and
Ito 1974). Complementing the chitosan synthesis route, Davis and Bartnicki-Garcia
(1984) found strong evidence that chitin deacetylase activity is highly efficient in
Chitosan 3

Fig. 1 Schematic overview of fungal cell wall composition. Chitin is represented by brown lines
located close to the cell membrane. Chitin is synthesized by transferring N-acetylglucosamine
residues from uridine diphosphate-N-acetylglucosamine (UDPGlcNAc; brown hexagon) to a grow-
ing fiber that is shuttled through the cell membrane by the transmembrane chitin synthase (light
blue). In this preformed chitin, there is a little incidence of chitin deacetylase action to form
chitosan. (Reprinted from Figure 1, Fesel PH, Zuccaro A. β-glucan: Crucial component of the
fungal cell wall and elusive MAMP in plants. Fungal Genet Biol 90:53–60, 2016, with permission
from Elsevier. Licensed by CC BY-NC-ND (https://creativecommons.org/licenses/by-nc-nd/4.0/)).
Licensed by CC BY-NC-ND (https://creativecommons.org/licenses/by-nc-nd/4.0/))

residues of a growing chitin chain, and it shows low action on preformed chitin
molecules. These findings suggest that changes in the culture medium in which fungi
grow can directly influence chitosan production (Batista et al. 2020; Yuan et al.
2021). The literature also explains that in all cases, the optimum temperature for
enzyme activity is 50
C while optimum pH varies from 4.5 to 8.5 (Jeraj et al. 2006).
At the moment, Mucorales is the most representative and studied order as it
contains most species that produce chitosan for commercial application, especially
those belonging to the genus Mucor and Rhizopus. This commercial interest is since
significant levels of beta-D-Glucan cannot be detected on their cell wall, the ease of
growth of these organisms in submerged systems, and their ability to use different
substrates as a source of carbon and nitrogen. Other orders have been reported and
described as chitosan producers (Table 1); however, some accumulate beta-D-Glu-
can, for example, Aspergillus sp. and Agaricus sp. The presence of beta-D-glucan
makes the purification process more expensive for subsequent application in the
biomedical field (Fesel and Zuccaro 2016).
The production based on the cultivation conditions described in Table 1 is guided
by the chitosan synthesis route in fungi, more precisely, in the enzymes related to its
4 A. C. de Lima Batista et al.

Table 1 Chitosan production by species from different orders depends on the cultivation condi-
tions. These conditions could interfere directly with yield and quality of the chitosan produced
Cultivation Physical-chemical
Species conditions characteristic References
Syncephalastrum Corn steep DD 88.14%; Low Batista
sp. (Mucorales) liquor molecular weight et al. (2020)
Rhizopus stolonifer YPD DD 84% Paiva et al.
(Mucorales) (2017)
Aspergillus niger (Eurotiales) Not described Not described Kitozyme ®
Agaricus bisporus (Agaricales) Not described DD  90%; low molecular ChiBio ®
and Aspergillus niger weight (for both)
(Eurotiales)
YPD media (Yeast Extract 10 g; Peptone 20 g; Dextrose 20 g per liter) (Davis and Bartinicki-Garcia
1984); Kitozyme (https://www.kitozyme.com/en/); ChiBio (https://www.chibiotech.com/)

Table 2 Substrates were directly influencing in enzyme action of chitin synthase and chitin
deacetylase extracted from different fungi. In all cases, they positively interfere. Other authors
checked the positive influence of substrates in chitosan production in vivo and reported that it
depends on the concentration and the fungus species
Influence Substrate Reference
In chitin Zn2+; Mn2+; Co2+; Ca2 Jaworska and Konieczena (2001)
deacetylase
In chitin synthase Trypsin; Mn2+; Co2+; Shehata et al. (2018)
Mg2+
On chitosa Co2+; Chitin; Fe2+ Jaworska and Konieczena (2001), Shehata et al.
production (2018)

production. Although the description of these enzymes occurred almost 50 years

ago, it was only in the last decade that the substrates influencing their action were
described. Jaworska and Konieczena (2001), among others, mainly report the
influence of ions on the synthase and deacetylase chitin enzyme action, with caveats
for the concentrations to be added so that there is no decrease in chitosan production
(Table 2). In vivo analysis of the influence of these substrates on chitosan production
showed that Co2+ and chitin depend on the concentration present in the culture
medium and the fungal species (Jaworska and Konieczena 2001; Batista et al. 2014).
These ions have also been described as important for manipulating the physico-
chemical properties of chitosan (Jaworska and Konieczena 2001; Shehata et al.
2018).

1.2 Extraction and Isolation

Although some authors report that there is a process of extracting chitosan from
crustaceans and insects (Mohan et al. 2020; Rasweefali et al. 2021), its nomenclature
is not entirely correct since extraction processes occur when the compound is taken
Chitosan 5

directly from the source. Here, there is only chitin extraction, a polymer present in
these organisms, crustaceans, and insects, in the approximate proportions of 20%–
30% and 10%–15%, respectively (Spranghers et al. 2017).
The extraction process occurs from the fungi cell wall, where chitosan is formed
from the enzymatic deacetylation of chitin residues. In these organisms, the structure
of chitosan was first described in 1954 by Kreger using the X-ray technique. At that
time, they analyzed the cell wall of the yeast Phycomyces blakesleeanus (Kreger
1954). In the nineteenth century, scientists White et al. (1979) developed a method-
ology that allowed chitosan isolation from fungal mycelium. Inspired by this study,
several scientists started developing adaptations to improve the efficiency of this
process (Synowiecki and Ali-Khateeb 1997; Hu et al. 1999). Among these, the
scientist Hu et al. (1999), at the end of the twentieth century, developed an extraction
protocol that is currently widely used in experiments with fungal chitosan. This
protocol describes each stage to be performed from the cultivation period to specific
pH values, essential data for successful extraction. Modifications in this protocol
have been made to adapt the process to each strain, to the culture medium, and
general conditions of the laboratory (Yuan et al. 2021; Kitozyme ®; ChiBio ®). By
using the most specific extraction process for each situation, researchers can obtain a
higher yield of fungal chitosan, as well as lower production costs. In this way, they
intend to compete commercially with the chitosan obtained from the shell of
crustaceans (Ghormade et al. 2017).
Despite the amount of chitosan extracted from the cell wall of fungi, it is difficult
to exceed 20% of dry biomass, profitability considered economically viable for some
companies in different countries (Kitozyme ®; ChiBio ®; InvivoGen ®; Inbiose ®).
For companies, production costs versus application area have been advantageous,
especially when the application area is biomedical. For this field, the chitosan
produced needs to be in a high degree of purification, and the physicochemical
and biological properties must be as stable as possible for the production line to be
continuous. Another factor to be analyzed is the production of chemical residues
generated when producing fungal chitosan and producing animal chitosan. The
fungal chitosan production is considered ecologically friendly (Ghormade et al.
2017; Batista et al. 2020; Paiva et al. 2021).

1.3 Purification and Production Advanced Processes

Purification of fungal chitosan occurs, in particular, for separating residual proteins,

lipids, and glucans from the matrix, and the literature frequently focuses on remov-
ing glucan residues from this matrix. These methodologies are mainly used when
producing chitosan from strains not belonging to the Zygomycetes class (see Sect.
1.1), as is the case for Aspergillus sp. (Heux et al. 2000).
Chitosan obtained from Zygomycetes strains, in general, have proteins and lipids
removed during the process of extraction by an alkali solution, and from this
substrate, modifications are made to increase the yield. Naghdi et al. (2014) suggest
using 0.1 N H2SO4 at 25
C/30 min to remove phosphate groups before
6 A. C. de Lima Batista et al.

precipitating the purified chitosan from Rhizopus oryzae. For the chitosan obtained
from A. niger, the suggested purification requires washing with NaOH solutions
(solution 1 - 0.1 M; solution 2 - 0.5 M) and chloroform/methanol (2/1 v / v) (Heux
et al. 2000).
Nevertheless, regardless of the purification treatment conducted while extracting
the fungal chitosan, there is a suggestion for checking the purity of the produced
chitosan, where the chitosan powder is solubilized into a solution of sulfuric acid and
sodium nitrite. In this solution, chitosan is converted into 2,5-Anhydro-D-mannose
and gaseous nitrogen. The percentage of conversion is an indication of the purity of
the fungal chitosan (Mohammadi et al. 2012).

2 Biochemical and Biological Characteristics

Chitosan is the usual and widely studied deacetylated form of chitin, which has three
distinct polymorphs. These polymorphs have been described in different species and
could appear without a standard. Cabib et al. (1988) found γ-chitin while Ifuku et al.
(2011) found α-chitin in the fungi cell wall. These authors described these poly-
morphs in different fungi species, and the fungi are capable of producing chitosan on
their walls in all polymorphs.
Structurally, chitosan is a polysaccharide heteropolymer formed from the
deacetylation of some glucopyranose residues present in the chitin chain (Annu
et al. 2017). It has 2-acetamido-2-deoxy-D-glucopyranose and 2-amino-2-deoxy-D-
glucopyranose units linked by β- (Ahmed et al. 2018; Almutairi et al. 2020;
Alshubaily 2019; Alshubaily and Al-Zahrani 2019) linkage. However, this molecule
is only considered chitosan when more than 50% of the polymer residues are present
in the form of 2-amino-2-deoxy-D-glucopyranose (Campos-Takaki 2005; Yuan et al.
2021). It is important to mention that the 50% proportion of the number of amino
residues in the chain is still controversial for some authors, who prefer to consider
chitosan only proportions above 75% (Li et al. 1997).
As a result of this structure, chitosan has physical, chemical, and biological
properties that define it as an advantageous polymer for the most diverse biotech-
nological applications.

2.1 Physicochemical Properties

Chitosan can be evaluated from different physical and chemical properties; however,
the most relevant are those that best define its biotechnological application. The
degree of deacetylation, molecular weight, crystallinity, thermogravimetry, and
solubility are described and characterized below.

2.1.1 Degree of Deacetylation

The determination of the degree of deacetylation (DD) can be performed using
different techniques, including infrared spectroscopy (FIT-IR) and proton nuclear
Chitosan 7

magnetic resonance (H1 NMR) (Brugnerotto et al. 2001; Martínez-Camacho et al.

2010). Its measurement is essential because its value will interfere with the chemical
bonds between chitosan and different adjuvants and other compounds that direct it to
the area to be applied, in addition to interfering with other chemical properties
(Franca et al. 2011; Yuan et al. 2021). However, the DD value is highly influenced
by the analytical method used for its measurement (Khan et al. 2002). For this
reason, attention should be paid to the description of the method used when
determining the DD for commercial chitosan purposes. Among the mentioned
above, FIT-IR and H1 NMR are the most applied techniques.
The Fourier-transform infrared spectroscopy (FT-IR) analysis is used to obtain
the degree of deacetylation as a function of the applied equation (Table 3). For this
technique, different researchers propose equations according to the approximate rate
of amide I (A1655 – A1630), amide II (A1560), hydroxyl groups (A3450 – A3430),
and presence of the groups OH, NH2, CO to A1320 (Dong et al. 2001; Khan
et al. 2002; Brugnerotto et al. 2001).
This technique has demonstrated high safety and low cost as advantages and does
not suffer interference from pH value or the degree of hydration of the sample.
However, changes in the accuracy of the results have been described when chitosan
from different chitin polymorphs was analyzed (Brugnerotto et al. 2001).
Another important technique for determining the degree of deacetylation is the
Hydrogen Nuclear Magnetic Resonance (H-NMR) which requires expensive equip-
ment, and the sample must be completely solubilized to avoid reading errors (Yang
and Montgomery 2000). However, similarly to FIT-IR, it needs a small amount of
sample without ultra-purification.
The H-NMR is indicated for chitosan characterization with a possible high degree
of deacetylation once it allows a more accurate analysis comparing to FT-IR or any
other technique (Dung et al. 1994). Its determination is based on NMR spectra
integrals, being the most common based on the H-1 liquid state spectrum. Several
equations have been described to calculate the DD, especially for the proton
spectrum in the liquid state of 1H-NMR (Table 4).
By using this technique, it is possible to analyze chitosan when solubilized in a
deuterated solvent, which can be D2O, when chitosan is soluble in water (Rinaudo
et al. 1992) or acid chemical agents when it is in its original composition:
CD3COOD (acetic acid deuterium) and DCl (chloride deuterium) (Dung et al.
1994); D2O/DCl (deuterium oxide/chloride deuterium) (Lavertu et al. 2003);
CD3OOD (Kumirska et al. 2010).

Table 3 Proposed equations for analyzing the degree of deacetylation of chitosan based on
Fourier-transform infrared spectroscopy (FTIR) analysis
Equation References
A1320/A1420 ¼ 0.3822 + 0.03133 DA Brugnerotto et al. (2001)
[(Abs1655/Abs3450) x 100]/1.33 Domszy and Roberts (1985)
A1560/A3430 ¼  0.0057 DD + 0.7375 Dong et al. (2001)
DD ¼ 100  [(A1655/A3450) x 115] Baxter et al. (1992)
8 A. C. de Lima Batista et al.

Table 4 The used equations to determine the degree of acetylation (DA) by analyzing the proton
spectrum 1H-NMR in the liquid state (Ahmed et al. 2018). ICH3(A) degree of intensity of the
presence of CH3 residues, and IH2-H6 (A + D), represents the sum of proton intensities from H2 to
H6; (Almutairi et al. 2020) IH1(D) represents the intensity of the deacetylated monomer, and ICH3
(A) the peak intensity of the three protons in the acetyl group. According to the author, this formula
is indicated for chitosan with a degree of deacetylation above 90%; (Alshubaily 2019) IH1
(D) represents the intensity of the deacetylated monomer and IH1(A) the intensity of the acetylated
monomers. According to the author, this equation is not indicated for chitosan with a high degree of
deacetylation, because the acetylated peak will not be visible in the spectrum
Equation Reference
1 %DA ¼ {[(1/3)  ICH3(A)]/[(1/6)  IH2  H6(A + D)]}  100 Hirai et al. (1991)
2 %DA ¼ 100 – {IH1(D)/[IH1(D) + (ICH3(A))/3]}  100} Lavertu et al. (2003)
3 %DA ¼ 100 – [IH1(D)/(IH1(D) + IH1(A))]  100} Lavertu et al. (2003)

Through the NMR-H1 spectrum, the characteristic signals for chitosan are
described in the anomeric region, with H-1 for glucosamine δ ~ 4.9 and H-1 for
N-acetylglucosamine δ ~ 4.6. The remaining protons of the H-2 ring of glucosamine
residues are shifted to lower values (δ ~ 3.2) because of the adjacent amino group.
The characteristic signal of protons in the N-acetyl group, from N-acetylglu-
cosamine, is at δ ~ 2.1 (Kumirska et al. 2010).
In addition to presenting varying degrees of deacetylation, chitosan molecules
can also be of different sizes, with oligosaccharides being the most used for
biomedical applications (Shehata et al. 2018; Luo et al. 2014; Liu et al. 2017).
Low weight chitosan has MW <50 kDa, medium weight has 50–150 kDa, and high
weight has >150 kDa (Tavaria et al. 2013).
In a production line, the size of the chitosan molecule can change according to
environmental characteristics to which the fungus is subjected (Batista et al. 2020).
Another possibility for obtaining chitosan with different molecular weights is by
applying physical, chemical, or enzymatic techniques to break the glycosidic bridges
that connect the N-glucosamine residues. This break will result in poligomers and
oligomers of different sizes. According to Benchamas et al. (2021), it is better to use
these techniques when chitosan has a degree of deacetylation above 80% (DD  80%).
For the profitable formation of these oligosaccharides, the used technique should
include environmental-friendly methods, low production cost, high degree of puri-
fication, and to be easily organizable in the production line at the industrial level
(Benchamas et al. 2021). Among the proposed techniques, the one that uses enzymes
has been the most adequate, but they have a high cost to be used on a large scale.
Similar to the degree of deacetylation, the molecular weight can be measured by
different techniques. Through viscometry analysis, the weight can be calculated
from the relation to the intrinsic viscosity. Using this technique, chitosan solutions
vary in concentration from 0.25 to 6.0 g/L (w/v) in a solvent formed by 0.3 M acetic
acid/0.2 M sodium acetate (Rinaudo et al. 1993). The formed solutions are passed
through Ubbelohde capillary viscometer in a water bath at 25
C, and the molecular
weight is determined according to the Mark-Houwink-Sakurada equation (Martínez-
Camacho et al. 2010; Rinaudo et al. 1993):
Chitosan 9

½η ¼ κMVa ð1Þ

In this equation, [η] is the intrinsic viscosity, MV is the viscosity-average molecular

weight, and “κ” and “a” are empirical constants that depend on the polymer, solvent,
and temperature. For that reference, the value of “κ” is 3.04  105 and “a” 1.26
(Rinaudo et al. 1993).
The crystallinity of chitosan molecules was first described by Clark and Smith
(1937) by measuring the structure of chitosan using X-ray diffraction and determin-
ing the density of electrons within the crystal. This density can be influenced by
intramolecular hydrogen bonding and hydrogen bonds in adjacent parallel chains
(Yui et al. 1994). The density is given as a percentage and measured using the
parameters λ ¼ 1542Ǻ, with range scanning of 4
–50
in intervals of 0.02
/min
(Batista et al. 2020).
According to the literature, this crystallinity varies slightly considering the total
percentage of deacetylated groups present in the molecules. These deacetylated
groups prevent the formation of intermolecular hydrogen bonds and break the lateral
packaging of the original chitin chains (Li et al. 1997). For this reason, the crystal-
linity of chitosan is a factor dependent on the original chitin polymorph (Yen and
Mau 2007; Kumirska et al. 2010) (Fig. 2).

1000

900

800

700
Intensity

600

500

400

300

200

100 20º
9º
0
10 20 30 40 50 60
2θ

Fig. 2 X-ray diffraction spectrum analysis of chitosan extracted from Cunninghamella elegans.
Diffractometer Shimadzu XRC6000, Kyoto, Japan, with copper tube (1 ¼ 1.54 Å). The voltage and
current used were 40KV and 30 mA, respectively. The measurements were performed in the range
of 3–50
C with a scanning rate of 1
C/minute in steps of 0.02
C. Here are marked characteristic
points of crystallinity for chitosan. (Paiva et al. 2021)
10 A. C. de Lima Batista et al.

Thermal gravimetric analysis (TGA) makes it possible to identify the changes that
heating can cause over time in the total mass of chitosan. By using TGA, it is
possible to establish the temperature range in which chitosan composition is con-
stant, the temperature at which starts to decompose, to follow the time of the
dehydration, oxidation, combustion, and decomposition reactions (Canevaloro Jr
2004). Complementing the analysis, Denari and Cavalheiro (2012) paid attention to
factors that can influence the response, such as intramolecular nature, thermal
conductivity, particle size, and reaction heat. The standards for the analysis vary
slightly depending on the manufacturer of the equipment to be used. Approximately
10 mg of the sample is used, with a temperature variation of 0
C–900
C, heating
rate of 10
C/min, and under a constant flow of dry nitrogen gas (Batista et al. 2020).
Chitosan has a pKa around 6.0–6.5, characterizing this polysaccharide with a
protonated amine group (–NH2) when the pH of the solution is below this value.
Chitosan is solubilized in acidic pH and this depends on physicochemical properties
such as molecule size, degree of deacetylation, and crystallinity (Chiappisi and
Gradzielski 2015). Another factor that interferes with solubility is the organization
by ordering acetylated and amine residues. The greater the quantity of the amine/
acetyl sequences, in that order, the greater the chain flexibility; while the higher
quantity of acetyl/acetyl, acetyl/amine, and amine/amine sequences higher the sta-
bility of the chain through the interchain hydrogen bridges (Skovstrup et al. 2010).

2.2 Biological Properties

Among the chitosan biological properties mentioned, low toxicity, high biodegrad-
ability, and biocompatibility are the ones that can be modified depending on the
chitosan molecular weight and degree of deacetylation (Wang et al. 2020). These
properties are common regardless of the origin of the chitosan (animal or fungal) but
each chitosan presents particularities. Animal chitosan has in its origin allergenic
proteins associated with its structure, for example, the tropomyosin. Because of this
associated molecule, a more expensive and careful purification of animal chitosan
should be performed before applied in the biomedical area (Ghormade et al. 2017).
Fungal chitosan also has particularities, such as the presence of carbohydrate
glucan in its wall structure; however, this molecule is not present in all fungal
species. This carbohydrate has been described as a cytokine expression stimulator,
among other molecules that activate the immune system in animals (Kozlowska et al.
2020). Also, when combined with chitosan, it can induce strong immune reactions.
Glucan is also related to fungal infections diagnostic because it stimulates the
immune system precisely (Jong et al. 2010).
The literature shows that glucan is present in species of the genus Aspergillus
sp. These have been widely used by biotechnological companies that sell fungal
chitosan and its derivatives (Heux et al. 2000; Kitozyme ®; ChiBio ®). However,
there are not significant amounts of glucan in the wall of species of the class
Chitosan 11

Zygomycetes (Ruiz-Herrera and Ortiz-Castellanos 2019), a widely used genus in

research in Brazil for the application of chitosan in different areas (Batista et al.
2018).

3 Biotechnology Applications

Chitosan has been widely used in the most diverse biotechnological applications, as
an example, in tissue engineering and the transport of medicines, due to its unique
characteristics (see Sect. 2).
These characteristics occur mainly because chitosan has a cationic group in
carbon 2 of its glucopyranose framework and because it can be of different sizes,
referencing the properties of deacetylation degree and molecular weight, respec-
tively. In the last decade, these two properties have been guiding some of the main
biotechnological applications in the biomedical area (Table 5).
In addition to the different products from both chitosan (animal and fungal), there
are several patents for applying these molecules in different areas of biotechnology.
This fact is promising for the consumer market. Although the patents of INPI-BR
(National Institute of Industrial Property), Espacenet (European Patent Office), and
WIPO (World Intellectual Property Organization) are mostly on animal chitosan, we
have noticed, in the last 5 years, an increase in the number of patent applications on
fungal chitosan, especially for the biomedical area
These applications in the biomedical area that follows the characterizations
described by Irastorza et al. (2021) as necessary for the polymer or composite
include low in vitro immune reaction (Sathiyaseelan et al. 2020), the generation of
nontoxic products (Islam et al. 2020); it can be sterilized without losing the charac-
teristics and be commercially viable. Observing these characteristics, companies that
produce fungal chitosan commercialize products for the most diverse biomedical
applications, such as hemostatic dressing, health supplements, excipient for
pharmaceutics products, chemotherapeutic agents, among others (Kitozyme ®;
ChiBio ®).
Below we describe some of the most common applications in the scientific
literature, in the last decade, considering only fungal chitosan.

Table 5 Some biotechnology applications of fungal chitosan in the last 5 years in biomedical areas
Application potential References
Antidiabetic; antioxidant; antibacterial; Sathiyaseelan et al. (2020), Alshubaily and
drug delivery system; antimycotic; Al-Zahrani (2019), Alshubaily (2019), El Rabey
antitumor et al. (2019), Chien et al. (2016), Almutairi et al.
(2020)
12 A. C. de Lima Batista et al.

3.1 Applications and Uses in the Pharmaceutical Industry

Studies in the pharmaceutical area are composed of highly challenging and expen-
sive processes because when reaching the final stages of clinical trials, most drugs
are unable to achieve favorable efficacy. This fact usually occurs due to the drug’s
inability to reach its target, leading to inadequate distribution of chemical formula-
tions and consequently to the appearance of many side effects (Islam et al. 2017). To
try to help with this problem, Yuan et al. (2021) initiated tests for the production of
deuterated fungal chitosan directly from the cultivation of filamentous fungi and
yeasts in a culture medium containing a deuterated carbon source. That’s way it
would be possible to detect chitosan along its path through the body in vivo tests. The
use of fungal chitosan has been intensively explored by some researchers since it is
possible to work with characteristics that add advantages to the entire chain of
production, commercialization, and consumption of the drug.
In the pharmaceutical area, some works stand out for bringing innovations with
potential for application to the consumer market. Rizeq et al. (2019) reveal a series of
results that show that chitosan-based nanomaterials have been gaining notoriety due
to satisfactory results. This study highlights the importance of chitosan-based nano-
particles in the delivery of drugs and genes, as well as in the therapeutic delivery for
cancer, for wound healing, and as bactericidal agents. Through the data, it is possible
to understand that by making some chemical modifications via hydroxyl and amino
groups, new nanoparticles can be improved concerning stability and biocompatibil-
ity, increasing their effectiveness for different actions.
The interest in drug release research is due to a growing concern in the use of
antibiotics in the accumulation of synthetic substances in the body, in addition to an
increase in the rate of selection of microorganisms resistant to commonly used drugs.
For this reason, different groups of researchers around the world have been testing
chitosan as a bactericidal and fungicidal agent. Initially, there was exclusivity in the
use of animal chitosan; however, in the last 5 years, there was an increase in the use
of fungal chitosan as a bactericidal and fungicidal agent (Batista et al. 2011;
Alshubaily and Al-Zahrani 2019).
Regarding the antimicrobial activity, four main mechanisms are proposed, regard-
less of the chitosan origin, since the activity has been mainly related to its degree of
deacetylation and molecular weight. For the antimicrobial action to occur, some
studies show a connection with the degree of deacetylation above 75% and low
molecular weight (Amorim et al. 2003; Paiva et al. 2014; Martinez-Camacho et al.
2010):

1. Formation of polyelectrolyte complexes: chitosan has a positive residual surface

charge present in the chain. These charges are due to the presence of an amine
group that selectively binds to the cell surface of microorganisms, favoring
changes in membrane permeability with consequent disruption, which may result
in inhibition or cell death. This event leads to the entry of calcium ions as has
been reported (Fig. 3).
Chitosan 13

Fig. 3 Scanning electron microscopy of interaction between fungi chitosan and Escherichia coli.
The arrows indicate rupture of cell wall. (a) untreated amplified 12,000x; (b) treated in presence of
0.5% of chitosan dissolved in 0.5% of acetic acid (v/v) 30,000x (Batista et al. 2011)

2. Chelation of ions necessary for the functioning of enzymes aimed at maintaining

cellular integrity and passing information, thus, compromising cell growth.
3. Interaction of low molecular weight chitosan with microbial cell DNA and
interference in mRNA activities, affecting protein synthesis.
4. The fungicidal mechanism involves penetration of chitosan molecule inside
hyphae fungus body, followed by disruption of the enzyme structure, essential
for fungus growth.

Along with research on antimicrobial activity, there are studies on drug delivery
about the composite formed based on chitosan. Alshubaily and Al-Zahrani (2019)
used the fungal chitosan extracted from Cunninghamella elegans to transport cef-
triaxone (semi-synthetic antibiotic widely applied for treating several bacterial
infections). They formed nanoconjugates by using the ionic reticulation method.
Their results showed that fungal chitosan/ceftriaxone nanoparticles were crosslink
and form particles with sizes of 56 nm, a charge of 54.37%, and 79.43% reticulation
efficiency. Thus, scientists confirmed the efficiency of the antibacterial activity of the
fungal chitosan/ceftriaxone nanocomposite against three strains of Staphylococcus
aureus (resistant to methicillin), using disk diffusion and scanning electron micro-
scope images.
Chitosan has also been used against fungal strains resistant to antimycotics
already commercialized. For example, Alshubaily (2019) reports that chitosan
nanocomposites extracted from Aspergillus niger, associated with costus extract
(Saussurea costus), act against Candida spp. strains, especially those resistant to
fungicides; El Rabey et al. (2019) used fungal chitosan extracted from Amylomyces
rouxii conjugated with fluconazole against Candida parapsilosis and Candida
glabrata strains and obtained increased antimycotic activity when using the
nanoconjugate.
In addition to the efficient discoveries of the drug delivery systems and antimi-
crobial activity of fungal chitosan, researchers have been trying to assess the
14 A. C. de Lima Batista et al.

effectiveness of the antitumor (Chien et al. 2016; Almutairi et al. 2020), antioxidant,
and antidiabetic (Sathiyaseelan et al. 2020) activity.
Chien et al. (2016) demonstrate a significant improvement in the anticancer
activity of compounds formed from fungal chitosan compared with those from
animal chitosan. Almutairi et al. (2020) produced fungal chitosan nanocomposites
from Amylomyces rouxii and curcumin, an active substance in the Curcuma
(Curcuma longa) that increases anticancer availability and bioactivity. It was possi-
ble to increase the antitumor activity on some types of colon cancer and human lung
adenocarcinoma after 96 h of exposure to this nanocomposite. Sathiyaseelan et al.
(2020) increased the antidiabetic, antioxidant, and antibacterial activity of a system
that synthesized silver nanoparticles encapsulated by fungal chitosan extracted from
Cunninghamella elegans. Also, good biocompatibility and less cytotoxicity in
healthy cells have been proved compared to cancer cells.
In addition to the direct action reported by the authors above, chitosan deserves
special mention because it also acts indirectly on tumor cells, and it has been
reported by:

1. Increased Caspase-3 protein expression, which induces cell apoptosis (Liu et al.
2017)
2. Increased production of antioxidant enzymes, suppression of expression of
pro-inflammatory cytokines, and production of nitric oxide (Nam et al. 2007)
3. Negative regulation of matrix metalloproteinase-2 (MMP-2) expression, indicat-
ing a possible suppression of the cancer cell metastasis process (Luo et al. 2014)

The enzyme immobilization technique applied in the cosmetic, food, and phar-
maceutical industries is another interesting characteristic. As an example, Amorim
et al. (2003) used fungal chitosan as a film to perform lipase immobilization. In this
experiment, the degree of deacetylation of fungal chitosan was 88.9%. This charac-
teristic provided a smaller number of acetamide clusters (reactive points), which
favored a decrease in the molecule density and enzyme immobilization. As a result,
it was found 47% of the initial catalytic activity after four reaction cycles and
efficiency statistically compared to the use of animal chitosan.

3.2 Applications and Uses in Biomedical Fields

Following the advancement of research worldwide, the interest and use of fungal
chitosan in medicine are also increasing. Currently, the majority of works still use
animal chitosan for biomedical applications, but fungal chitosan has been gaining
ground mainly because of companies producing their products in the area and selling
chitosan powder for manufacturing by others (Ding et al. 2020; Kitozyme ®; ChiBio ®).
Here, it is essential to emphasize that chitosan has effective properties in the
manufacture of dressings, production of hydrogels and membranes, scaffold con-
struction, and ideal structures for cell regeneration. These frameworks are usually
built for tissue engineering and have compatible physicochemical and biological
Chitosan 15

properties, allowing good adhesion of new cells to them. The properties required for
an effective framework include biodegradability, biocompatibility, biomineraliza-
tion, porosity, expansion capacity, protein absorption, wettability, and mechanical
strength, all of them present in fungal chitosan materials (Ahmed et al. 2018; Paiva
et al. 2021).
Sathiyaseelan et al. (2017, 2018) revealed the potential of fungal chitosan from
Cunninghamella elegans in the formation of nanocomposites added with Aloe vera
extract to obtain a product for wound dressing applications. The nanocomposite
produced showed satisfactory results against different pathogenic bacteria in vitro,
and cell viability against human dermal fibroblasts (HDF cells) was proved in vitro.
The results were significantly important and complemented the following year with
other cell lines and associating the release of tetracycline hydrochloride antibiotic.
Also, the literature describes the importance of fungi chitosan as a catalyst in the
wound healing process, stimulating inflammatory cells, macrophages, and fibro-
blasts, thus speeding up the inflammatory phase. Its effectiveness is even more
evident in mitigating diabetic damage, as it starts the proliferative phase earlier
(Matica et al. 2019).

4 Conclusion

The use of chitosan, fungal or animal, has several advantages for biotechnological
processes and has been highly demanded in the biomedical industry. Even with the
large-scale use of animal chitosan, the advantages of applying fungal chitosan are
noticeable, especially for the biomedical area. It can be produced in an environmen-
tally friendly way and does not require expensive purification processes. Although
the presence of glucan in extracts of some filamentous fungi is reported, the literature
describes members of the Zygomycetes class as low glucan and good chitosan
producers.
Thus, considering the advantages and descriptions of successful applications
mentioned here, we hope that new research groups will be interested in working
with fungal chitosan for the biomedical area, among others.

References
Ahmed S, Annub AAA, Sheikh J. A review on chitosan centred scaffolds and their applications in
tissue engineering. Int J Biol Macromol. 2018;116:849–62.
Almutairi FM, El Rabey HA, Tayel AA, et al. Augmented anticancer activity of curcumin loaded
fungal chitosan nanoparticles. Int J Biol Macromol. 2020;155:861–7.
Alshubaily FA. Enhanced antimycotic activity of nanoconjugates from fungal chitosan and
Saussurea costus extract against resistant pathogenic Candida strains. Int J Biol Macromol.
2019;141:499–503.
Alshubaily FA, Al-Zahrani MH. Appliance of fungal chitosan/ceftriaxone nanocomposite to
strengthen and sustain their antimicrobial potentiality against drug resistant bactéria. Int J Biol
Macromol. 2019;135:1246–51.
16 A. C. de Lima Batista et al.

Amorim RVS, Melo ES, Carneiro-da-Cunha MG, et al. Chitosan from Syncephalastrum
racemosum used as a film support for lípase immobilization. Bioresour Technol. 2003;89:35–9.
Annu SA, Ahmed S, Ikram S. Chitin and chitosan: history, composition and properties. In:
Ahmed S, Ikram S, editors. Chitosan: derivatives, composites and applications. Beverly:
Scrivener Publishing LLC; 2017.
Araki Y, Ito E. A pathway of chitosan formation in Mucor rouxii: enzymatic deacetylation of chitin.
Biochem Biophys Res Commun. 1974;55:669–75.
Baker LG, Specht CA, Donlin MJ, Lodge JK. Chitosan, the Deacetylated form of chitin, is
necessary for cell wall integrity in Cryptococcus neoformans. Eukaryot Cell. 2007;6:855–67.
Batista ACL, Dantas GC, Santos J, et al. Antimicrobial effects of native chitosan against opportu-
nistic gram-negative bacteria. Microbiol J. 2011;1:105–12.
Batista ACL, Bandeira MGL, Souza Neto FE, et al. Obtenção de quitosana fúngica crescida em
meio alternativo constituído com farinha de carapaça de camarão. Revista Saúde e Ciência On
Line. 2014;3:11–7.
Batista ACL, Souza Neto FE, Paiva WS. Review of fungal chitosan: past, present and perspectives
in Brazil. Polímeros [online]. 2018;28:275–83.
Batista ACL, Melo TBL, Paiva WS. Economic microbiological conversion of agroindustrial waste
to fungi chitosan. Acad Bras Cienc. 2020;92:e20180885.
Baxter A, Dillon M, Taylor K, et al. Improved method for i.r. determination of the degree of
N-acetylation of chitosan. Int J Biol Macromol. 1992;4:166–9.
Benchamas G, Huang G, Huang S, et al. Preparation and biological activities of chitosan oligosac-
charides. Trends Food Sci Technol. 2021;107:38–44.
Brugnerotto J, Lizardi J, Goycoolea FM, et al. An infrared investigation in relation with chitin and
chitosan characterization. Polymer. 2001;42:3569–80.
Cabib E, Bowers B, Sburlati A, et al. Fungal cell wall synthesis: the construction of a biological
structure. Microbiol Sci. 1988;5:370–5.
Campos-Takaki GM. The fungal versatility on the copolymers chitin and chitosan production. In:
Dutta PK, editor. Chitin and chitosan opportunities and challenges. Allahabad: Dubey Printers
and Graphics; 2005. p. 69–94.
Campos-Takaki GM, Beakes GW, Dietrich SM. Electron microscopic X-ray microprobe and
cytochemical study of isolated cell walls of mucoralean fungi. Trans Br Mycol Soc. 1983;80:
536–41.
Canevaloro SV Jr. Técnicas de Caracterização de Polímeros. ArtLiber Associação Brasileira de
Polímeros. SP: São Carlos; 2004.
Chiappisi L, Gradzielski M. Co-assembly in chitosan–surfactant mixtures: thermodynamics, struc-
tures, interfacial properties and applications. Adv Colloid Interf Sci. 2015;220:92–107.
Chien RC, Yen MT, Mau JL. Antimicrobial and antitumor activities of chitosan from shiitake stipes,
compared to commercial chitosan from crab shells. Carbohydr Polym. 2016;138:259–64.
Clark GL, Smith AF. X-ray diffraction studies of chitin, chitosan and derivatives. J Phys Chem.
1937;40:863–79.
Davis LL, Bartinicki-Garcia S. Chitosan synthesis by the tandem action of chitin synthetase and
chitin deacetylase from Mucor rouxii. Biochemist. 1984;23:1065–73.
Denari GB, Cavalheiro ETG. Princípios e aplicações de análise térmica. São Carlos: IQSC/
USP; 2012.
Ding S, Wang Y, Li J, et al. Progress and prospects in chitosan derivatives: modification strategies and
medical applications. J Mater Sci Technol. 2020; https://doi.org/10.1016/j.jmst.2020.12.008.
Domszy JG, Roberts G. Evalution of infrared spectroscopic techniques for analyzing chitosan.
Macromol Chem Phys. 1985;186:1671–7.
Dong Y, Xu C, Wang J, et al. Determination of degree of substitution for N-acylated chitosan using
IR spectra. Sci China Ser B. 2001;44:216–24.
Dung P, Milas M, Rinaudo M, et al. Water soluble derivatives obtained by controlled chemical
modifications of chitosan. Carbohydr Polym. 1994;24:209–14.
Chitosan 17

El Rabey HA, Almutairi FM, Alalawy AL, et al. Augmented control of drug-resistant Candida spp.
via fluconazole loading into fungal chitosan nanoparticles. Int J Biol Macromol. 2019;141:511–6.
Fesel PH, Zuccaro A. β-glucan: crucial component of the fungal cell wall and elusive MAMP in
plants. Fungal Genet Biol. 2016;90:53–60.
Franca EF, Freitas LCG, Lins RD. Chitosan molecular structure as a function of N-acetylation.
Biopolymers. 2011;95:448–60.
Ghormade V, Pathan EK, Deshpande MV. Can fungi compete with marine sources for chitosan
production? Int J Biol Macromol. 2017;104:1415–21.
Heux L, Brugnerotto J, Desbrieres J, et al. Solid state NMR for determination of degree of
acetylation of chitin and chitosan. Biomacromolecules. 2000;1:746–51.
Hirai A, Odani H, Nakajima A. Determination of degree of deacetylation of chitosan by 1H NMR
spectroscopy. Polym Bull. 1991;26:87–94.
Hu KJ, Yeung KW, Ho KP, et al. Rapid extraction of high-quality chitosan from mycelia of Absidia
glauca. J Food Biochem. 1999;23:187–96.
Ifuku S, Nomura R, Morimoto M, et al. Preparation of chitin nanofibers from mushrooms.
Materials. 2011;4:1417–25.
Irastorza A, Zarandona I, Andonegi M, et al. The versatility of collagen and chitosan: from food to
biomedical applications. Food Hydrocoll. 2021; https://doi.org/10.1016/j.foodhyd.2021.106633.
Islam S, Bhuiyan MAR, Islma MN. Chitin and chitosan: structure, properties and applications in
biomedical engineering. J Polym Environ. 2017;25:854–66.
Jaworska MM, Konieczna E. The influence of supplemental components in nutrient medium on
chitosan formation by the fungus Absidia orchidis. Appl Microbiol Biotechnol. 2001;56:220–4.
Jeraj N, Kunic B, Lenasi H, et al. Purification and molecular characterization of chitin deacetylase
from Rhizopus nigricans. Enzym Microb Technol. 2006;39:1294–9.
Jong MAWP, Vried LEM, Theelen B, et al. C-type lectin Langerin is a β-glucan receptor on human
Langerhans cells that recognizes opportunistic and pathogenic fungi. Mol Immunol. 2010;47:
1216–25.
Khan TA, Peh KK, Ch’ng HS. Reporting degree of deacetylation values of chitosan: the influence of
analytical methods. J Pharm Pharmacol. 2002;5:205–12.
Kozlowska E, Brzezinska-Blaszczyk E, Rasmus P, et al. Fungal β-glucans and mannan stimulate
peripheral blood mononuclear cells to cytokine production in Syk-dependent manner.
Immunobiology. 2020;225:151985.
Kreger DR. Observations on cell walls of yeast and some other fungi by X-ray diffraction and
solubility tests. Biochim Biophys Acta. 1954;13:1–9.
Kumirska J, Czerwicka M, Kacznski Z, et al. Application of spectroscopic methods for structural
analysis of chitin and chitosan. Mar Drugs. 2010;8:1567–636.
Lavertu M, Xia Z, Serreqi AN, et al. A validated 1H NMR method for the determination of the
degree of deacetylation of chitosan. J Pharm Biomed Anal. 2003;32:1149–58.
Li J, Revol JF, Marchessault RH. Effect of degree of deacetylation of chitin on the properties of
chitin crystallites. J Appl Polym Sci. 1997;65:373–80.
Liu L, Xin Y, Liu J, et al. Inhibitory effect of chitosan oligosaccharide on human hepatoma cells
in vitro. Afr J Tradit Complement Altern Med. 2017;14:272–7.
Luo Z, Dong X, Ke Q, et al. Downregulation of CD147 by chitooligosaccharide inhibits MMP-2
expression and suppresses the metastatic potential of human gastric cancer. Oncol Lett. 2014;8:
361–6.
Martínez-Camacho AP, Cortez-Rocha MO, Ezquerra-Brauer JM. Chitosan composite films: ther-
mal, structural, mechanical and antifungal properties. Carbohydr Polym. 2010;82:305–15.
Matica MA, Aachmann FL, Tøndervik A, et al. Chitosan as a wound dressing starting material:
antimicrobial properties and mode of action. Int J Mol Sci. 2019;20:5889.
Mohammadi M, Zamani A, Karimi K. Determination of glucosamine in fungal cell walls by high-
performance liquid chromatography (HPLC). J Agric Food Chem. 2012;60:10511–15.
Mohan K, Ganesan A, Muralisankar T, et al. Recent insights into the extraction, characterization,
and bioactivities of chitin and chitosan from insects. Trends Food Sci Technol. 2020;105:17–42.
18 A. C. de Lima Batista et al.

Naghdi M, Zamani A, Karimi K. A sulfuric-lactic acid process for efficient purification of fungal
chitosan with intact molecular weight. Int J Biol Macromol. 2014;63:158–62.
Nam KS, Kim MK, Shon YH. Inhibition of proinflammatory cytokine induced invasiveness of
HT-29 cells by chitosan oligosaccharide. J Microbiol Biotechnol. 2007;17:2042–5.
Paiva WS, Souza Neto FE, Batista ACL. Avaliação da atividade antibacteriana da quitosana
fúngica. Perspect Online, Biol Saúde. 2014;13:37–43.
Paiva WS, Souza Neto FE, Batista ACL. Characterization of polymeric biomaterial chitosan
extracted from Rhizopus stolonifer. J Polym Mater. 2017;34:115–21.
Paiva WS, Souza Neto FE, Paiva ES, Batista ACL. Fungal chitosan as membranous material
modified by atmospheric plasma. Res Soc Dev. 2021;10:9210111543.
Rasweefali MK, Sabu S, Sunooj KV, et al. Consequences of chemical deacetylation on physico-
chemical, structural and functional characteristics of chitosan extracted from deep-sea mud
shrimp. Carbohydr Polym Technol Appl. 2021;2:100032.
Rinaudo M, Le Dung P, Gey C, et al. Sustituent distribution on O,N-carboxymethylchitosa by 1H
and 13C n.m.r. Int J Biol Macromol. 1992;14:122–8.
Rinaudo M, Milas M, Le Dang P. Characterization of chitosan: influence of ionic strength and
degree of acetylation on chain expansion. Int J Biol Macromol. 1993;15:281–5.
Rizeq BR, Younes NN, Rasool K, et al. Synthesis, bioapplications, and toxicity evaluation of
chitosan-based nanoparticles. Int J Mol Sci. 2019;20:5776–800.
Ruiz-Herrera J, Ortiz-Castellanos L. Cell wall glucans of fungi. A review. Cell Surf.
2019;5:100022.
Sathiyaseelan A, Shajahan A, Kalaichelvan PT. Fungal chitosan based nanocomposites sponges –
an alternativemedicine for wound dressing. Int J Biol Macromol. 2017;104:905–1915.
Sathiyaseelan A, Saravanakumar K, Mariadoss AVA, et al. Biocompatible fungal chitosan encap-
sulated phytogenic silver nanoparticles enhanced antidiabetic, antioxidant and antibacterial
activity. Int J Biol Macromol. 2020;153:63–710.
Shehata AN, El Aty AA, Darwish DA, et al. Purification, physicochemical and thermodinamic studies
of antifungal chitinase with production of bioactive chitosan-oligosaccharide from newly isolated
Aspergillus griseoaurantiacus KX010988. Int J Biol Macromol. 2018;107:990–9.
Skovstrup S, Hansen SG, Skrydstrup T. Conformational flexibility of chitosan: a molecular
modeling study. Biomacromolecules. 2010;11:3196–207.
Spranghers T, Ottoboni M, Klootwijk C, et al. Nutritional composition of black soldier fly
(Hermetia illucens) prepupae reared on different organic waste substrates. J Sci Food Agric.
2017;97:2594–600.
Synowiecki J, Ali-Khateeb NAAQ. Mycelia of Mucor rouxii as a source of chitin and chitosan.
Food Chem. 1997;60:605–10.
Tavaria FK, Costa EM, Pina-Vaz I, et al. A quitosana como biomaterial odontológico: estado da
arte. Braz J Biom Eng. 2013;29:110–20.
Wang W, Xue C, Mao X. Chitosan: structural modifications, biological activity and application. Int
J Biol Macromol. 2020;164:4532–46.
White SA, Farina PR, Fulto I. Production and isolation of chitosan from Mucor rouxii. Appl
Environ Microbiol. 1979;38:323–8.
Yang BY, Montgomery R. Degree of acetylation of heteropolysaccharides. Carbohydr Res.
2000;323:156–62.
Yen MT, Mau JL. Selected physical properties of chitin prepared from shiitake stipes. LWT–Food
Sci Technol. 2007;40:558–63.
Yuan Y, Li H, Leite W, et al. Biosynthesis and characterization of deuterated chitosan in filamen-
tous. Carbohydr Polym. 2021;257:117637.
Yui T, Imada K, Okuyama K. Molecular and crystal structure of the anhydrous form of chitosan.
Macromolecules. 1994;27:7601–5.